Unraveling GLUT-mediated transcytosis pathway of glycosylated nanodisks

Abstract

Glucose transporter (GLUT)-mediated transcytosis has been validated as an efficient method to cross the blood-brain barrier and enhance brain transport of nanomedicines. However, the transcytosis process remains elusive. Glycopeptide-modified nanodisks (Gly-A7R-NDs), which demonstrated high capacity of brain targeting via GLUT-mediated transcytosis in our previous reports, were utilized to better understand the whole transcytosis process. Gly-A7R-NDs internalized brain capillary endothelial cells mainly via GLUT-mediated/clathrin dependent endocytosis and macropinocytosis. The intracellular Gly-A7R-NDs remained intact, and the main excretion route of Gly-A7R-NDs was lysosomal exocytosis. Glycosylation of nanomedicine was crucial in GLUT-mediated transcytosis, while morphology did not affect the efficiency. This study highlights the pivotal roles of lysosomal exocytosis in the process of GLUT-mediated transcytosis, providing a new impetus to development of brain targeting drug delivery by accelerating lysosomal exocytosis.

Keywords: Blood-brain barrier; Glucose transporter; Glycosylation; Lysosomal exocytosis; Transcytosis.

Graphical abstract

Fig. 1

Characterization of Gly-A7R-NDs. (A) Schematic diagram of Gly-A7R-NDs. Spatial structures: upper; Planar structures: lower. (B) Cryo-EM micrograph of Gly-A7R-NDs. The arrow and arrowhead indicated the frontal and lateral views of Gly-A7R-NDs, respectively. The hydrodynamic diameter (C) and zeta potential (D) of Gly-A7R-NDs measured using DLS. Gly-A7R-NDs displayed a hydrodynamic diameter around 60 nm and slightly negative surface charge (−4.86 mV). (E) SDS-PAGE and fluorescence image of Gly-A7R-NDs-FITC. Gly-A7R-NDs-FITC were incubated with (1) PBS at 4 °C, (2) PBS at 37 °C, (3) 10% FBS at 37 °C, (4) 10% ICR mice serum at 37 °C or (5) 5% Triton at 37 °C for 4 h. In triton, the nanodisks were disrupted and the dye was shifted forward. Gly-A7R-NDs were stable under all other conditions.

Fig. 2

Endocytic pathways of Gly-A7R-NDs by bEnd.3 cells. (A) Confocal micrographs of cells incubated with Gly-A7R-NDs/DiO/DiI/DiD or Gly-A7R-NDs/DiO at 37 °C for 12 h. Gly-A7R-NDs efficiently internalized into bEnd.3 cells and remained intact. Scale bar = 10 µm. (B) The effect of a variety of inhibitors that can block different endocytic pathways on cellular uptake using a flow cytometer. Cells treated with the blank medium represented the control and the intracellular fluorescence intensity was set as 100%. All data were statistically analysed using ANOVA (ns indicates non-significant, **P < 0.01, ***P < 0.001); Mean ± SD, n = 3.

Fig. 3

Intracellular trafficking of Gly-A7R-NDs in bEnd.3 cells. Co-localization of Gly-A7R-NDs with early endosomes marked with EEA1 at 1 h (A), late endosomes marked with M6PR at 4 h (B), lysosomes at 12 h (C), recycling endosomes marked with Rab11 at 1 h (D), Golgi apparatus at 4 h (E) and GLUT4 vesicles marked with Rab8 at 4 h (F), respectively. Blue: nuclei; Red: Gly-A7R-NDs/DiD; Green: Different intracellular compartments or markers. Scale bar = 10 µm.

Fig. 4

Exocytic pathway of Gly-A7R-NDs from bEnd.3 cells. (A) Co-localization of Gly-A7R-NDs and recycling endosomes treated with monensin at 37 °C for 2 h. (B) Co-localization of Gly-A7R-NDs and Golgi apparatus treated with brefeldin A at 37 °C for 4 h. (C) Co-localization of Gly-A7R-NDs and lysosomes treated with nocozazole at 37 °C for 8 h. Scale bar = 10 µm. The influence of exocytosis related inhibitors on intracellular (D) Gly-A7R-NDs, (E) A7R-NDs, A7R-LSs and Gly-A7R-LSs during co-incubation. Cells treated with blank medium represented the control and the intracellular fluorescence intensity was set as 100%. All data were statistically analysed using ANOVA (*P < 0.05, ***P < 0.001). Data are mean ± SD, n = 3.

Fig. 5

Effect of lysosomal exocytosis on efflux of Gly-A7R-NDs by bEnd.3 cells. Co-localization of Gly-A7R-NDs with lysosomes treated with chloroquine (A) or LY294002 (B) at 37 °C for 8 h. (C) The influence of lysosomal exocytosis related inhibitors on intracellular Gly-A7R-NDs during co-incubation. Cells treated with blank medium represented the control and the intracellular fluorescence intensity was set as 100%. (D) Cytotoxicity of free lysosomal exocytosis-related inhibitors on bEnd.3 cells. (E) The effect of lysosomal exocytosis related-inhibitors on cytotoxicity of Gly-A7R-NDs/PTX on bEnd.3 Cells for 72 h incubation (*P < 0.05, ***P < 0.001 v.s. Control, mean ± SD, n = 3).

Fig. 6

Schematic representation of the transcytosis pathways of Gly-A7R-NDs in bEnd.3 cells.