STING-inhibitor-phenamil polymeric nanocarriers for the in vitro modulation of macrophage polarization.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation and polyarthritis. RA has no cure and imposes a significant burden on individuals because current standard-of-care anti-inflammatory treatments reduce inflammation by regulating lymphocyte proliferation and pro-inflammatory cytokine function. However, recent studies have shown that macrophages play a pivotal role in the progression of RA and therefore, we hypothesize that co-loaded nanocarriers (NCs) containing a STING inhibitor and phenamil can reduce inflammation by repolarizing macrophages from a pro-inflammatory (M1-like) to an anti-inflammatory (M2-like) phenotype. Using Flash NanoPrecipitation (FNP), both compounds were encapsulated into polymeric nanocarriers which were then tested for their ability to induce an anti-inflammatory phenotype in vitro. Macrophage NF-κB activation levels were assessed using a reporter cell line, gene expression via qRT-PCR, and polarization state via flow cytometry. NF-κB activity was monitored using a cell line that secretes embryonic alkaline phosphatase upon activation; reductions in this signal indicate suppression of inflammatory signaling. Key markers examined for an anti-inflammatory phenotype for macrophages included IL-1β, IL-6, IL-10, and TGF-β. Finally, by assessing the ratio of M2-like to M1-like macrophages, we sought to determine if these formulations could successfully repolarize macrophages from M1-like to M2-like, and/or help macrophages resist polarization to an M1-like state following exposure to a pro-inflammatory stimulus. Through this work, we aimed to investigate how nanotechnology can be leveraged to improve therapeutic outcomes by regulating macrophage polarization states.