Abstract Body:

Background, Motivation and Objective

Focused ultrasound (FUS) combined with microbubbles is a promising non-invasive approach for delivering therapeutics to the brain by temporarily opening the blood-brain barrier (BBB). There is an unmet clinical need for effective brain delivery of disease-modifying drugs for neurodegenerative disorders. Clinical trials are underway to establish the safety and feasibility of large-volume blood-brain barrier opening in humans combined with antibody-based therapies [1] [2]. While this approach shows promise for small molecules, delivery of larger molecules like antibodies (IgG, MW ~165 kDa) across the BBB remains a unique challenge. Hence, it is important to understand the cellular effects associated with FUS-BBB opening in large brain regions, especially when attempting delivery of larger molecular-weight therapeutics such as antibodies. While increasing ultrasound pressure is generally known to enhance drug delivery to the brain, especially with smaller molecules, it is still unclear whether this holds for larger molecules such as immunoglobulins. [3] [4]. We hypothesized that a healthy endothelium is required for transient permeabilization of the BBB to facilitate the delivery of large molecules like IgG antibodies. To test this hypothesis, we conducted a parametric study using large-volume sonication in the striatum to assess the delivery of endogenous IgG antibodies.

Statement of Contribution/Methods

Antibodies hold promise for a wide range of neurodegenerative disorders including Parkinson’s disease [5]  Alzheimer’s disease [6] and Huntington’s disease [7]. Over the last 15 years, over 50 drugs of this category have been approved for clinical use, with dozens more in the pipeline [8]. Improved delivery with focused ultrasound of antibodies to large volumes of the striatum could lead to the development of effective therapies for movement disorders.

A 1.1MHz single-element transducer (Sonic Concepts, WA, USA) was mounted on a stereotactic frame using a customized 3D-printed holder. C57BL/6 mice underwent unilateral sonication of the striatum under general anesthesia for a total duration of 1 minute. The following sonication parameters were used: 10 ms burst duration, 1 Hz pulse repetition frequency, and increasing acoustic pressures of 0.30, 0.35, and 0.40 peak negative pressure (PNP). A microbubble bolus was applied via the tail vein before sonication at a dose of 1 μl/g/BW (1:50 dilution in PBS).  Animals were perfused at 24 hours with aldehydes and brains were collected for histological analysis. Brains were sectioned at 40μm in the transverse plane and processed for free-floating immunohistochemistry.

IgG antibody deposition throughout the rostrocaudal axis of the dorsal striatum was quantified using the Cavalieri probe (StereoInvestigator, MBF Biosciences). Adjacent sections were assessed for cell structure (Nissl staining), cell death (Cleaved caspase-3), and blood vessel integrity (collagen-IV). Statistical analysis was performed using GraphPad Prism 10.

Results

All three tested acoustic pressures successfully delivered endogenous antibodies throughout the striatum (Fig. 1a), with a single sonication session achieving penetration in up to 90% of the striatal volume (Fig. 1b). However, higher pressures did not further increase antibody deposition. Histological analysis revealed that higher pressure-induced BBB disruption limits antibody passage due to endothelial barrier damage and hemorrhage, associated with increased apoptosis (Fig 1d). Despite these detrimental effects, stereological quantification revealed no significant volume loss in the high-pressure treated striatum compared to the untreated striatum, although elevated levels of cleaved caspase-3 were observed (Fig. 1c).

Discussion

Taken together, our findings suggest an optimal acoustic pressure range exists for achieving uniform antibody distribution within the brain using FUS-BBBO. Preserving endothelial cell health is essential for effective antibody delivery using FUS.  Higher pressures, while potentially damaging to the endothelium, may be suitable in neuro-oncology where the blood-tumour barrier hinders antibody delivery and cell death is a desired outcome.

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