(563e) Breast Cancer Specificity Assessment of Tumor Targeted Nano-, Encapsulated Manganese Oxide (NEMO) Particles

Gadolinium chelates are the gold standard contrast agents for breast magnetic resonance imaging (MRI), but their constant MRI signal and lack of specificity results in high background and misdiagnosis. This lack of targeting makes it challenging to differentiate between benign and malignant breast masses, resulting in high false-positive rates of up to 25%. We have developed an enhanced contrast agent that utilizes tumor-targeted pH-sensitive manganese oxide (MnO) nanoparticles, which in physiological pH, produce low signal (“OFF”). After cancer cell uptake and digestion within low pH endosomes/lysosomes, the MnO dissociates into Mn²⁺, turning the MRI signal “ON.”

Nano-, Encapsulated Manganese Oxide (NEMO) particles were synthesized by the encapsulation of nanocrystalline MnO within poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) via single emulsion. The nanocrystals and nanoparticles were chemically and physically characterized by TEM, XRD, FTIR, SEM, and DLS. After characterizing the particles, a fluorescent tumor-targeting peptide against underglycosylated mucin-1 (uMUC-1) was attached through click chemistry using copper (I) as the catalyst. MUC-1 was chosen as a tumor target, as it is overexpressed and underglycosylated on breast cancer cells, but not on benign or healthy cells. The uMUC-1 conjugated NEMO particles were characterized with DLS to assess hydrodynamic size (~190 nm) and fluorescence to ensure attachment of the targeting peptide. Before evaluating the cellular uptake of the NEMO particles, different control and breast cancer cell lines were characterized for expression of MUC-1 and uMUC-1 via flow cytometry. HEK293T (control), BT20 (triple negative), and T47D (luminal A) were selected for particle specificity studies, as their expression varied from no MUC-1 or uMUC-1 (HEK293T) up to 85% uMUC-1+ cells (T47D).

Cells were exposed to targeted NEMO particles (5 µg of Mn/mL) for 2 hours, followed by an MRI scan of the cell pellet and collected media. The change of R₁ (∆R₁) between the control group (only exposed to media) and the nanoparticles-exposed group was calculated post imaging. In the case of cell pellets, HEK293T had a larger ∆R₁ than BT20 or T47D (266% vs. 116% vs. 162%). This was not expected based on relative MUC-1 and uMUC-1 expression. However, when scanning media alone, the opposite results were observed where HEK293T had a lower ∆R₁ than BT20 and T47D (13% vs. 67% vs. 56%), meaning that higher amounts of Mn²⁺ were found in BT20 and T47D media. Increased Mn²⁺ in the media could indicate that cancer cells internalized NEMO particles faster, dissolved MnO, and then released it. Therefore, further characterization of the time course of cellular uptake was performed by exposing the cells to NEMO particles (1.625 µg of Mn/mL) for 0.5, 1, and 2 hr, and again calculating ∆R₁. Lower expression of uMUC-1 resulted in a smaller change in MRI signal at shorter time points. Already at 30 min, T47D had over 200% ∆R₁ due to its high uMUC-1 expression and thus fast NEMO particle uptake, while it took 2hrs for the other two cell lines to reach that level. These results showed the dependency of uMUC-1 expression on NEMO particle uptake and, consequently, MRI signal.

Lastly, T47D tumor-bearing mice were injected with targeted NEMO particles (7.5 mg/kg), and MRI scans were performed pre-injection (baseline) and 1, 2, 4, and 24hr post-injection to assess the variation in MRI signal in the tumor. After 1hr and 2hr, the MRI signal intersity increased by ~37% compared to baseline, followed by a slight decrease to ~18% above baseline at 24hr. Additionally, through abdominal MRI scans, signal intensity was visualized in the liver and kidneys after NEMO particle injection, highlighting possible elimination via these routes. Future biodistribution studies will be performed to correlate MRI signal to Mn organ content. Overall, targeted NEMO particles caused an increase in T₁ MRI signal in breast cancer cells expressing uMUC-1+ in vitro and in vivo, and further study is warranted to compare its sensitivity and specificity to Gd-chelates.