Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis

Abstract

Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

Routing Nanomolar Protein Cargoes to Lipid Raft‐Mediated/Caveolar Endocytosis through a Ganglioside GM1‐Specific Recognition Tag

“…As expected for the lipid raft‐mediated/caveolar pathway, the progression of the internalized caveolae to early endosomes and later to lysosomes was absent or very slow; therefore, no colocalization with lysosomes was observed, sparing the cargo from early degradation . This may have promoted partial escape of the cargo from the caveosomes, which could be observed in our experiments as diffuse intracellular fluorescence.…”

“…Mammalian cells exert strict control over macromolecular traffic through the cell membrane to cellular compartments. Lipid raft‐mediated/caveolar endocytosis is the most promising method for delivering cargo proteins in their functional form, as exemplified by viruses and bacterial toxins . Our concept was therefore to steer the macromolecular cargo toward lipid raft‐mediated/caveolar endocytosis and to avoid the clathrin‐mediated and macropinocytosis pathways.…”

“…Selection of the ganglioside‐mediated pathway addresses many of the challenges associated with the endocytic delivery of therapeutic macromolecules by allowing carrier‐triggered internalization at low concentrations and avoiding endosomal entrapment. For this reason, there is increasing interest in reading the glycan code through which gangliosides trigger lipid raft‐mediated/caveolar endocytosis . The referenced studies identified peptide sequences (13 and 16 residues) that bind to various gangliosides.…”

A Tf-modified tripterine-loaded coix seed oil microemulsion enhances anti-cervical cancer treatment

“…Modifying the surface of nanomolecules with a tumortargeted ligand can improve drug accumulation at the desired sites, a commonly used and effective method for enhancing efficacy and reducing toxicity of the drug. [34][35][36][37][38][39] Transferrin receptor (TfR) is a protein that is overexpressed on the surface of many cancer cells at a density that is significantly higher than that of normal cells, making TfR a potential target for specific recognition of tumor cells. As reported previously, transferrin (Tf) acts as an iron ion shuttle in the human body and delivers iron ion payloads into the cells and then locates back to the extracellular environment after transporting iron.…”

Self-delivering prodrug-nanoassemblies fabricated by disulfide bond bridged oleate prodrug of docetaxel for breast cancer therapy

“…Confocal laser scanning microscopy (CLSM) images of 4T1 cells incubated with C-6solution or C-6labeled prodrug NPs for 0.5 h or 2 h, respectively (A,B); (C) Quantitative analysis for the fluorescent intensity of confocal laser scanning microscopy ( Ã p < .05 and ÃÃ p < .01, n ¼ 3); (D) Flow cytometry results of cellular uptake of 4T1 cells after incubation for 2 h with (Blank control, C-6 solution, DTX-OA/DSPE2K NPs and DTX-S-S-OA/DSPE2K NPs); Cellular uptake results of pretreating 4T1 cells with several endocytosis inhibitors after incubation with DTX-OA/DSPE2K NPs (E) or DTX-S-S-OA/DSPE2K NPs (F) ( Ã p < .05, ÃÃ p < .01 and ÃÃÃ p < .001 vs. control group, n ¼ 3). the prodrug NPs was an energy-dependent process (Matsubara et al, 2017). Besides, chlorpromazine and indometacin could also significantly reduce the cellular uptake of the prodrug NPs, suggesting that both clathrin-mediated and caveolae-mediated endocytosis contributed to the cellular uptake of prodrug NPs.…”