(161o) In-House v. Commercialized Cores for Nano-, Encapsulated, Manganese Oxide (NEMO) Particles As MRI Contrast Agents

Over the last 40 years, gadolinium (Gd)-based contrast agents have served as the gold-standard for contrast enhancement in magnetic resonance imaging (MRI). Unfortunately, Gd chelates retain high false-positive rates and can lead to harmful side effects, such as nephrogenic systemic fibrosis and Gd-deposition in the brain. Thus, there has been a shift in research toward the use of polymeric metal oxide nanoparticles (NPs) because of their biocompatibility, biodegradability, and surface functionalization. Although there has been substantial research using superparamagnetic iron oxide nanoparticles (SPIONs) as contrast agents due to their robust dark T₂* MRI signal, these particles suffer from some limitations. SPIONs produce a constant dark signal that is always “on” and can be easily confounded by the negative contrast produced by natural iron stores in the liver, bone marrow and in areas of hemorrhage. Here, we propose poly(lactic-co-glycolic acid) (PLGA) Nano-, Encapsulated, Manganese Oxide (NEMO) particles due to their ability to convert from an “off” to “on” state in low-pH environments, such as tumor microenvironments, rendering the particles pH-activatable. Three types of manganese oxide cores (MnO, Mn₂O₃, and Mn₃O₄) were either synthesized through thermal decomposition of manganese(II) acetylacetonate or purchased from Company A or Company B. Note, we will uphold each company’s anonymity and remain objective when analyzing the data.

The composition of the nanocrystals was confirmed using X-Ray diffraction (XRD), which showed impurities in cores from Company A that corresponded to calcium, sodium, and zinc. Transmission electron microscopy (TEM) results showed that the true nanocrystalline sizes directly contradict the reported diameters with up to a 30nm and 60nm difference than reported for Company A and Company B, respectively. Company A’s nanocrystals presented with a consistent, non-uniform distribution with some rod-like figures, while Company B’s nanocrystals presented with an unknown substance creating a persistent film across all TEM images. The diameters for in-house nanocrystals were ~35nm and ~11nm for MnO and Mn₃O_4, respectively, with uniform distributions.

All manganese oxide cores were then encapsulated within PLGA, a biocompatible and biodegradable polymer, to form NEMO particles using a single emulsion technique. To produce bright signal on MRI, MnO must degrade to produce Mn²⁺ ions at low pH. To assess Mn²⁺ release and resulting MRI signal, NEMO particles were incubated at pH 7.4, 6.5, and 5 to mimic pH in blood, tumor extracellular space, and intracellular endosomes, respectively. Supernatants were collected regularly over 24h and analyzed by inductively coupled plasma - optical emission spectrometry (ICP-OES) for released Mn content and 1T MRI for T₁ signal enhancement. After encapsulation of the first of three replicates, controlled release showed greater than 100% release of Mn²⁺ at 24h in pH 5 buffer for Company A Mn₂O₃, Company B MnO, and Company B Mn₃O_, which was attributed to impurities and poor encapsulation efficiency. According to scanning electron microscopy (SEM), micron-size particles were synthesized when encapsulating cores from both Company A and Company B. In-house NEMO particles showed a uniform distribution with SEM, and there was ~94% Mn²⁺ release for MnO and ~41% Mn²⁺ release for Mn₃O₄ after 24h at pH 5. In general, the T₁MRI signal of particle supernatants increased with decreasing pH as expected due to greater release of Mn²⁺ under acidic conditions. Specifically, in-house MnO and Company A Mn₃O₄ presented with the maximum T₁ MRI signal. Contrarily, both samples of Company B Mn₂O₃ presented with no discernable signal change with varying pH indicating that these particles were very poor contrast agents.

Additional analyses will be performed including but not limited to Fourier Transform Infrared (FTIR) spectroscopy and dynamic light scattering (DLS) to determine polymer coating of nanocrystals and hydrated particle size, respectively. Based on our preliminary assessment, it appears that in-house synthesis of smaller batches of manganese oxide nanocrystals is currently preferable to commercialized sources to ensure sample purity, a tight size distribution, and maximum T₁ MRI signal. Ultimately, determining a precise and reliable way to synthesize commercially available manganese oxide nanocrystals at large scales will allow for reproducibility of NEMO particles bringing them one step closer to being used in a clinical setting.