Info
Background:
Emerging therapeutic agents for brain injuries are promising, such as neuroprotective noble gases and drugs. Inhalation of xenon, a well-known anesthetic gas, has shown promising therapeutic potential for preserving brain health after TBI in small-animal models. Even with validation in TBI animal models, however, there are major obstacles to implementing xenon inhalation therapy in humans: it is expensive, inconvenient (requiring up to 24 hrs of xenon inhalation), and systemically delivered (i.e., to the entire body) with low therapeutic yield to the brain. Similarly, systemic delivery of experimental drugs, such as ferrostatin-1, is inefficient due to rapid metabolism by the kidneys and liver, limited delivery across the blood-brain barrier, and unwanted side effects.

Hypothesis/Objective:
To overcome these challenges, ultrasound-responsive theranostic microbubbles (MBs) loaded with neuroprotective noble gases or drugs were developed for ultrasound image-guided therapy. The benefits of this technique are owed to the use of MBs as a carrier and ultrasound stimulation for controlled release. The sub-10 µm theranostic MBs encapsulate large quantities of therapeutic agents and protect them from systemic degradation, thereby enhancing the therapeutic payload. The MBs exhibit excellent ultrasound responsiveness, enabling simple in vivo tracking via ultrasound imaging. In this work, two formulations of theranostic MBs were fabricated and assessed: xenon-loaded MBs (XeMBs) and ferrostatin-1-loaded MBs (Ferr-1 MBs).

Methods:
XeMBs and Ferr-1 MBs were fabricated by thin film hydration and ultrasonication. The composition of the MB lipid shells was optimized to ensure stability and high therapeutic loading. The MBs were characterized using light microscopy for size distribution and mass spectrometry to validate therapeutic encapsulation. The ultrasound responsiveness and therapeutic-release profiles were evaluated in vitro using ultrasound and a flow phantom. The XeMBs were then assessed in a pediatric porcine TBI model for treatment feasibility, and Ferr-1 MBs were assessed in a healthy rat model for general biosafety.

Results:
Both XeMBs and Ferr-1 MBs were reproducibly fabricated with size distributions < 5 μm. The lipid shell formulation was optimized for the Ferr-1 MBs to achieve high drug loading (88% encapsulation efficiency). Both MBs were highly echogenic in an in vitro flow phantom. The MBs could be successfully cavitated at a mechanical index (MI) of ~0.68, which meets FDA standards for use in diagnostic ultrasound.

In a healthy rat model, continuous ultrasound at an MI of 0.68 induced cavitation in the Ferr-1-MBs. All animals demonstrated stable, with normal heart rate, oxygen saturation, and temperature during the MB infusion. The injection of Ferr-1 MBs was feasible, and they were successfully visualized via contrast-enhanced ultrasound at the carotid artery.

Preliminary in vitro experiments under conditions to simulate hypoxic-ischemic injury were conducted on the XeMBs. Under oxidative stress, XeMBs enhanced cell viability, even without ultrasound exposure. In the pediatric porcine model of TBI, XeMBs, compared with control MBs, drastically reduce injury size from Day 1 to Day 5, as visualized on MRI. Histological evaluation of the brains also showed significantly lower endothelial proliferation and perivascular inflammation in the XeMBs treatment group.

Conclusion:
XeMBs and Ferr-1 MBs show great promise for image-guided drug delivery to the brain. The sub-10 µm theranostic MBs are highly stable, encapsulate neuroprotective therapeutics, and exhibit excellent ultrasound responsiveness in vivo. Future studies will explore the efficacy of this approach in additional disease models, paving the way for novel therapeutic strategies for the brain.

Author
Kathrine Curtin, Ph.D.
Perelman School of Medicine at the University of Pennsylvania