Researcher: Robert Nikolai
Introduction
Gene therapy offers transformative potential for treating spinal cord disorders, but safe and effective delivery of viral vectors into the spinal cord remains a major challenge. Intrathecal injection enables broad spinal cord distribution with reduced immune response and lower off-target risk compared to systemic delivery. However, most viral vectors cannot penetrate the glia limitans, the cellular barrier that separates the intrathecal space from the spinal cord, sharply limiting therapeutic reach.
Laser-activated perfluorocarbon nanodroplets (PFCnDs) have emerged as a promising tool for non-invasive glia limitans opening. PFCnDs are ultrasound (US) and photoacoustic (PA) contrast agents that undergo repeatable phase transitions from liquid nanodroplets to gas microbubbles when triggered by optical or acoustic pulses. The formation of microbubbles, called cavitation, induces mechanical vibrations that can permeabilize cells and multi-cell barriers. While this technology has previously been applied to blood-brain barrier opening, its potential for spinal cord drug delivery is still in development.
In our studies, we advance the application of laser-activated PFCnDs for spinal cord therapeutics by evaluating their ability to non-invasively open the glia limitans following intrathecal delivery. PFCnDs are injected into the cerebrospinal fluid where they distribute throughout the intrathecal space. Next, they are remotely activated at desired locations with directed laser pulses. Finally, the dynamics of barrier opening are monitored in situ using US/PA imaging by tracking changes in PFCnD signal within the spinal cord.
This work expands the understanding of PFCnD-mediated barrier modulation and offers a promising strategy for enhancing gene delivery for patients with spinal cord injuries, diseases, or disorders.
Materials and Methods
Lipid-shell perfluorohexane nanodroplets were fabricated and loaded with Epolight 3072 dye (Epolin Inc.), which has peak absorption at 1064 nm for optical activation (Figure A). Nanodroplets had a diameter of ~300 nm, measured by a Zetasizer Nano ZS (Malvern Panalytical). The lipid shell was synthesized via thin-film hydration, and PFCnDs were assembled by mechanical emulsification. To evaluate their ability to open the glia limitans, 30 μL of PFCnD solution was intrathecally injected via lumbar puncture into adult Sprague-Dawley rats (Figure B). After 30 minutes, non-invasive laser irradiation was applied transdermally using a fiberoptic system coupled to a Tempest pulsed Nd:YAG laser (New Wave Research), delivering 1064 nm light at 10 Hz, 4 ns pulse duration, and 80 mJ/cm² fluence.
Following treatment, a laminectomy was performed for in situ imaging. Ultrasound (US) imaging (Vevo 2100, FUJIFILM VisualSonics Inc.) provided anatomical maps of the spinal cord and intrathecal space, while photoacoustic (PA) imaging at 1064 nm (Vevo LAZR system) visualized PFCnD signal within the spinal cord. Imaging timepoints were varied to qualitatively assess temporal distribution of PFCnDs.
Results
US imaging showed the surface of the spinal cord as a hyperechoic structure, with occasional visualization of intrathecal space boundaries (Figure C). PA imaging confirmed the presence of PFCnDs within the intrathecal compartment. Intrathecal PFCnDs generated PA signal at 30 minutes post-injection (Figure C.1), but this signal largely cleared by 24 hours (Figure C.2). In contrast, when laser irradiation was applied, the PA signal was retained for at least 24 hours and showed deep penetration into the spinal cord (Figure C.3). These findings indicate that PFCnDs flow freely within the intrathecal compartment to the target location, but do not penetrate the spinal cord and eventually clear out over time. Then, the application of laser irradiation permits deep tissue penetration nanodroplets to penetrate the spinal cord at a focal section and be retained there. This behavior is consistent with a mechanism of glia limitans disruption mediated by nanodroplet cavitation.
Conclusion
Laser-activated PFCnDs enable localized, non-invasive opening of the spinal cord glia limitans and significantly enhance delivery of intrathecal agents into the spinal parenchyma. This delivery strategy has the potential to facilitate treatment of spinal cord pathologies by addressing the key limitations of inadequate therapeutic delivery encountered in many prior intrathecal gene therapy studies.