(684d) Ultrasound-Responsive Microbubbles As Carriers of Xenon and Argon Gas for the Treatment of Traumatic Brain Injury

Mild-to-moderate traumatic brain injury (TBI) is a serious public health concern, accounting for over 90% of known TBI cases. This includes injuries suffered on a regular basis by military personnel, athletes in certain sports, victims of domestic violence, and day-to-day falls, especially in children and the elderly. Growing research shows that the adverse effects of mild-to-moderate TBI persists and often present after days or months, sometimes even years. Effects can include post-concussion syndrome and cognitive and behavioral deficits. Existing drug candidates under research require high doses to compensate for drug loss due to partial or complete blood brain barrier (BBB) impermeability, which can cause adverse side-effects. There is in fact no clinically approved drug to directly treat TBI in the market, with the inability to deliver a drug into the brain being a major challenge. In this talk, we will discuss a novel drug delivery platform wherein the noble gases xenon or argon are delivered individually or synergistically to the brain after a mild or moderate brain injury.

Therapeutic gases like xenon (Xe) and argon (Ar) have been shown to be an effective treatment for arresting the progression of acute TBI. Xenon (Xe) is known to be an antagonist of NMDA and AMPA receptors that are key in excitotoxic signaling following TBI. Argon (Ar) is postulated to upregulate PI3/Akt and ERK1/2 pathways to arrest neuronal degeneration. Importantly, both gases freely diffuse across the blood brain barrier and have excellent safety profiles. The problem remains that while Xe treatment especially has shown great promise in both pre-clinical and clinical settings, delivery is via inhalation. Inhalation is systemic, may cause side effects due to high dosage, and leads to low and uncontrolled gas concentration in the brain.

The most effective method would be to locally deliver high quantities of Xe via a standard IV methods, while being able to image the delivery vehicle through a low cost, noninvasive technique like ultrasound. To that end, we use microbubbles (MBs) as a solution to these issues since bubbles have high gas payload while being extremely echogenic under clinical ultrasound. Microbubbles (MBs) are 1-10 µm gas particles, stabilized by a biocompatible phospholipid, polymer, or protein shell. Most commonly, the core is a water-insoluble, inert perfluorocarbon (PFC) gas, while the shell is a phospholipid-lipopolymer mixture which reduces the surface tension at the aqueous/gas interface. MBs are mainly used for contrast-enhanced ultrasound (CEUS) imaging due to their exceptional scattering and non-linear volumetric oscillation properties under clinical ultrasound frequencies. In recent years, MBs have been used to carry and deliver therapeutic gases like oxygen and nitric oxide, or xenon stabilized by perfluorocarbons. However, attempts to formulate pure xenon microbubbles (XeMBs) by different labs yielded MBs that were too large and non-echogenic in vivo, while alternative PFC-doped bubbles have limited release profile due to finite solubility of Xe in the PFC. Moreover, due to the high water-solubility and diffusivity of Xe, XeMBs made with common stabilizers such as short-chain phospholipids dissolve rapidly, limiting their efficacy in Xe delivery. To address that, we have developed the first examples of stable, echogenic MBs encapsulating pure xenon or argon without any solubilizing agents. Through optimization of the phospholipid composition of the microbubble shell, we demonstrated that a long-chain phospholipid dibehenoylphosphatidylcholine (DBPC, C22:0), can formulate storage-stable sub-10 µm Xe and Ar MBs – these MBs shown consistent echogenicity in vivo in a mouse and a porcine model. 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 tested the neuroprotective ability of XeMBs in a porcine model of moderate TBI. Focal controlled cortical impact was used to create injury in 1 month old, 10 kg pigs. IV administration of bubbles (both xenon and control gas) at 1, 3, and 24 hrs after injury. A clinical ultrasound scanner was used to visualize MBs at the common carotid artery of the pig, followed by increasing the ultrasound pressure to release the gas from the MBs locally near the brain. Magnetic resonance imaging (MRI) scans of the pig brain revealed significantly reduced perilesional edema in the xenon-treated group as compared to the control-group. Immunohistochemical staining (H&E) was done with coronal brain sections showed lower endothelial proliferation and perivascular inflammation near the injury site for the xenon treated group. Interestingly, we also observed the protection of the cerebral vasculature, a therapeutic effect of xenon that hasn’t been explored in detail previously. Fibrinogen staining showed lower protein extravasation outside the cytoplasm in brain tissue, indicating greater vascular integrity post-XeMB treatment. Preliminary trials with an in vitro model of the BBB composed of benD.3 cells showed the positive effect of XeMBs in preserving tight junction protein (ZO-1) expression after excitotoxic shock as an indication of potential BBB protection.

The newly established Chattaraj Lab is currently exploring the efficacy of both XeMBs and ArMBs on reduction of injury progression following mild and moderate TBI in a rat model. To ascertain therapeutic efficacy, both histological responses and behavioral/cognitive responses will be recorded. We are focusing on the effect of MBs on cerebral vasculature – specifically, injured 4–6-week Sprague-Dawley rats are being treated with MBs, followed by testing the integrity of the BBB with tight junction and gap junction protein expression and the activation of glial cells in the short and long-term. In summary, this work shows the feasibility of neuroprotection using Xe MBs, thus providing a promising theranostic agent for targeted therapeutic gas delivery for ultrasound image-guided treatment of mild-to-moderate TBI and its effects on the cerebral vasculature. We will also talk about the potential of Xe and Ar MBs as theranostic agents for other types of injury including cardiac and ischemic injuries and paths forward to carry out such treatment.