Introduction

In this study, we aimed to increase intravenous delivery of peptides to the spinal cord by modulating the blood-spinal cord barrier (BSCB). Although magnetic resonance imaging-guided focused ultrasound in combination with microbubbles is being clinically tested for blood-brain barrier modulation, this technique cannot be used as easily for the BSCB due to the unique geometry of the spinal bone, which hampers ultrasound capabilities. Instead, we used a combination of tight junction-targeting plasmonic nanoparticles with ultrashort pulsed lasers to temporarily increase BSCB permeability in the area of illumination.

Methods

Briefly, we fabricated gold nanoparticles by reducing gold hydrochloride and growing gold seeds to 45-nm diameter particles. Then, we functionalized these nanoparticles by conjugating polyethylene glycol (PEG) with anti-junctional adhesion molecule A (JAM-A) antibodies, then backfilling the rest of the gold surface with PEG. Next, we would inject these functionalized nanoparticles intravenously into mice before using an ultrashort, pulsed picosecond laser to induce photoacoustic stress on spinal cord endothelial cells to temporarily and locally increase BSCB permeability. After laser exposure, we administered tracer molecules or the peptide bombesin.

We first determined the degree of permeability modulation using Evans blue dye to visualize the penetration of intravenous agents within the light-stimulated region. Next, we used a combination of immunohistochemistry (IHC) analysis, rotarod tests, and infrared imaging to determine whether our technique negatively impacts cellular structure within the spinal cord, motor function, or peripheral inflammation, respectively. Under IHC, high-resolution confocal images were taken of tissues stained for neurons (NeuN) and sensory neurons (IB4 and CGRP). Fluorescence intensities were compared to determine differences in signal between laser and no-laser regions of the spinal cord cross sections. Rotarod testing involved timing mice running on a rotating rod before falling as a measure of motor function. Forward-Looking Infrared (FLIR) imaging was used to determine levels of peripheral inflammation, using thermal temperature as a proxy. For each thermogram image, an ROI was drawn on the plantar surface of both hind paws, and the mean temperature was recorded from the average of each pixel by an analyst blinded to the conditions.

With the intravenous administration of the itch-inducing bombesin peptide after mBSCB, we recorded the number of itch bouts within mBSCB-treated and control groups over time.

Results

Targeting plasmonic nanoparticles can be used to locally enhance BSCB permeability with ultrashort pulsed lasers, allowing for the delivery of large molecules such as Evans blue or small peptide molecules. We visualized a notable penetration of Evans blue into spinal cord tissue within the region of light stimulation, in contrast with all other regions of the spinal cord that did not have Evans blue signal. As shown from IHC image analysis, there was no significant impact to cells within the spinal cord from the mBSCB procedure. No significant differences were found between mBSCB-treated and control mice with regards to motor function and inflammation.

Within mice treated by mBSCB and intravenously injected with bombesin, we found a significant increase in itching behaviors compared with the group that only received intravenous bombesin. This itching decreased after 10 minutes, correlating with the peptide’s short clearance time. Thus, mBSCB is a novel and effective approach to increase molecular delivery to the spinal cord and modulate behavioral change.

Conclusion

From this work, we determined that our method of BSCB modulation could be used to deliver molecules of different sizes with high spatiotemporal resolution and modify behavior. Going forward, we aim to apply this method towards enhancing therapeutic delivery to the spinal cord for cancer applications.