In vivo therapeutic gas delivery for neuroprotection with echogenic liposomes

Abstract

Background: Ischemia-related neurological injury is a primary cause of stroke disability. Studies have demonstrated that xenon (Xe) may have potential as an effective and nontoxic neuroprotectant. Xe delivery is, however, hampered by lack of suitable administration methods. We have developed a pressurization-freeze method to encapsulate Xe into echogenic liposomes (Xe-ELIP) and have modulated local gas release with transvascular ultrasound exposure.

Methods and results: Fifteen microliters of Xe were encapsulated into each 1 mg of liposomes (70% Xe and 30% argon). Xe delivery from Xe-ELIP into cells and consequent neuroprotective effects were evaluated with oxygen/glucose-deprived and control neuronal cells in vitro. Xe-ELIP were administered into Sprague-Dawley rats intravenously or intra-arterially after right middle cerebral artery occlusion. One-megahertz low-amplitude (0.18 MPa) continuous wave ultrasound directed onto the internal carotid artery triggered Xe release from circulating Xe-ELIP. Effects of Xe delivery on ischemia-induced neurological injury and disability were evaluated. Xe-ELIP delivery to oxygen/glucose-deprived neuronal cells improved cell viability in vitro and resulted in a 48% infarct volume decrease in vivo. Intravenous Xe-ELIP administration in combination with the ultrasound directed onto the carotid artery enhanced local Xe release from circulating Xe-ELIP and demonstrated 75% infarct volume reduction. This was comparable to the effect after intra-arterial administration. Behavioral tests on limb placement and grid and beam walking correlated with infarct reduction.

Conclusions: This novel methodology may provide a noninvasive strategy for ultrasound-enhanced local therapeutic gas delivery for cerebral ischemia-related injury while minimizing systemic side effects.

Figure 1

In vitro setup for ultrasound-triggered Xe release from Xe-ELIP under physiologic flow conditions.

Figure 2

Spontaneous Xe release profiles from Xe-ELIP in PBS and HSP over the first hour and up to 18 hours. Data are demonstrated using normalized Xe release as a percentage with respect to the initial amount of Xe in the Xe-ELIP.

Figure 3

Effects of ultrasound on Xe release from Xe-ELIP under physiological flow conditions. Low-amplitude continuous ultrasound (0.18 MPa) increased Xe release (44%) from Xe-ELIP compared to no ultrasound (5%).

Figure 4

Effects of Xe-ELIP on cell viability during hypoxia. Using the MTT cell viability assay, this demonstrates the capability of Xe-ELIP to protect cells from hypoxic damage with 90% cells remaining viable.

Figure 5

Effect of intravenous Xe-ELIP treatment in combination with ultrasound application on neurologic disability in animal model of ischemia. (A) Limb placement, (B) Beam walking, (C) Grid walking.

Figure 6

Effects of intravenous Xe-ELIP delivery in combination with ultrasound application on cerebral infarct volume in an animal model. (A) Representative TTC-stained coronal brain sections demonstrating brain infarction in rats at 3 days after MCA occlusion with no treatment, Xe-saturated saline treatment, and Xe-ELIP treatment, (B) Infarct size comparison between treatment groups demonstrating a 48% infarct volume reduction with Xe-ELIP alone and 75% reduction with Xe-ELIP combined with ultrasound activation. IA – intraarterial, IV – intravenous, US – ultrasound.

Figure 7

Velocity profile of cerebral blood flow after the MCA occlusion, reperfusion, and Xe-ELIP delivery estimated by using a laser Doppler flowmeter.

Figure 8

Diagram of a strategy to apply ultrasound treatment over the common carotid artery, a site that is distant from the cerebral infarct site, for both imaging of Xe-ELIP and ultrasound-enhanced therapeutic gas delivery.