Perfluorohexane Emulsions Induce Macrophage-Stimulating Effects through iNOS Expression

Abstract

Despite advances in cancer immunotherapy, efficacy has been limited to a subset of patients, highlighting the need for continued development of innovative therapeutic approaches. Perfluorocarbon-filled (PFC) particles, due to unique physicochemical properties, biological stability, and quantifiability as 19F-MRI tracers, have been intensely investigated as in vivo macrophage imaging agents. Despite this, little is known about the effect of PFC particle uptake on macrophage behavior. Here, phospholipid-stabilized perfluorohexane (PFH) nanodroplets (NDs) were used to treat bone-marrow-derived macrophages (BMDMs) in vitro and tumor-implanted mice in vivo. Physiological response was assessed by using biochemistry, flow cytometry, fluorescence imaging, and tumor measurements. The incubation of alternatively-activated (M2) BMDMs with PFH NDs led to a dose- and time-dependent increase of iNOS expression but not that of other common M1 or M2 markers. PFH ND-induced iNOS expression was further enhanced when treated M2 BMDMs were co-cultured with MC38 murine colon carcinoma cells, resulting in more effective tumoral cell apoptosis. Similarly, intratumoral injection of PFH NDs in a murine colon carcinoma model led to increased iNOS expression within the tumors and caused a substantial decrease in tumor size. This size reduction was also unexpectedly accompanied by the formation of peritumoral gas pockets, which, when aspirated, abrogated tumoral effects. Conversely, intratumoral injection of emulsions made with high-boiling-point (BP = 142 °C) perfluorooctyl bromide (PFOB) did not produce gas pockets or inhibit tumor growth. Our data collectively show the immunogenic potential of PFH NDs through iNOS expression and promote the potential development of theranostic PFC-based platforms.

Keywords: cancer; immunomodulation; macrophage; nanomedicine; perfluorohexane.