Figure 6: Uptake of NPs exposing DY-635 on their surface in vitro.

(a) A microfluidic-assisted ‘organoid’ composed of co-cultured HepaRG and HUVEC demonstrates an increased uptake by the hepatocytes preferentially if cells are subjected to flow conditions (‘dynamic’). Under flow conditions, this organoid better recapitulates the function of the liver in vivo by circulating fluid containing NP through the equivalent of the space of Disse. (b) Representative overlay of brightfield and fluorescence images of HepaRG cells differentiated to hepatocytes (stars) or endothelia like (arrow head) cells cultured under static or dynamic conditions reflect increased uptake of DY-635[NP](−) (red) after 90 min of incubation if cells are subjected to flow. This uptake can be inhibited by cyclosporine A as well as Pitstop-2. DY-635[NP](−) uptake was assessed by epi-fluorescence microscopy (Cy5 channel); (scale bar 100 μm). To characterise cell selectivity and uptake mechanisms, further experiments were conducted. (c) Uptake of DY-635[NP](−) is inhibitable by cyclosporine A, a ligand for OATPs and NTCP in HepaRG but not in HUVEC that do not express these transporters; inhibition by Pitstop-2 suggests clathrin-mediated endocytosis as molecular mechanism of cellular uptake. (d) Cyclosporine A failed to affect the overall less pronounced basal uptake of [NP](nile red) that are not exposing DY-635 on their surface. For the statistical analysis a generalised mixed model was applied, taking into account dependent (time, flow condition) and independent (cell type) data. Post hoc analysis was performed using Tukey’s test. In panel (a) * indicates P<0.05 comparing respective static versus dynamic conditions; # indicates P<0.05, ## indicates P<0.01 for the comparison between HepaRG and HUVEC. In panel (d), * and ** indicate P<0.01 and P<0.01 respectively, for the uptake of DY-635[NP](−) in HepaRG or HUVEC in the absence of a co inhibitor (w/o) compared with the uptake in presence of cyclosporine A or Pitstop-2.