Sonoselective transfection of cerebral vasculature without blood–brain barrier disruption

Significance Focused ultrasound (FUS) is a targeted and noninvasive technique that can be used to activate gas-filled microbubbles (MBs) to oscillate within the bloodstream. This technique has been used previously to open the blood–brain barrier (BBB) to facilitate the delivery of therapeutics to the surrounding brain tissue. However, disruption of the BBB may be contraindicated in certain disease contexts. Here, we utilize low-pressure FUS to oscillate the MBs just enough to transfect endothelial cells, without opening the BBB. The low-pressure FUS regimen results in enhanced gene delivery to endothelial cells, with none of the inflammatory or immune pathway up-regulation observed at higher FUS pressures.

On the morning of experiments, the appropriate purified plasmid was thawed and added to the cleaned MB solution at a ratio of 1.5 ug plasmid per 10 7 microbubbles. This ratio is consistent with prior studies of DNA binding to cationic MBs(1, 2). This mixture was allowed to incubate at room temperature for 10 min to permit the electrostatic coupling of the positively-charged bubbles and negatively-charged DNA, and was then stored on ice until use. Roughly 20-25% of the plasmid added to the MBs bound, for a total of 0.03 to 0.035 pg per MB. We did not observe significant changes to the size distributions of the MBs after conjugation to plasmid ( Figure S1).

Passive Cavitation Detection
Acoustic emissions were detected with a 2.5 mm wideband unfocused hydrophone mounted in the center of the transducer. Acoustic signal was captured using a scope card (ATS460, Alazar, Pointe-Claire, Canada) and processed using an in-house built MATLAB algorithm. Acoustic emissions at the fundamental frequency, harmonics (2f, 3f, 4f), sub harmonic (0.5f), and ultra-harmonics (1.5f, 2.5f, 3.5f) were assessed by first taking the root mean square of the peak spectral amplitude (Vrms) in each frequency band after applying a 200 Hz bandwidth filter, and then summing the product of Vrms and individual sonication duration over the entire treatment period. Broadband emissions were assessed by summing the product of Vrms and individual sonication duration for all remaining emissions over the entire treatment period.

Histological Processing
Immediately following euthanasia via an overdose of pentobarbital sodium and phenytoin sodium, animals were perfused via the carotid arteries with 10 ml of 2% heparinized 0.9% saline followed by 5 ml of 4% paraformaldehyde. The brains were suffusion-fixed in 4% paraformaldehyde for 24 hours at 4 degrees Celsius, followed by desiccation in 30% sucrose for 24 hours at 4 degrees Celsius. The desiccated brains were then equilibrated in OCT compound for 1 hour prior to flash freezing and storage at -80 degrees Celsius. The brains were then mounted with OCT and sectioned using a cryostat (Leica, Buffalo Grove, Illinois) into 5 um thick sections.

Immunofluorescence -GLUT1 Staining of Endothelium
To assess endothelial selectivity of transfection using GLUT1 as an endothelial marker, mounted sections were washed 3× for 10 min in PBS with 0.1% Tween 20 and Sections were imaged on a Nikon Eclipse TE2000 confocal microscope equipped with a 20× oil objective. Endothelial selectivity was assessed using ImageJ by manually comparing co-localization of mCherry expression with GLUT1 expression. At least three representative fields of view were counted from the FUS-treated region of the brain, as well as three fields of view from the contralateral side of the brain.
Sections were imaged on a Nikon Eclipse TE2000 confocal microscope equipped with a 20× oil objective. Endothelial selectivity was assessed using ImageJ by manually comparing co-localization of mCherry expression with GLUT1 expression. At least three representative fields of view were counted from the FUS-treated region of the brain, as well as three fields of view from the contralateral side of the brain.

Bioluminescence Measurements
To assess biodistribution of the transgene and off-target transfection, mice received MBs conjugated to a luciferase plasmid and were treated with FUS in the right hemisphere of the brain. One day later, the mice were sacrificed and their organs (brain, lungs, heart, kidneys, and liver) were harvested and placed in a solution of D-Luciferin (150 ug/ml; Gold Biotechnology, St. Louis, MO) in PBS for 5 minutes. The organs were then imaged using an IVIS100 imaging system (Xenogen, Alameda, CA). Photons were collected and integrated for a period of 1 minute. Images were then processed using Xenogen's Living Image software.

Bulk RNA Sequencing and Analysis
Immediately following euthanasia, the mouse brains were harvested and the front right quadrants (FUS-treated region) were excised, placed in RNAlater (Qiagen), and stored at -80 °C. RNA extraction was performed using the RNeasy Mini Kit (Qiagen).     Bioluminescence images of heart, lungs, liver, kidneys, and brains from mice (n=3) taken 24h after luciferase plasmid was coupled to cationic microbubbles, injected I.V., and delivered to cerebrovascular endothelium under stereotactic guidance using 1 MHz FUS with a PNP of 0.1 MPa. Luciferase was robustly expressed in FUS-treated brains (arrows), but was undetectable in off-target organs (asterisks). The brain in the middle row was inadvertently bisected during processing.  Table S1. FDR adjusted p-values for relative expression levels of "anti-inflammatory" transcripts. Transcript expression significance levels are shown for FUS+MB groups at all 3 tested peak-negative pressures in comparison to the "MB only" control group. No significant differences were observed.