Heparan sulfate proteoglycan-mediated dynamin-dependent transport of neural stem cell exosomes in an in vitro blood-brain barrier model

Abstract

Drug delivery to the brain is greatly hampered by the presence of the blood-brain barrier (BBB) which tightly regulates the passage of molecules from blood to brain and vice versa. Nanocarriers, in which drugs can be encapsulated, can move across the blood-brain barrier (BBB) via the process of transcytosis, thus showing promise to improve drug delivery to the brain. Here, we demonstrate the use of natural nanovesicles, that is, exosomes, derived from C17.2 neural stem cells (NSCs) to efficiently carry a protein cargo across an in vitro BBB model consisting of human brain microvascular endothelial cells. We show that the exosomes are primarily taken up in brain endothelial cells via endocytosis, while heparan sulfate proteoglycans (HSPGs) act as receptors. Taken together, our data support the view that NSC exosomes may act as biological nanocarriers for efficient passage across the BBB. Nanomedicines that target HSPGs may improve their binding to brain endothelial cells and, possibly, show subsequent transcytosis across the BBB.

Keywords: blood-brain barrier; cargo; endothelial cell; exosomes; extracellular vesicles; heparan sulfate proteoglycans; nanocarriers; transcytosis.

FIGURE 1

Characterization of exosomes derived from C17.2 neural stem cells. (a) Methodology used for exosome isolation. Cell culture supernatant 48 hours post seeding of C17.2 neural stem cells is collected and subjected to a series of centrifugations at different speeds to get rid of contaminants. In the end, high‐speed ultracentrifugation is used to collect exosomes. (b) Western blotting analysis of the exosome fraction obtained from procedure in (a) using exosome markers TSG101 and CD9. ß‐actin is used as a loading control. Cell and Exo correspond to parent cell and exosome lysates, respectively. 30 μg protein was loaded for both conditions. Exosome markers are enriched in the exosome fraction compared to the parent cells, while the ß‐actin amount is slightly lower in the exosome fraction. (c) Size distribution of exosomes measured by dynamic light scattering. Exosomes show a size of approximately 120 nm

FIGURE 2

Transport of DiI‐labeled exosomes across an in vitro BBB model. (a) Cartoon depicting the spontaneous incorporation of the lipophilic DiI into the exosome membrane, generating DiI‐labeled exosomes (Exo‐DiI). (b) Schematic representation of the In vitro BBB model, composed of a hCMEC/D3 cell monolayer grown on a Transwell filter, incubated with DiI‐labeled exosomes. (c) Quantification of transcytosis of DiI labeled exosomes across the BBB model. 10 μg Exo‐DiI was added apically and incubated with the BBB model for 18 hours at 370C. DiI fluorescence associated with the apical, basolateral and cellular fractions was measured and is expressed relative to the combined DiI fluorescence of the three fractions (mean ± SD, n = 3). (d) Quantification of the paracellular permeability for 70 kDa dextran‐TRITC in the BBB model in the absence and presence of exosomes, to assess the integrity of the endothelial monolayer. Note that the endothelial monolayer integrity is not altered upon incubation with exosomes (mean ± SD, n = 3)

FIGURE 3

Transport of mCherry‐containing exosomes across an in vitro BBB model. (a) Schematic representation of the XP‐mCherry construct used in this study. Proteins tagged with the XP peptide are expressed at the cytosolic side of the plasma membrane and become concentrated in exosomes as they localize to multivesicular bodies, where ILVs (future exosomes) are generated. (b) Western blots of cell and exosome lysates show that XP‐mCherry is present in cells and exosomes. Exosome marker TSG101 is enriched in exosome fractions. (c) Dot blots of permeabilized (+detergent) and non‐permeabilized (−detergent) exosomes, demonstrating that XP‐mCherry is present at the exosome interior. Exosomes were dot‐blotted on nitrocellulose membrane in different quantities followed by anti‐mCherry immunostaining, in presence or absence of a detergent. (d) Schematic representation of the transcytosis assay. Exosomes containing XP‐mCherry are added to the apical compartment. After 18 hours, apical and basal media are collected and ultracentrifuged to collect exosomes. Collected exosomes are permeabilized and dot‐blotted, followed by mCherry immunostaining. (e) Dot blots of apical and basal fractions obtained from the in vitro BBB model after incubation with wildtype exosomes and XP‐mCherry exosomes, demonstrating XP‐mCherry signal in the basal fraction, which indicates effective transport of exosomes across the in vitro BBB model. The experiment was performed in triplicate, with apical fractions collected and pooled and basolateral fractions collected and pooled in order to obtain a detectable signal after dot blotting. XP: XPack; Exo: exosome lysate; Cell: whole cell lysate

FIGURE 4

Endothelial cells internalize exosomes via dynamin‐dependent endocytosis. hCMEC/D3 cell monolayers were pre‐incubated with DMA (macropinocytosis inhibitor) or Dynasore (dynamin inhibitor) for 30 minutes at 37°C followed by incubation of exosomes in the continued presence of the inhibitor for 2 hours. Graph shows the relative number of exosomes per cell following incubation with Exo‐DiI in the absence (control) or presence of inhibitor. Exosome uptake in hCMEC/D3 cells is significantly reduced in presence of Dynasore (n = 4; ≥300 cells analyzed, *p < 0.05, ANOVA Tukey's post hoc test)

FIGURE 5

Exosomes interact with HSPGs in hCMEC/D3 cells. (a) Schematic representation of the possible effects of heparin and HSase on the interaction of exosomes with hCMEC/D3 cells. (b) SDC2 antibodystaining for assessing the effect of HSase to remove HSPGs enzymatically. Note that SDC2 immunostaining signal is almost absent in cells treated with HSase. Scale bar = 10 μm. (c) Fluorescence images of hCMEC/D3 images incubated with Exo‐DiI in absence (control) or presence of 50 μg/ml heparin or 100 U/ml HSase. Exosome interaction with hCMEC/D3 cells is nearly abolished in presence of heparin and HSase. Scale bar = 25 μm. (d) Quantification of Exo‐DiI uptake in hCMEC/D3 cells in absence (control) or presence of 1, 10 or 50 μg/ml heparin (n = 4; ≥300 cells analyzed per time point, *p < 0.05, ****P < 0.0001, ns – nonsignificant, ANOVA Tukey's post hoc test for comparison of each treatment condition with control, unpaired t‐test for comparison between treatment conditions). (e) Quantification of Exo‐DiI uptake in hCMEC/D3 cells in the absence (control) or presence of 50, 75 or 100 U/ml HSase (n = 4; ≥ 300 cells analyzed per time point, *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA Tukey's post hoc test for comparison of each treatment condition with control, unpaired t‐test for comparison between treatment conditions)