N28 - Stimuli-Responsive Contrast Agents for Low-Field, Theranostic Magnetic Resonance Imaging Applications

Introduction: : Magnetic resonance imaging (MRI), among imaging modalities, is widely utilized for its noninvasiveness. However, high-field MRI systems are immobile and require extensive infrastructure, which limits their accessibility in low-resource or urgent settings such as war fields or rural clinics. In these scenarios, portable low-field systems offer a practical alternative. While reduced in resolution and sensitivity, these systems can still provide meaningful anatomical and functional data when paired with an effective contrast agent (CA). CAs enhance signal intensity at the target site, thereby compensating for the lower sensitivity of MRI systems in general. Unlike techniques that rely on ionizing radiation, MRI allows for repeated imaging, making it particularly well-suited for tracking tissue response over time. We hypothesize that we can create a CA system simultaneously capable of delivering a therapeutic, as a theranostic tool, which can be tracked in near real time using low-field MRI.
To develop a theranostic platform compatible with low-field MRI, hyaluronic acid (HA)-poly(lactic acid) (PLA) polymersomes (PSs), previously optimized for protein encapsulation and responsive degradation, were co-loaded with iron oxide nanoparticles (IONPs) serving as contrast agents with different MRI orientations. With optimized contrast agents and further investigation into their in vivo behavior, this platform may enable more precise, noninvasive imaging of disease progression and tracking of nanoparticle-based therapeutics in low resource settings.

Materials and

Methods: : HA(5 kDa)-PLA(15 kDa) PSs were prepared using a self-assembly method, optimized to encapsulate hydrophilic proteins for controlled release under pathophysiological conditions1–3. During formation, iron oxide nanoparticles (IONPs), one with a polyacrylic acid coating (PH160) and one without (P169), were incorporated into the aqueous core to produce responsive PSs. Transmission electron microscopy (TEM) confirmed morphology and nanoparticle loading. To assess pathological responsiveness and confirm maintenance of HA-PLA polymersome degradation behavior, release studies were performed in various neutral (HEPES, Water, PBS) and acidic+enzymatic (citrate + hyaluronidase-enriched, acetate hyaluronidase-enriched). Fluorescein Isothiocyanate-Bovine Serum Albumin (FITC-BSA) was used as a model protein cargo to monitor release kinetics and confirm co-loading of IONPs (diagnostic) with proteins (therapeutic).
For imaging evaluation, MRI phantom studies were performed using a 0.5T benchtop MRI system. PS-PH160s and PS-P169s were incubated under relevant buffer conditions and scanned at multiple timepoints (0h, 48h, and 72h) to monitor signal retention or enhancement. T1 and T2 contrast intensities were analyzed to verify whether pathological triggers elicited contrast enhancement. Polymersomes with PH-160s (PS-PH160s) and with P169s (PS-P169s) were then directly injected into postmortem brain tissue and evaluated under a 0.25T MRI system and respective orientation for confirmation of delivery and visualizing localization.

Results, Conclusions, and Discussions:: TEM images visually confirmed successful formation of spherical HA-PLA polymersomes with encapsulation of IONPs within both PS-PH160 and PS-P169 formulations (Figure 1A, 2A). Release studies revealed distinct degradation and release profiles under different buffer conditions. PS-PH160s showed minimal early FITC-BSA release differences between neutral (HEPES, water) and acidic+enzymatic (citrate+hyaluronidase) buffers, with prologed incubation leading to increased release in acidic+enzymatic buffer, indicating time-dependent permeability but reduced early-stage pathological specificity (Figure 1B). PAA can swell in aqueous conditions, which is likely causing rupture of polymersome and leading to burst release in physiologic conditions. In contrast, PS-P169s, which have no PAA coating, demonstrated enhanced pathological responsiveness. Release was significantly accelerated in acidic+enzymatic buffers (Figure 2B). Rapid increases in release at 36 hours (citrate) and 72 hours (acetate) were consistent with potential UV interference caused by IONP breakdown. Despite these fluctuations, PS-P169s consistently maintained a distinct release profile between pathological and physiological conditions, supporting their potential use in site-responsive delivery.
MRI phantom studies conducted with 0.5T benchtop system demonstrated preserved contrast functionality of two formulations. Consistent relaxivity ratio of PS-PH160s after its initial spike shows PS-PH160 has similar contrast enhancement for both orientations, which was reflected through T1 phantom images (Figure 1C, 1D). In contrast, T1 relaxation time and r1 relaxation rate of PS-P169s stayed consistent at 0h, 48h, and 72h in both HYAL and HEPES buffers (Figure 2C, 2D), while T2 relaxation time in the HYAL buffer increased from 1500 ms to 2100 ms and r2 relaxation rate decreased over time, indicating lowered level of magnetic field inhomogeneity (Figure 2E, 2F). T2 phantom images also showed increased contrast intensity over time as the magnetic field became more uniform (Figure 2G, 2H). In vitro MRI image with 0.25T MRI system of a healthy brain with NPs injected showed PS diffused locally around the injection point.
HA-PLA PSs co-loaded with contrast agents, PS-PH160s and PS-P169s, demonstrated enhanced MRI signal in pathological environments. However, only PS-P169s exhibited tunable degradation and release behavior. This responsive imaging-drug delivery system highlights PS-P169s as a promising platform for accessible, controlled, and repeatable theranostic tool for brain pathology using low-field MRI.

Acknowledgements and/or References (Optional):: 1. Foster, D., Cakley, A. & Larsen, J. Optimizing Enzyme-Responsive Polymersomes for Protein-Based Therapies. Nanomedicine 19, 213–229 (2024).
2. Foster, D., Shah, N., Cakley, A., Beyers, R. & Larsen, J. Multilamellar hyaluronic acid-b-poly(lactic acid) polymersomes for pathology-responsive MRI enhancement. Biomater Sci (2025). doi:10.1039/D4BM01583E
3. Paruchuri, B. C., Smith, S. & Larsen, J. Enzyme-responsive polymersomes ameliorate autophagic failure in a cellular model of GM1 gangliosidosis. Frontiers in Chemical Engineering 4, (2022).