Intrathecal Therapeutic Delivery to the Spinal Cord by Laser-Activated Perfluorocarbon Nanodroplets

Introduction:: Viral vector mediated gene therapy for the treatment of spinal cord disorders is an active area of research and a promising strategy for a wide variety of diseases, including motor neuron disease, demyelinating conditions, and traumatic spinal cord injury. However, the delivery of viral vectors to the spinal cord parenchyma is challenged by a lack of non-invasive and direct administration routes. Intrathecal delivery of viral vectors is an attractive route for gene vector delivery because it allows more global delivery to the spinal cord compared to direct intraparenchymal injection. Compared to intravascular infusion, it elicits a lower immune response to the viral vectors, requires lower doses, and has lower risk of off-target effects. However, many viral vectors cannot penetrate through the cerebrospinal fluid-spinal cord barrier (CSB) rendering them ineffective. We propose, the use of laser-activated perfluorocarbon nanodroplets (PFCnDs) for CSB opening. PFCnDs are ultrasound (US) and photoacoustic (PA) contrast agents loaded with an optical dye for remote, non-invasive laser light activation and have been previously used for applications including blood-brain barrier opening and neuroimaging. The key physical principle powering PFCnDs is their ability to repeatably phase-change between liquid nanodroplets and gas microbubbles when remotely activated by acoustic or optical pulses. In this study, PFCnDs are intrathecally injected, allowed to distribute throughout the sub-arachnoid space in spinal fluid, and then are laser-activated in specific regions to focally open the CSB. We analyze the extent of CSB opening using US/PA imaging of PFHnDs signal contrast within the spinal cord.

Materials and Methods:: PFCnDs were synthesized with a perfluorohexane core, Zonyl shell, and a dye with a peak optical absorbance of 1064 nm. Nanodroplet size (215 nm diameter) was measured using a dynamic light scattering instrument (Zetasizer Nano ZS, Malvern Instruments Ltd.) (Fig. 1A). Adult Sprague-Dawley rats were intrathecally injected with nanodroplets via direct lumbar puncture. A control group received no injection or laser treatment (n = 2). Thirty minutes after injection, animals, laid in a prone position, received focal non-invasive laser stimulation treatment to specific segments of the spinal cord for PFHnDs induced CSB opening. Treatment groups received either no stimulation, or stimulation in one or two regions (n = 2 per group, 6 total). Laser stimulation treatment utilized fiber optics to direct a laser beam with a wavelength of 1064 nm to track 1064 nm optical dye and a pulse repetition frequency of 10 Hz for two minutes (Tempest laser system) (Fig. 1B). Four hours after the treatment session, animals were sacrificed, spinal cords were harvested and fixed. The lamina was removed for US/PA imaging (Fig. 1C). Ex vivo US/PA imaging (Vevo Lazer, 1064 nm) was performed across the transverse plane and a translation stage shifted the US/PA probe to generate reconstructed sagittal sections.

Results, Conclusions, and Discussions:: Results and Discussion: US imaging showed the spinal cord as having a hyperechoic surface and a hypoechoic core (Figs. 1D-E). Reconstructed sagittal images (Fig. 1D) showed the length and characteristic curves of the spinal cord and transverse images showed its cross-section (Fig. 1E). US/PA imaging showed PA signal contrast in animals treated with PFCnDs, whereas the control groups had negligible signal (Figs. 1D-E). In all samples treated with PFCnDs, there was high PA signal on the surface of the spinal cord, consistent with the anatomy of the intrathecal space, where PFCnDs were originally injected. In the reconstructed sagittal images (Fig. 1D), areas that received laser stimulation showed PA signal deep within the spinal cord parenchyma. This was further visualized in the transverse images (Fig. 1E) where the PA signal in the lased segments penetrated through the area of the spinal cord.

Conclusions: By leveraging the dynamic oscillatory capabilities of laser-activated PFCnDs, CSB opening was achieved and visualized using US/PA imaging. This proof-of-concept study demonstrates the potential for laser-activated PFCnDs to enhance intrathecal delivery of therapeutics to the spinal cord. This delivery strategy has the potential to facilitate treatment of many spinal cord pathologies by addressing the key limitation of inadequate therapeutic delivery encountered in many prior intrathecal gene therapy studies.


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