Developing Ultrasound Contrast Agents to Deliver siRNA to Spinal Cord Injury
Authors:   , Oeffinger Brian E. , Wheatley Margaret A. 
Published: October 2024

Spinal cord injury (SCI) initiates a complex biological response and leads to the disruption of motor and sensory signals between the brain and the peripheral body at and below the level of damage. SCI can lead to a host of morbidities such as respiratory infections, and result in a reduced quality of life. There are no established regeneration methods to heal spinal cord damage, and treatment is limited to attempting to minimize secondary damage to the spinal cord. SCI repair is impeded by both the poor regenerative capacity of adult spinal cord and the formation of inhibitory scar tissue around a large cystic lesion. A successful healing strategy must simultaneously overcome the negative cues from the scar while providing positive cues to allow axons to grow across the lesion to connect with their targets. Our long-term goal is to develop a multi-component biocompatible platform that will enable a diversity of synergistic healing strategies at the site of SCI. This strategy includes a novel ultrasound-based system for targeted delivery of small interfering RNA (siRNA). Inhibiting the upregulation of neuronal proteins such as RhoA using siRNA could help mitigate the negative effects of the glial scar. However, the usefulness of naked siRNA is limited by its extreme instability in vivo and inability to penetrate the cell with ease. Both drawbacks can be addressed with the use of ultrasound and contrast agents. Ultrasound contrast agents (UCAs) are injectable, stabilized gas microbubbles that increase tissue image contrast, and can be loaded with therapeutics for use in directed delivery to desired sites, including SCI. The overall goal of this thesis was the development of UCAs loaded with RhoA siRNA for future delivery to SCI to initiate the downregulation of overexpressed RhoA protein that inhibits neuronal growth. A novel poly (lactic acid) (PLA) UCA, developed previously in our lab, was first investigated. It was hypothesized that these PLA microbubbles, when subjected to ultrasound, would burst using a frequency and pressure appropriate for use in the ultrasound-sensitive spinal cord, and fragmentation would facilitate delivery of siRNA. It was determined that the PLA microbubble burst best using an insonating frequency of 2.25 MHz, close to its resonance frequency, which was above the 1 MHz typically used to facilitate delivery. Bursting thresholds of approximately 0.3 MPa peak negative pressure (PNP) were found, lower than the pressure reported in the literature to be damaging. No evidence was found of the PLA UCA fragmenting or creating particles less than 0.5 [mu]m due to ultrasound destruction, in contrast with previous results. A model siRNA was successfully combined with the PLA microbubbles. This was aided with the addition of the cationic polymer polyethylenimine (PEI), which allowed for a loading up to 9.73 ± 0.30 [mu]g /mg UCA, better than the calculated target of 7.5 [mu]g/mg PLA UCA. The addition of PEI, however, was also found to prevent the release of the model siRNA during ultrasound induced microbubble bursting, rendering this solution ineffective. A surfactant based UCA developed ion our lab, SE61, was also investigated as it was believed that it could be modified to include a cationic species to facilitate loading anionic therapeutics. A new fabrication method that better allows for cationic additions was developed and was determined to create microbubbles with equivalent properties to prior methods. The cationic surfactant CTAB and the lipids DSTAP and DMTAP were found suitable for inclusion into SE61 without disruption to the microbubble properties. Inclusion of these cationic species increased the zeta potential of unmodified SE61 from -31.6 ± 5.5 mV to above +30 mV, onto which anionic therapeutics such as DNA and rose Bengal were loaded. SE61 microbubble ultrasound mediated destruction and fragmentation was found to be best with 1 MHz, which is optimal for potential siRNA delivery. Thes ultrasound parameters were utilized for SE61-DMTAP mediated siRNA transfection in HeLa cells, which was found to be as effective and less toxic than the gold standard Lipofectamine 2000 when using a reverse transfection protocol. Transfection using SE61-DMTAP loaded with RhoA siRNA and burst with ultrasound resulted in a successful 63.5% knockdown in RhoA protein in HeLa cells. Developing new therapeutic approaches to overcome the inability of the spinal cord to repair after injury remains an important goal. These findings indicate that the developed SE61-DMTAP microbubbles have significant potential to safely improve RhoA siRNA delivery to SCI using targeted ultrasound bursting. Overall, this work has shown the potential of utilizing ultrasound-induced destruction of contrast agents for the delivery of therapeutics to SCI.