Clathrin-mediated endocytosis of quantum dot-peptide conjugates in living cells.
Efficient intracellular delivery of quantum dots (QDs) and unravelling the mechanism underlying the intracellular delivery are essential for advancing the applications of QDs toward in vivo imaging and therapeutic interventions. Here, we show that clathrin-mediated endocytosis is the most important pathway for the intracellular delivery of peptide-conjugated QDs. We selected an insect neuropeptide, namely, allatostatin (AST1, APSGAQRLYG FGL-NH(2)), conjugated it with CdSe-ZnS QDs, and investigated the intracellular delivery of the conjugate in living cells such as human epidermoid ovarian carcinoma cells (A431) and mouse embryonic fibroblast cells (3T3). We selected AST1 to investigate the intracellular delivery of QDs because we recently found it to be efficient for delivering QDs in living mammalian cells. Also, the receptors of AST1 in insects show functional and sequence similarity to G-protein-coupled galanin receptors in mammals. We employed flow cytometry and fluorescence microscopy and investigated the contributions of clathrin-mediated endocytosis, receptor-mediated endocytosis, and charge-based cell penetration or transduction to the intracellular delivery of QD-AST1 conjugates. Interestingly, the intracellular delivery was suppressed by approximately 57% when we inhibited the regulatory enzyme phosphoinositide 3-kinase (PI3K) with wortmannin and blocked the formation of clathrin-coated vesicles. In parallel, we investigated clathrin-mediated endocytosis by colocalizing QD560-labeled clathrin heavy-chain antibody and QD605-AST1. We also estimated galanin receptor-mediated endocytosis of QD-AST1 at <10% by blocking the cells with a galanin antagonist and transduction at <30% by both removing the charge of the peptide due to arginine and suppressing the cell-surface charge due to glycosaminoglycan. In short, the current work shows that multiple pathways are involved in the intracellular delivery of peptide-conjugated QDs, among which clathrin-mediated endocytosis is the most important.
Anas
Abdulaziz
A
Nanobioanalysis Team and Glycolipid Function Analysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-Cho, Takamatsu, Kagawa 761-0395, Japan.
Okuda
Tetsuya
T
Kawashima
Nagako
N
Nakayama
Kenichi
K
Itoh
Tamitake
T
Ishikawa
Mitsuru
M
Biju
Vasudevanpillai
V
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D007301:Insect Hormones / Q000737:chemistry / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D045663:Quantum Dots; D000077191:Wortmannin
D000730:Androstadienes; D002966:Clathrin; D007301:Insect Hormones; D019476:Insect Proteins; D009479:Neuropeptides; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; C061164:allatostatin 1; D000077191:Wortmannin