SALT LAKE CITY—A new method of drug delivery, encapsulating Xenon (Xe) in echogenic liposomes (ELIPs) and then releasing the gas by ultrasound triggering, may provide effective neuroprotection against cerebral hypoxic-ischemic injury after stroke, according to findings presented at the 133rd Annual Meeting of the American Neurological Association.
“To date, research findings have found Xe to be an ideal anesthetic gas with neuroprotective properties by acting as a noncompetitive N-methyl-d-aspartate antagonist, therefore blocking the excitoxicity pathway leading to apoptosis,” lead researcher George L. Britton Jr., a Research Assistant in the Department of Internal Medicine at the University of Texas Health Science Center in Houston, told Neurology Reviews. “However, its exuberant associated costs and lack of suitable administration methods have crippled its clinical use.”
The researchers therefore developed gas-encapsulated liposomes to facilitate targeted release. “These gas-encapsulated liposomes are formulated to burst and release Xe gas locally when a low-power ultrasound is applied near the site of disease,” Mr. Britton explained.
A Delivery Strategy for Xenon
The researchers prepared Xe-containing liposomes—which comprised phosphatidylcholine, positively charged phospholipids, and cholesterol—by lipid film hydration, sonication, pressurization with a gaseous mixture of Xe and argon gases, and freezing under pressure, followed by thawing. This method contrasts with the conventional freeze-thawing method of lipid film hydration, sonication, freezing, and then thawing.
Experiments were performed in a device that consisted of a transwell insert with a semipermeable polyester membrane base (pore size, 0.4 µm) resting on a Rho-C rubber block. The investigators placed a thin layer of water between the membrane and the rubber to exclude air and air bubbles. “The opening of the transwell insert allowed the introduction of Xe-containing liposomes and placement of the 1-MHz ultrasound probe (diameter, 1.2 cm) delivering ultrasound at 1 W/cm2 and 50% duty cycle for 20 seconds,” Mr. Britton and coauthors stated.
Neuroprotection Measured With Cell Cultures
PC-12 cortical cell cultures, without hippocampus and basal ganglia cells, were prepared from early postnatal rat pups. The cells were plated in Eagle’s minimum essential medium after trypsination and resuspension and reached confluence in five days.
Cell cultures were then deprived of oxygen and glucose in deoxygenated phosphate-buffered saline and transferred into an anoxic chamber containing 100% Argon at 37°C. After three hours of deprivation, Xe-containing liposomes were added to the anoxic cells. Neuronal injury was quantified by measuring lactate dehydrogenase and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] release.
Effective Encapsulation and Delivery of Xenon
According to the investigators, 10 µL of Xe was encapsulated into 1 mg of liposomes. Xe-containing liposomes demonstrated higher echogenicity than identically concentrated air-containing liposomes.
“Delivery of 20 µg of 70% Xe-containing liposomes [by ultrasound] inhibited cytotoxic events in neuronal cell lines when compared with nontreatment groups by as much as 40%,” the researchers reported. The Xe-ELIP group retained 90% cell viability, compared with the hypoxic nontreatment group.
Clinical Applications for Xe-ELIP
“Xe-ELIP may be used in any surgical setting where a loss of blood flow to tissue may occur,” Mr. Britton commented. “During a heart transplant, it is common for patients to have minor strokes because of an inherent loss of blood or perfusion to the brain. To protect against any tissue damage that may occur during this window, Xe-ELIP may be applied to inhibit the progressive excitotoxicity cascade.”
Additional studies are needed to improve the stability of Xe encapsulation and evaluate the therapeutic efficiency of Xe-liposomes for cerebral hypoxic-ischemic insult in animal models, the study authors concluded.
—Marguerite Spellman