Manganese oxide (MnO) nanoparticles (NPs) can serve as robust pH-sensitive contrast agents for magnetic resonance imaging (MRI) due to Mn²⁺ release at low pH, which generates a ~30 fold change in T₁ relaxivity. Strategies to control NP size, composition, and Mn²⁺ dissolution rates are essential to improve diagnostic performance of pH-responsive MnO NPs. We are the first to demonstrate that MnO NP size and composition can be tuned by the temperature ramping rate and aging time used during thermal decomposition of manganese(II) acetylacetonate. Two different temperature ramping rates (10°C/min and 20°C/min) were applied to reach 300°C and NPs were aged at that temperature for 5, 15, or 30 min. A faster ramping rate and shorter aging time produced the smallest NPs of ~23 nm. Shorter aging times created a mixture of MnO and Mn₃O₄ NPs, whereas longer aging times formed MnO. Our results indicate that a 20°C/min ramp rate with an aging time of 30 min was the ideal temperature condition to form the smallest pure MnO NPs of ~32 nm. However, Mn²⁺ dissolution rates at low pH were unaffected by synthesis conditions. Although Mn²⁺ production was high at pH 5 mimicking endosomes inside cells, minimal Mn²⁺ was released at pH 6.5 and 7.4, which mimic the tumor extracellular space and blood, respectively. To further elucidate the effects of NP composition and size on Mn²⁺ release and MRI contrast, the ideal MnO NP formulation (~32 nm) was compared with smaller MnO and Mn₃O₄ NPs. Small MnO NPs produced the highest amount of Mn²⁺ at acidic pH with maximum T₁ MRI signal; Mn₃O₄ NPs generated the lowest MRI signal. MnO NPs encapsulated within poly(lactide-co-glycolide) (PLGA) retained significantly higher Mn²⁺ release and MRI signal compared to PLGA Mn₃O₄ NPs. Therefore, MnO instead of Mn₃O₄ should be targeted intracellularly to maximize MRI contrast.

Novel metal oxide nanoparticle (NP) contrast agents may offer safety and functionality advantages over conventional gadolinium-based contrast agents (GBCAs) for cancer diagnosis by magnetic resonance imaging. However, little is known about the behavior of metal oxide NPs, or of their effect, upon coming into contact with the innate immune system. As neutrophils are the body’s first line of defense, we sought to understand how manganese oxide and iron oxide NPs impact leukocyte functionality. Specifically, we evaluated whether contrast agents caused neutrophils to release web-like fibers of DNA known as neutrophil extracellular traps (NETs), which are known to enhance metastasis and thrombosis in cancer patients. Murine neutrophils were treated with GBCA, bare manganese oxide or iron oxide NPs, or poly(lactic-co-glycolic acid) (PLGA)-coated metal oxide NPs with different incorporated levels of poly(ethylene glycol) (PEG). Manganese oxide NPs elicited the highest NETosis rates and had enhanced neutrophil uptake properties compared to iron oxide NPs. Interestingly, NPs with low levels of PEGylation produced more NETs than those with higher PEGylation. Despite generating a low rate of NETosis, GBCA altered neutrophil cytokine expression more than NP treatments. This study is the first to investigate whether manganese oxide NPs and GBCAs modulate NETosis and reveals that contrast agents may have unintended off-target effects which warrant further investigation.

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in magnetic resonance imaging (MRI). While iron oxide nanoparticles provide constant negative contrast on MRI over typical experimental timeframes, MnO generates switchable positive contrast on MRI through dissolution of MnO to Mn²⁺ at low pH within cell endosomes to ‘turn ON’ MRI contrast. This protocol describes a one-pot synthesis of MnO nanoparticles formed by thermal decomposition of manganese(II) acetylacetonate in oleylamine and dibenzyl ether. Although running the synthesis of MnO nanoparticles is simple, the initial experimental setup can be difficult to reproduce if detailed instructions are not provided. Thus, the glassware and tubing assembly is first thoroughly described to allow other investigators to easily reproduce the setup. The synthesis method incorporates a temperature controller to achieve automated and precise manipulation of the desired temperature profile, which will impact resulting nanoparticle size and chemistry. The thermal decomposition protocol can be readily adapted to generate other metal oxide nanoparticles (e.g., iron oxide) and to include alternative organic solvents and stabilizers (e.g., oleic acid). In addition, the ratio of organic solvent to stabilizer can be changed to further impact nanoparticle properties, which is shown herein. Synthesized MnO nanoparticles are characterized for morphology, size, bulk composition, and surface composition through transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. The MnO nanoparticles synthesized by this method will be hydrophobic and must be further manipulated through ligand exchange, polymeric encapsulation, or lipid capping to incorporate hydrophilic groups for interaction with biological fluids and tissues.

Triple-negative breast cancer (TNBC) develops in hypoxic, nutrient-limited tumors enriched with macrophages and cell death. We show that metabolically distinct TNBCs differentially exploit macrophage-derived nutrients, influencing tumor growth and therapeutic response. Prolonged hypoxia reprogrammed mouse and human macrophages, enabling them to release metabolites that rescued the growth of select TNBC cell lines during glutamine deprivation or glutamine metabolism inhibition. Hypoxic macrophages reduced glutamine consumption, increased arginine utilization, and secreted higher levels of ornithine, an intermediate of arginine metabolism. Exogenous ornithine, but not arginine, restored the growth of responsive TNBC cells. Mechanistically, TNBC cells diverted ornithine into proline biosynthesis, supporting oxidative pentose phosphate pathway activity. In vivo, depletion of tumor-associated myeloid cells reduced tumor growth and impaired proline synthesis in glutaminase inhibitor–resistant TNBC. These findings identify hypoxia-driven metabolic cross-talk between macrophages and TNBC cells, revealing ornithine-dependent proline metabolism as a mechanism by which macrophages sustain tumor growth under nutrient stress and contribute to resistance to glutamine-targeted therapies.