Effect of flow on targeting and penetration of angiopep-decorated nanoparticles in a microfluidic model blood-brain barrier
Fig 2
Binding of Ang2-liposomes to brain ECs in static fluid or in the presence of flow.
A) Total binding (ie. surface bound + internalized liposomes) of Ang2-liposomes or non-functionalized liposomes in static fluid was visualized by incubation for 90 minutes at 37°C in cell culture plates (N ≥ 2). Cells incubated with non-functionalized liposomes showed negligible fluorescence compared with Ang2-liposomes. Scale bars are ~15 μm. Cell nuclei were labelled with DAPI (blue signal), while liposomes were labelled with rhodamine (red signal). B) Total binding of Ang2-liposomes or non-functionalized liposomes (55 pM) was quantified after 45 or 90 minutes static incubation at 37°C (N ≥ 3). C) Competitive inhibition of Ang2-liposomes binding was performed by pre-incubation with an anti-LRP antibody for 45 minutes at 37°C, then co-incubated with 55 pM Ang2-liposomes (37% of the predicted Kd) for 60 minutes at 4°C (N ≥ 5). D) A saturation binding study at 4°C (i.e. surface binding only) of Ang2-liposomes was performed by static incubation for 60 minutes (N ≥ 2). Following the incubation period in B-D, cells were lysed in 1% Triton/1M NaOH and fluorescence detected in a spectrophotometer. E) Ang2-liposome binding to brain ECs after incubation in static fluid or in the presence of FSS. The concentration of Ang2-Liposomes was ~120 pM in each condition (N = 2). Scale bars are ~10 micron. * compares Liposomes to Ang2-liposomes, ^ compares 45 minutes to 90 minutes, # compares Ang2-liposomes to Ang2-liposomes + anti-LRP1. p < 0.05 was depicted with one symbol in graphs, and p < 0.01 was depicted with two symbols. Data are expressed as mean ± SEM.