Breast cancer is the second most common cancer worldwide, with 670,000 deaths in 2022. The diagnostic stage has a significant influence on patient prognosis, with early cancer detection of localized disease enabling prompt treatment for improved survival. Mammography is the current gold standard for breast cancer screening; however, it misses 20% of breast cancers present and misdiagnoses 50% of benign masses as malignant over 10 years of annual screening, particularly in younger women with dense breasts^2,3. Magnetic resonance imaging (MRI) offers superior soft tissue contrast to view structural changes in greater detail and thus detects more breast cancers than mammography^4,5. Unfortunately, MRI still misdiagnoses 40% of benign tumors as malignant masses, despite increased sensitivity. False positives on MRI emerge due to the intravenous contrast agent currently utilized (e.g., gadolinium chelates)⁶. Gadolinium (Gd) chelates are always “ON,” creating a signal wherever the agent accumulates in the body. Moreover, Gd-chelates lack specificity⁷ and enter highly vascularized tissues of both benign and malignant tumors.

Therefore, we have developed novel receptor-targeted, pH-sensitive, Nano-Encapsulated Manganese Oxide (NEMO) particles as alternative T₁ MRI contrast agents which turn “ON” in acidic environments^8–10 such as endosomes/lysosomes inside cancer cells. MnO nanocrystals were encapsulated within poly(lactic-co-glycolic acid) and poly(ethylene glycol) with a fluorescent Cy5 dye and conjugated to a breast cancer biomarker, underglycosylated mucin-1 (uMUC-1) targeting peptide (EPPT) or control scrambled peptide via click chemistry. EPPT NEMO particles or scrambled NEMO particles were incubated at 37°C with benign control (MCF10A) and breast cancer cells (T47D) at different time points over 1 hour. Labeled cells were evaluated for differential NEMO particle intracellular localization into endosomes or lysosomes using confocal microscopy, MRI signal activation, and total cellular Mn content by ICP-MS. As uMUC-1 receptors are present on malignant mammary cells but not benign mammary cells, we hypothesized that uMUC-1 targeted EPPT NEMO particles will preferentially attach and be endocytosed by T47D cells over MCF10A to promote specific activation of MRI signal inside low pH endosomes/lysosomes of breast cancer cells.

The T₁ weighted MRI contrast of EPPT NEMO in T47D breast cancer cells significantly peaked to ~275% signal enhancement at 30 min, which was significantly greater than T47D cells labeled with scrambled NEMO or MCF10A cells (control) labeled with EPPT NEMO. Despite MRI signal in T47D cells decreasing to ~150% enhancement at 45 min and 60 min, it was still significantly brighter than MCF10A cells + EPPT NEMO at these time points. Mn content analysis by ICP-MS confirmed that T47D cells labeled with EPPT NEMO had significantly greater Mn levels than MCF10A cells labeled with EPPT NEMO at all time points other than 15 min, confirming the specificity of the peptide targeting. Interestingly, we observed an increased colocalization of EPPT NEMO particles with (1) early endosome and late endosome compared to lysosome at 15 and 30 min, and (2) late endosome and lysosomes compared to early endosomes in T47D cells after incubation at 37°C for 60 minutes. In summary, EPPT NEMO particles are promising alternative MRI contrast agents that produce significantly brighter signal in breast cancer cells compared to benign mammary cells after just 30 min of incubation, which supports clinically relevant timeframes for signal activation. Future studies include in vivo investigation of EPPT NEMO biodistribution, toxicity, and MRI signal activation in breast cancer mouse models versus Gd-chelates.

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