Poster Y14 - Advancements in Inflammation Imaging: Development of an Oxidative Stress-Activated MR Contrast Agent

Introduction: Chronic inflammation underlies numerous debilitating diseases, rendering their early detection and precise monitoring crucial yet challenging due to the limitations in current medical imaging technologies. Particularly, magnetic resonance (MR) imaging, while highly valuable and diagnostically crucial, is non-specific for biochemical activity associated with inflammation. This limitation necessitates advancements in MR contrast agents that can provide direct, specific visualization of inflammatory processes. Addressing this, we developed Interpolymer-Complexed Superparamagnetic Iron Oxide Nanoparticles (IPC-SPIOs). These nanoparticles are uniquely engineered to activate in the presence of oxidative stress, specifically superoxide radicals, a common marker of inflammation. By remaining dormant under normal physiological conditions and activating only in response to excess oxidative stress, IPC-SPIOs offer a novel approach to enhance the specificity and utility of MR imaging for diagnosing and monitoring inflammatory diseases.

Materials and

Methods: Poly(ethylene glycol) (PEG)-coated iron oxide nanoparticles (IONPs) were synthesized via co-precipitation. A solution of ferrous (FeCl2) and ferric chloride (FeCl3) in deoxygenated DI water, with a 1:2 molar ratio, was heated to 80°C. 300kDa PEG was added to the iron solution under nitrogen flow. Then, 1M ammonium hydroxide was added dropwise to yield PEG-coated IONPs. These nanoparticles then underwent complexation with poly(gallol), which forms hydrogen bonds with the PEG layer, creating an interpolymer complex responsive to superoxide.
There are different allotropes of iron oxide, with magnetite (Fe3O4) demonstrating optimal magnetic properties. X-ray diffraction crystallography was used to confirm the material type.
The core size and morphology of the nanoparticles were analyzed using Transmission Electron Microscopy (TEM), ensuring they were within the required range of under 50nm to elicit optimal superparamagnetic properties. The steps of formation and activation of IPC-SPIOs result in a corresponding increase in particle size during each step. Due to altered absorbance from the change in particle size UV/Vis spectroscopy was used to verify their formation and function.
The responsiveness of IPC-SPIOs to superoxide was assessed using a nitro blue tetrazolium. 3 different concentrations of IPC-SPIOs were exposed to a controlled concentration of superoxide for 3 lengths of time to determine dose-response.
In future studies, we will evaluate whether IPC-SPIOs activate in response to superoxide radicals within a murine inflammation model. This research aims to verify the in vivo functionality of IPC-SPIOs as a responsive MR contrast agent.

Results, Conclusions, and Discussions: Results
TEM analysis confirmed that the core size of our IPC-SPIOs falls within the required range for eliciting superparamagnetic properties necessary for effective MR imaging. The nanoparticles displayed a consistent spherical morphology with an average diameter of approximately 12.87 ± 5.16nm for n=100 as shown in Figure 1.
X-ray diffraction (XRD) analysis was conducted on the samples to identify characteristic peaks shown in Figure 2. These peaks were then compared to known standards to confirm the successful synthesis of magnetite (Fe3O4).
UV-Vis spectroscopy indicated successful complexation and decomplexation of the IPC-SPIOs. During complexation, there was a marked increase in absorbance, confirming the formation of the nanoparticle complexes. Upon decomplexation, a further increase in absorbance was observed, aligning with the activation of IPC-SPIOs in response to oxidative stress.
The NBT assay demonstrated a significant decrease in superoxide concentrations following exposure to IPC-SPIOs, indicating successful activation of the nanoparticles.
Discussion
Results demonstrate that IPC-SPIOs are effectively activated in response to superoxide as indicated by the NBT assay showing a significant reduction in superoxide concentrations upon exposure to IPC-SPIOs. Particle size and material composition of IPC-SPIOs are essential for their function as contrast agents in MR imaging and were confirmed through TEM and XRD analyses respectively.
Complexation and decomplexation kinetics of IPC-SPIOs, monitored through UV/Vis spectroscopy, revealed significant changes in absorbance, indicative of successful formation, and activation of the nanoparticles in response to oxidative environments. This activation mechanism is particularly pertinent given the increasing interest in precision medicine in the field.
The planned animal studies will be pivotal in validating the in vivo functionality of IPC-SPIOs. These studies will aim to assess the responsiveness of IPC-SPIOs to pathological levels of superoxide radicals. The outcomes will help refine the application of IPC-SPIOs, ensuring they meet the efficacy standards necessary for clinical use.
Conclusions
The results confirmed that IPC-SPIOs effectively respond to oxidative stress markers which will potentially result in detectability through MR imaging. This specificity could significantly enhance the diagnostic capabilities of MR imaging for inflammatory diseases by providing direct visualization of biochemical activity at the inflammation sites.

Acknowledgements (Optional): Dr. Amber Doiron, PI
Ian Stender - Undergraduate Assistant