Engineered Nanoceria with Tuned Surface Valence for Macrophage Modulation

Introduction:: Abnormal inflammatory states are involved in many diseases ranging from rheumatoid arthritis to solid tumors. There is a strong need for a drug capable of modulating the immune environment to overcome these conditions. Cerium oxide nanoparticles (nanoceria) provide a unique opportunity to treat abnormal inflammatory states. Their small size (< 5 nm) and unique surface chemistry (mixed surface valences of Ce³⁺ and Ce⁴⁺) allow them to act as multi-enzyme mimetics (catalase, peroxidase, and superoxide dismutase (SOD)) and to reversibly scavenge reactive oxygen species (ROS). The ROS scavenging nanoceria influences the differentiation of macrophage cells, which are a key part of primary immune response, from an M0 undifferentiated state to an M1 pro- or M2 anti-inflammatory subtype. When differentiated, these cells can modulate the overall immune microenvironment through chemical signaling (e.g., cytokine, chemokine). Nanoceria can be synthesized with tuned surface valence through substrate selection, such as single-walled carbon nanotubes (SWCNTs) and albumin protein, or catalyst choices. This tuned surface chemistry can be used to optimize the immune-modulative activity of the nanoceria. The engineered nanoceria with varied substrates can retain additional functionality, such as immune cell targeting and imaging potential. This study will lead to the optimization of nanoceria as a new class of drugs that can effectively modulate inflammatory disease conditions.

Materials and Methods:: Synthesis of albumin-nanoceria (albumin-coated nanoceria) was carried out by combining albumin protein, cerium (III) hexahydrate, and sodium hydroxide with and without the addition of H₂O₂ at 40°C for 2 hours. SWCNT-ceria (nanoceria bound with SWCNT) was synthesized with the substitution of PEGylated CNT (< 300 nm) for albumin protein. The particles were then washed by centrifugal filtration and underwent dialysis to remove impurities. Cerium content was evaluated via Inductively Coupled Plasma Spectroscopy (ICP), Surface morphology and crystallinity of the particle were characterized via Transmission Electron Microscopy (TEM), and surface valence was validated by X-ray Photoelectron Spectroscopy (XPS). The particles were then tested for their enzymatic behaviors using SOD, Peroxidase, and Catalase assay kits. Raw 264.7 cells were seeded at 50K cells/well in 24-well plates for DCFDA assay and Flow Cytometry (FACS; Accuri C6) experiments. The cells were then treated with LPS or LPS/IFN- γ to induce ROS generation and M1-like pro-inflammatory state and treated by either albumin-nanoceria 1 or 2 (30 µg Ce/mL) and incubated overnight. For DCFDA assay, cells were washed and then incubated for 30 minutes with DCFDA and fluorescence was quantified via UV-vis spectroscopy. For FACS, the following day, cells were washed with PBS, stained with Zombie Green viability dye, and stained with anti-CD80 and anti-CD206 antibodies to measure pro and anti-inflammatory behavior, respectively. The results were evaluated with FCS Express. DCFDA and FACS experiments are performed with SWCNT-nanoceria as well. Statistical analysis was performed using a student’s t-test and considered significant at p< 0.05.

Results, Conclusions, and Discussions:: Albumin-nanoceria were successfully synthesized via bio-mineralization. SWCNT-nanoceria were synthesized via assembly method through two-step synthesis. Both particles were found to be highly crystalline and monodisperse ceria particles (< 5 nm) with coated substrates (albumin, SWCNT). The albumin-nanoceria were found to be enzymatically active (catalase and SOD) and capable of scavenging reactive oxygen species. Particles synthesized with H₂O₂ (albumin-nanoceria 2) were highly anti-inflammatory, both reducing the expression of CD80 pro-inflammatory markers and increasing anti-inflammatory CD206. They were Ce³⁺ dominated in their surface valence states by XPS analysis and orange in color. On the other hand, albumin-nanoceria 1 exhibited more SOD-like activity than albumin-nanoceria 2 and did not increase anti-inflammatory CD206 marker. They had a more even ratio of Ce³⁺/Ce⁴⁺ and were yellow in color. This indicates that enhanced Ce³⁺ on the surface of the particles is associated with enhanced anti-inflammatory M2-like phenotype while enhanced Ce⁴⁺ is associated with enhanced pro-inflammatory M1-like phenotype. We also investigated the nanoceria surface valence states (Ce³⁺/Ce⁴⁺ ratio) tuned by use of a different substrate (SWCNT). Much like albumin-nanoceria 1, the increased presence of Ce⁴⁺ on the surface of the SWCNT-nanoceria will lead to enhanced pro-inflammatory behavior of macrophages, where additional experiments are needed to validate the hypothesis. Experiments are ongoing to investigate the specific targeting effect of SWCNT-nanoceria to immune cells in comparison with albumin-coated nanoceria and to validate their immune-modulative efficacy (either pro-inflammatory or anti-inflammatory) in experimental animals. Nanoceria with surface valence states (Ce³⁺/Ce⁴⁺ ratio) tuned through a selection of catalysts or substrate choices can be used to alter the immune microenvironment and ultimately overcome barriers to the simple and effective treatment of inflammatory conditions.


Acknowledgements (Optional): : The authors would like to acknowledge Dr. Matthew Bernard and the MSU flow cytometry core for their valuable contributions to the design and implementation of flow cytometry experiments.


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