Fig. 6: Post-capillary venules are the key locus for transcytosis-mediated nanoparticle entry to the brain.

a Nanoparticle movement is restricted to the vessel boundary in capillaries but not in venules. b Real-time imaging of nanoparticle transcytosis to the brain. See also Supplementary Movie 7. c Nanoparticle progression in the brain following transcytosis. See also Supplementary Movie 8. d Nanoparticle movement traces in the brain following laser extravasation at capillaries (yellow) and venules (orange). Inset illustrates vessel hierarchy and color-coding. See also Supplementary Movie 9. e Nanoparticles exhibit a relatively high degree of perivascular movement at venules (orange) compared to capillaries (yellow), where the nanoparticle progression is restricted (n = 64 vs. 64 nanoparticles across 2 mice, ****p < 0.0001, two-tailed Mann–Whitney tests with Bonferroni post hoc correction). Boxplots show medians with IQR, whiskers extend between 5–95th percentile range. f Upper drawing: Craniotomy for chronic two-photon imaging. Lower drawing: Microscope stage with a movement-unrestricted awake animal. The objective is stationary, and the air-pressurized pad reacts reciprocally to the mouse movement. g Long-term imaging of nanoparticle delivery to the brain parenchyma. Upper rows: Nanoparticles are transcytosed to the brain at venular segments; Lower row: No apparent nanoparticle presence in the brain at capillaries and arterioles. h High-resolution images of nanoparticles after transcytosis in post-capillary venules 2 days post-injection. i Measurement of nanoparticles distances (y) to the closest vessel with a given diameter (d). j Nanoparticle distribution in the brain 2 days post-injection. Inset illustrates vessel hierarchy and color-coding. Right panel: nanoparticles distribution with respect to the vessel type, distance from the nearest vessel, and the vessel diameter. Each point represents a single nanoparticle, n = 1256 nanoparticles across 2 mice. Left panel: corresponding percentage distribution of nanoparticles. Dashed lines separate nanoparticles found in the brain at capillaries (yellow), post-capillary and ascending venules (orange), and pial venules (red); from nanoparticles in the endothelium at capillaries (dark yellow), and post-capillary and ascending venules (brown). All panels: ‘zs’ inset indicates Z-stack maximum intensity projection. pA pial artery, pV pial venule, ascV ascending venule, pcV post-capillary venule, cap capillary.