Fig. 1.

Experimental setup of co-culture model mimicking the human intestinal mucosa (A) and co-culture treatment using siRNA-based nanomedicine for localization studies (B) and JAK/STAT pathway inhibition (C). Co-culture model composed of three different cell types was arranged in a physiologically relevant microenvironment and placed in a Transwell® system. PMA-differentiated THP-1 cells (dTHP-1) were embedded together with dendritic-like cells (MUTZ-3) in a collagen matrix in a ratio of 2:1. After solidification, epithelial cells (Caco-2) were seeded on top and cultivated for 6 days to represent a sub-confluent epithelial barrier (Leaky gut model) instead of previous 11 days comprising a tight epithelium (Tight barrier model). Inflammation of the co-culture was induced by LPS-stimulation (indicated red flash). For localization studies of siRNA-loaded nanomedicine, co-culture was treated with fluorescently-labeled nanocarrier (DiI-LNPs) and siRNA (AF647-siJAK1) and analyzed by confocal microscopy. For JAK/STAT pathway inhibition, co-culture was pre-treated with siJAK1-loaded LNPs or TOFA and subsequently stimulated with IFN-γ. The downregulation of JAK/STAT signaling was evaluated by downstream analysis of STAT1 protein phosphorylation using phospho-specific antibody staining and flow cytometry. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)