(497b) Image-Guided, Bioactive Gas Delivery with Ultrasound-Activatable Microbubbles for Treatment of Ischemic and Traumatic Injuries

Therapeutic gases like xenon (Xe), argon (Ar), nitric oxide (NO), oxygen, have been shown to be an effective treatment for acute onset conditions of hypoxia and ischemia reperfusion injuries. Xenon is especially promising due to its otherwise inert nature thus precluding harmful side effects. Xenon is postulated to block the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors on cell surfaces, preventing a cascade that results from an injury and ultimately leads to apoptosis. Argon, another noble gas is postulated to stop an injury cascade by interfering with the PI3k/Akt apoptotic pathway and can serve as a less expensive and more widely available alternative to xenon. Clinical trials with xenon use inhalation for administration: this method is systemic, wasteful, and not cost effective.

Microbubbles containing a gas core offer an excellent option to local, safe, image-guided delivery of bioactive gases instead of systemic administration. Microbubbles, commercially used for diagnostic imaging, usually encapsulate a water-insoluble fluorous gas like perfluorocarbons or sulfur hexafluoride to prevent efflux from the bubble in aqueous media. We have developed microbubbles (MBs) encapsulating pure xenon gas as a first in the field. While there are limited examples of Xe MBs in literature, these have used a stabilizing fluorocarbon gas in the core to keep the otherwise water-soluble gas trapped inside the bubble, since therapeutic gases are tens of time more water-soluble than fluorocarbon gases. Through optimization of the phospholipid composition of the microbubble shell, we demonstrated that a long-chain phospholipid dibehenoylphosphatidylcholine (DBPC, C22:0), can stabilize 1-10 µm Xe and Ar microbubbles and keep them stable for several days in storage. This is likely due to the greater intermolecular van der waals cohesive energy for long-chain lipids which provides rigid packing to reduce gas efflux. We have then showed that both Xe and Argon microbubbles Xe and Ar microbubbles elicit significant and persistent non-linear, bubble-specific, ultrasound signal, in both a tissue-mimicking phantom and in vivo in a small (mouse) and large (pig) animal model. On intravenous injection, both as bolus of ~10⁹MBs/m and as an infusion at 0.6 mL/min of ~10⁸MBs/mL, we saw sustained ultrasound contrast for pure Xe MBs in a pig heart and carotid artery.

To establish the therapeutic effects of these Xe MBs in injury protection, we carried out trials for neuroprotection using Xe MBs in a large animal model of traumatic brain injury
(TBI). A well-established, focal controlled cortical impact porcine model of TBI was used with 1 month old, ~10 kg pigs, whereupon Xe MBs were injected through infusion for ~20 min at 1, 3, and 24 hrs after injury onset. Fluorocarbon bubbles were used as Control. MBs were visualized using a commercial clinical scanner in the carotid artery followed by an increase in ultrasound power to burst the bubbles and release the gas near the brain. After 1 and 5 days, magnetic resonance imaging (MRI) scans of the pig brain revealed significantly reduced perilesional edema in the xenon-treated group. In the Control group treated with fluorocarbon bubbles with the same shell composition,core/hemorrhage size was seen to increase. Histopathological H&E staining studies carried out with the 5 mm coronal brain sections showed lower endothelial proliferation and perivascular inflammation (lower migration of lymphocytes and macrophages) near the injury site for the xenon treated group. In summary, this study presents a first demonstration of pure xenon and argon microbubbles that are echogenic in vivo. It also shows the feasibility of neuroprotection using Xe MBs, thus providing a highly promising theranostic agent for local, non-systemic, gas delivery for ultrasound image-guided treatment of different kinds of injury. Ongoing work is exploring the lowered migration of cells near injury site in vitro in the presence of Xe MBs. Additionally, we are testing argon and nitric oxide bubbles for treating ischemia reperfusion injuries in vitro, where cells have been exposed to oxygen-glucose deprivation. Together, this presentation will focus on a variety of microbubbles with bioactive gases like xenon, argon, and nitric oxide as image-guided gas delivery agents for non-invasive, non-systemic, cytoprotective treatments of acute ischemic/traumatic injuries.