Fig 1.
Microfluidic BBB model for evaluation of Ang2-liposome binding and penetration in static or flow condition.
A) Cartoon of Ang2-Liposomes. Ang2-Liposomes were fabricated using the lipid film hydration method and sequentially extruded to reach a final size of 80–95 nm. Angiopep-2 was conjugated to the end of PEG chains using maleimide-thiol chemistry. B) Structure of the microfluidic BBB model. Brain ECs (bEnd.3 cells) were grown in the upper channel of devices for 3–6 days to enable barrier formation. Ang2-Liposomes were added to the upper channel and incubated in static fluid or in the presence of flow to assess binding and BBB penetration. Fluid in the lower channel was kept static for the duration of experiments. C) Cartoon depicting layers of the microfluidic BBB model. During the experiment the upper channel was opened to incubate Ang2-Liposomes with flow or in static fluid, while the lower channel was closed to limit advection between the upper and lower channels. After the experiment, the upper channel was closed, and the lower channel was opened to collect samples for analysis. D) After 6 days static culture on pit-patterned membrane, TEER of the microfluidic BBB model reached 172 ohm x cm2 (N = 5 devices), and FITC dextran permeability decreased as a function of molecular weight (N ≥ 3 devices per condition). Data are expressed as mean ± SEM.
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.
Fig 3.
Internalization of Ang2-liposomes by brain ECs in static condition or in presence of flow.
A) Internalization of Ang2-liposomes (250 pM) in static fluid after 90 minutes at 37°C vs 4°C. Z-stacks (0.6–0.9 microns/slice) through the entire cell (~6–10 microns) showed perinuclear localization of Ang2-liposomes at 37°C (i) compared with greater localization at the cell periphery at 4°C (iii). A cross-sectional view shows Ang2- liposomes within the cell at 37°C (ii) vs at the cell surface at 4°C (iv) (N ≥ 2). Cell nuclei were labelled with DAPI (blue signal), while liposomes were labelled with rhodamine (red signal). Scale bars are ~10 micron. B) Internalization kinetics and magnitude in static fluid were quantified by comparing binding of Ang2-liposomes at 37°C vs 4°C after 45 and 90 minutes incubation in cell culture plates (N ≥ 3). Following the incubation period, cells were lysed in 1% Triton/1M NaOH and fluorescence detected in a spectrophotometer.* compares 4°C vs 37 oC, and ^ compares 45 minutes vs 90 minutes. The use of 3 symbols for each comparison reflects that p < 0.001. Data are expressed as mean ± SEM. C) Internalization of Ang2-Liposomes (110 pM) in the presence of FSS was assessed after 90 minutes at 37°C. Scale bars are ~10 micron.
Fig 4.
Ang2-liposome penetration of brain ECs in the presence or absence of FSS.
A) Penetration of Ang2-liposomes incubated in static fluid or in the presence of FSS was evaluated at 37 oC after 120 minutes incubation. Fluid in the lower channel was kept static during the experiment (N ≥ 4 devices per condition). * compares static to FSS with one symbol where p < 0.05 and two symbols where p < 0.01. B) Penetration of Ang2-functionalized vs. non-functionalized liposomes in the presence of FSS in the upper channel. Fluid in the lower channel was kept static during the experiment (N ≥ 3).C) Induction of barrier disruption by Ang2-liposomes was evaluated by comparing dextran penetration in the presence or absence of Ang2-liposomes at 37°C or trypsin as a positive control (N ≥ 2). Brain EC barriers were incubated with HBSS (Ca2+, Mg2+) only, Ang2-liposomes, or trypsin for 120 minutes in static fluid in the upper and lower channels. After this period, penetration of 4 kDa dextran was measured after 15–30 minutes static incubation. * compares Dex + Trypsin to Dex only where p < 0.05. D) Competitive inhibition of Ang2-liposome penetration was evaluated with an anti-LRP antibody with static fluid in the upper and lower channels. Anti-LRP was preincubated for 45 minutes followed by co-incubation with Ang2-Liposomes for 120 minutes at 37°C (N ≥ 7). Data are expressed as mean ± SEM.
Fig 5.
Characterization of microfluidic BBB model function.
A) Claudin-5 staining of brain ECs cultured for 6 days in static cell culture plates. B-C) Claudin-5 staining after 6 days static culture followed by 2 hours flow in the upper channel of the microfluidic BBB model (brain ECs cultured on pit-patterned membrane, N = 2 per condition). Scale bars are ~ 10m micron.