Graduate Student Award Session: Development of Stable Targeted Nano-, Encapsulated Manganese Oxide (NEMO) Particles for Early Breast Cancer Diagnosis By MRI

The ultimate goal of this research project is to develop a novel contrast agent to reduce the number of false-positive and false-negative imaging results associated with current breast cancer imaging techniques. Gadolinium chelates are the current clinically approved contrast agents for breast magnetic resonance imaging (MRI), but they are always “on” and highlight any vascularized structure. Due to their lack of targeting, both benign and malignant breast tumors are enhanced with gadolinium, resulting in high false-positive rates up to 25%. By utilizing tumor-targeted pH-sensitive manganese oxide (MnO) nanoparticles, a contrast agent can be developed that will only turn “on” after internalization into cancer cells and dissolution within low pH endosomes/lysosomes.

Nanocrystalline MnO was synthesized through thermal decomposition of Mn(II) acetylacetonate in dibenzyl ether and oleylamine at 280°C. After confirmation of nanocrystalline MnO size and chemistry with TEM, XRD, and FTIR, MnO nanocrystals were encapsulated within poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) through single emulsion to form Nano-, Encapsulated, Manganese Oxide (NEMO) particles. Particles with different PEG percentages (2.5, 5, and 10% w/w) and PLGA terminations (carboxylic acid vs. ester) were synthesized and incubated in water over 24 hours to assess particle stability. The hydrodynamic diameter and charge of NEMO particles were measured pre- and post-incubation with dynamic light scattering (DLS) and zeta potential, respectively. For carboxylic acid terminated PLGA, a higher PEG % resulted in enhanced particle stability due to a more negative zeta potential that favored particle-particle repulsion (2.5% PEG: -12.89 mV; 5% PEG: -14.31 mV; 10% PEG: -16.33 mV). Surprisingly, increased PEG % had the opposite effect with ester terminated PLGA; particles became less stable with higher PEG due to a less negative zeta potential that reduced interparticle repulsion (2.5% PEG: -16.51 mV; 5% PEG: -12.61 mV; 10% PEG: -8.557 mV). As higher PEG density can reduce nanoparticle cell uptake, NEMO particles with 2.5% PEG ester terminated PLGA were chosen as the optimal particle for further experiments.

To enable specific uptake by cancer cells, a tumor-targeting peptide against underglycosylated mucin-1 (uMUC-1) was attached through click chemistry using copper (I) as the catalyzer. The uMUC-1 targeting peptide conjugated NEMO particles were characterized with DLS and a fluorometric assay to assess hydrodynamic size (~180 nm) and correct targeting attachment, respectively. For NEMO particles to cause a positive T₁ signal on MRI, MnO must dissolve and release Mn²⁺ at low pH. To evaluate the Mn²⁺ release and MRI properties, NEMO particles were incubated at 3 different pHs over time including pH 7.4 (blood pH), 6.5 (pH of tumor extracellular space), and 5 (endosome/lysosome pH), and supernatants were evaluated using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and 1T MRI. NEMO particles released the most Mn²⁺ at pH 5 (~93% at 24 hr), which produced the lowest T₁ value with the brightest MRI signal at the first two time points (848 ms at 1 hr, and 886 ms at 2hr, pH 5). The substantial MRI contrast generated after just 1-2 hours indicates that NEMO particles can result in a measurable contrast change within clinically relevant time frames.

To assess in vivo stability and toxicity, different dosages of tumor-targeted NEMO particles (15, 7.5, 3, and 1.5 mg Mn/kg) were injected once a week for three weeks into BALB/c female mice. Weight, behavior, and appearance were scored daily to measure toxic effects. Only the highest dosage, 15 mg Mn/kg, had an impact on mouse weight during the first 24 hr (~1 g weight loss), and resulted in a slight decrease in natural and provoked behavior; mice regained weight and normal behavior within 2 to 3 days post-injection. Based on these results, overall NEMO particles were well tolerated in vivo over 3 weeks. In future studies, 7.5 mg Mn/kg will be utilized to maximize Mn dose for enhanced MRI signal while avoiding the weight and behavioral effects associated with 15 mg Mn/kg. Future work includes further in vitro studies of NEMO particle cell labeling and toxicity, along with in vivo MRI of tumor-bearing mice to assess the specific detection of malignant versus benign breast tumors with NEMO particles.