Localized anesthesia of a specific brain region using ultrasound-responsive barbiturate nanodroplets

Abstract

Background: Targeted neuromodulation is a valuable technique for the study and treatment of the brain. Using focused ultrasound to target the local delivery of anesthetics in the brain offers a safe and reproducible option for suppressing neuronal activity. Objective: To develop a potential new tool for localized neuromodulation through the triggered release of pentobarbital from ultrasound-responsive nanodroplets. Method: The commercial microbubble contrast agent, Definity, was filled with decafluorobutane gas and loaded with a lipophilic anesthetic drug, before being condensed into liquid-filled nanodroplets of 210 ± 80 nm. Focused ultrasound at 0.58 MHz was found to convert nanodroplets into microbubbles, simultaneously releasing the drug and inducing local anesthesia in the motor cortex of rats (n=8). Results: Behavioral analysis indicated a 19.1 ± 13% motor deficit on the contralateral side of treated animals, assessed through the cylinder test and gait analysis, illustrating successful local anesthesia, without compromising the blood-brain barrier. Conclusion: Pentobarbital-loaded decafluorobutane-core Definity-based nanodroplets are a potential agent for ultrasound-triggered and targeted neuromodulation.

Keywords: focused ultrasound; phase-change emulsion; triggered drug delivery.

Figure 1

Schematic of (A) LP100 focused ultrasound system targeting the right motor cortex of a supine rat with (B) MRI guidance selecting three FUS targets.

Figure 2

Behavioral testing using gait analysis measuring (A) stride length, base width and (B) paw angle. (C) Schematics of gait analysis and cylinder test apparatus, and (D) example images from the cylinder test showing simultaneous paw use pre-treatment and ipsilateral paw use post-treatment.

Figure 3

UV-vis measurements to quantify pentobarbital concentration for (A) standard curve and (B) final droplet solution measured at a 1:2 dilution. Pentobarbital dilutions were used to quantify pentobarbital loading, showing 20 - 24 % loading efficiency of pentobarbital into Definity-based lipid droplets, achieving 22 ± 3 μg/mL.

Figure 4

Nanodroplet characterization: (A) schematic of drug-loaded lipid shell composed of 1,2-dihexadecanoyl-sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoryl-sn-glycero-3-phosphoethanolamine conjugated polyethylene glycol (MPEG5000 DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), (B) nanodroplet size distribution by nanoparticle tracking analysis at 1:10 dilution (n=3) with example brightfield image (scale bar 10 μm), (C) resultant microbubble size distribution measured by Coulter Counter (n=3) with example brightfield image (scale bar 10 μm), (D) spontaneous drug release measured in storage conditions of 4°C, 23°C, 37°C, (E) acoustic vaporization threshold in vitro shown through ultraharmonic emissions (black) alongside percentage of pentobarbital released (red) at increasing sonication pressures (n=6), (F) and comparison of vaporization pressures in vitro and in vivo (n=8), error bars show one standard deviation.

Figure 5

In vivo (A) feedback-controlled sonication pressure and (B) detected ultraharmonic emissions during a 180 s duration treatment, with droplet bolus injection at 10 s, indicated with arrows. Persistence of ultraharmonic emissions for sham and pentobarbital-loaded (PB-loaded) droplets in circulation in vivo at (C) 1 hour, (D) 3 hours and (E) 5 hours post-fabrication (time of nanodroplet bolus injection indicated with arrow). Each plot shows repetitions from 3 animals, each with an independent batch of droplets. Error bars show one standard deviation.

Figure 6

Behavioral changes induced by localized anesthesia in 8 treated animals and 5 animals in each control group. Change in paw angle is shown for (A) each treated animal individually and (B) summarized for each treatment group (**p<0.01, assessed by paired t-test). Ipsilateral paw preference is significantly increased for treated animals compared to control groups (C) (D), error bars show one standard deviation and statistical significance is defined by p < 0.05 (*p<0.05, ***p<0.001, assessed by analysis of variance).

Figure 7

MR images of (A) pre-treatment (T1-weighted) and (B, C) post-treatment (T1-weighted, T2*-weighted) axial slices showing no detectable BBB-opening or damage. Histology sections (D) with H&E staining and 5 μm slice thickness also confirm no tissue damage.

Figure 8

Assessment of gross histology and H&E stained sections from major organs - kidney (Ki), spleen (Sp), brain (Br), heart (He), lung (Lu) and liver (Li) - showed no signs of toxicity or necrosis (scale bars 100 μm).