Rapid and Widespread Distribution of Intranasal Small Extracellular Vesicles Derived from Mesenchymal Stem Cells throughout the Brain Potentially via the Perivascular Pathway

Abstract

Intranasal administration is a promising strategy to enhance the delivery of the sEVsomes-based drug delivery system to the central nervous system (CNS). This study aimed to explore central distributive characteristics of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) and underlying pathways. Here, we observed that intranasal MSC-sEVs were rapidly distributed to various brain regions, especially in the subcortex distant from the olfactory bulb, and were absorbed by multiple cells residing in these regions. We captured earlier transportation of intranasal MSC-sEVs into the perivascular space and found an increase in cerebrospinal fluid influx after intranasal administration, particularly in subcortical structures of anterior brain regions where intranasal sEVs were distributed more significantly. These results suggest that the perivascular pathway may underlie the rapid and widespread central delivery kinetics of intranasal MSC-sEVs and support the potential of the intranasal route to deliver MSC-sEVs to the brain for CNS therapy.

Keywords: distribution; extracellular vesicles; intranasal administration; perivascular space.

Figure 1

Characterization of sEVs from huc MSC. (A) Representative morphology of huc MSC-sEVs under TEM (scale bar 100 nm). (B) NTA analysis showing the size range of sEVs. (C) Western blot analysis showing classic EV proteins, including ALIX, TSG101, and CD63.

Figure 2

Biodistribution of MSC-sEVs following intranasal delivery. (A) Representative images of Cy7-MSC-sEVs fluorescence in the brain at 0.5, 2, 6, and 24 h after intranasal delivery. (B) Quantification of radiant efficiency of Cy7-MSC-sEVs administered. The treated group exhibited more significant radiant efficiency in the brain area at 0.5, 2, and 6 h while there was no significant difference at 24 h. (C) Representative images of Cy7-MSC-sEVs fluorescence in different organs at 2 h after delivery. (D) Quantification of relative radiant efficiency of Cy7-MSC-sEVs administered. (E) Representative images of Cy3-MSC-sEVs fluorescence in brain slice at 2 h after administration. (F) Quantification of normalized fluorescence intensity of Cy3-MSC-sEVs in all brain regions. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significance. Data are presented as mean  ±  SD, n = 4/group, (B) two-way ANOVA with Bonferroni’s posthoc testing, and (D,E) two-tailed unpaired t-test. OB: olfactory bulb, Ctx: cortex, CC: corpus callosum, Str: striatum, Hip: hippocampus, Pn: pons, and Cb: cerebellum.

Figure 3

Uptake of MSC-sEVs by multiple cells in various brain regions. Representative images showing the uptake of Cy3-MSC-sEVs (red/yellow dots) by microglia, astrocytes, and neurons in the OB, Ctx, CC (A), and other subcortex structure (SCtx), including Hip, Str, Pn, and Cb (B) (scale bar 25 µm). The white arrowheads indicate the presence of Cy3-MSC-sEVs within the cytoplasm of cells. Quantification of the percentage of cells containing sEVs in the OB, Ctx, CC (C), and SCtx (D). * p < 0.05, ** p < 0.01. Data are presented as mean  ±  SD, n = 3/group, (C,D) one-way ANOVA with Bonferroni’s posthoc testing. OB: olfactory bulb, Ctx: cortex, CC: corpus callosum, Str: striatum, Hip: hippocampus, Pn: pons, and Cb: cerebellum.

Figure 4

Localization of intranasal MSC-sEVs concerning the PVS. (A) Representative image of Cy3-MSC-sEVs captured in the PVS located in the CC, Str, and Hip (scale bar 10 µm). (B,C) Representative images of TEM showing the location of GNP-MSC-sEVs within the PVS. The white arrows indicated GNP-MSC-sEVs near the basement membrane and in pericytes (left) (scale bar 200 nm), and the white arrowheads showed magnified views of loaded GNPs (right). CC: corpus callosum, Str: striatum, Hip: Hippocampus, PVS: perivascular space, EC: endothelial cells, P: pericytes, L: capillary lumen, and *: basement membrane.

Figure 5

Intranasal administration of MSC-sEVs increased CSF tracer influx. (A) Representative image showing the distribution of CSF tracer OA-647 30 min after intracisternal injection in the control and treated group (scale bar 1 mm). (B,D) Quantification of CSF tracer influx into all brain regions (B), anterior (C), and posterior (D) brain regions. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as mean  ±  SD, n = 6/group, (B,D) two-way ANOVA with Bonferroni’s posthoc testing. Ctx: cortex, CC: corpus callosum, and SCtx: subcortex structure.

Figure 6

Schematic of a distributive pattern of MSC-sEVs following intranasal administration. (A) Intranasal MSC-sEVs (red dots) can reach the OB or Pn along the axons of the olfactory nerve and trigeminal nerves and have the potential to enter the olfactory subarachnoid space full of CSF. Subsequent distribution of MSC-sEVs in the brain may be mediated by the perineuronal pathway, which may result in local diffusion of MSC-sEVs near OB and Pn and perivascular pathway by which MSC-sEVs can be rapidly transported to various brain regions. (B) Intranasal MSC-sEVs with CSF influx can reach every level of PVS where CSF–ISF exchange occurs. (C) Through the exchange within pericapillary space, MSC-sEVs can be incorporated into nearby pericytes or astrocytes and distributed into the brain parenchyma, which is facilitated by the polarization of AQP 4 toward the PVS. OB: olfactory bulb, Ctx: cortex, CC: corpus callosum, Str: striatum, Hip: hippocampus, Mb: midbrain, Pn: pons, My: medulla, Cb: cerebellum, PVS: perivascular space, CSF: cerebrospinal fluid, ISF: interstitial fluid. Green and purple arrows showed the olfactory and trigeminal pathways respectively while blue arrows depicted the perivascular pathway.