Tracking macrophages in diabetic neuropathy with two-color nanoemulsions for near-infrared fluorescent imaging and microscopy

Background: The incidence of diabetes and diabetic peripheral neuropathy continues to rise, and studies have shown that macrophages play an important role in their pathogenesis. To date, macrophage tracking has largely been achieved using genetically-encoded fluorescent proteins. Here we present a novel two-color fluorescently labeled perfluorocarbon nanoemulsion (PFC-NE) designed to monitor phagocytic macrophages in diabetic neuropathy in vitro and in vivo using non-invasive near-infrared fluorescent (NIRF) imaging and fluorescence microscopy. Methods: Presented PFC-NEs were formulated with perfluorocarbon oil surrounded by hydrocarbon shell carrying two fluorescent dyes and stabilized with non-ionic surfactants. In vitro assessment of nanoemulsions was performed by measuring fluorescent signal stability, colloidal stability, and macrophage uptake and subsequent viability. The two-color PFC-NE was administered to Lepr^db/db and wild-type mice by tail vein injection, and in vivo tracking of the nanoemulsion was performed using both NIRF imaging and confocal microscopy to assess its biodistribution within phagocytic macrophages along the peripheral sensory apparatus of the hindlimb. Results: In vitro experiments show two-color PFC-NE demonstrated high fluorescent and colloidal stability, and that it was readily incorporated into RAW 264.7 macrophages. In vivo tracking revealed distribution of the two-color nanoemulsion to macrophages within most tissues of Lepr^db/db and wild-type mice which persisted for several weeks, however it did not cross the blood brain barrier. Reduced fluorescence was seen in sciatic nerves of both Lepr^db/db and wild-type mice, implying that the nanoemulsion may also have difficulty crossing an intact blood nerve barrier. Additionally, distribution of the nanoemulsion in Lepr^db/db mice was reduced in several tissues as compared to wild-type mice. This reduction in biodistribution appears to be caused by the increased number of adipose tissue macrophages in Lepr^db/db mice. Conclusions: The nanoemulsion in this study has the ability to identify phagocytic macrophages in the Lepr^db/db model using both NIRF imaging and fluorescence microscopy. Presented nanoemulsions have the potential for carrying lipophilic drugs and/or fluorescent dyes, and target inflammatory macrophages in diabetes. Therefore, we foresee these agents becoming a useful tool in both imaging inflammation and providing potential treatment in diabetic peripheral neuropathy.