Conclusion
These findings shed light on the mechanism of QD655-COOH uptake in DCs and that cellular uptake pathways are dependent on cell type and cell differentiation.
Methods
Peripheral blood mononuclear cells were isolated from porcine blood by gradient centrifugation, and monocytes, which are CD14 positive, were purified. Monocytes were differentiated into dendritic cells (DCs) with GM-CSF and IL-4.
Results
Monocytes showed cellular uptake of QD655-COOH, while lymphocytes did not. Monocyte differentiation into DCs increased the cellular uptake by sixfold when dosed with 2 nM of QD655-COOH. Transmission electron microscopy depicted QD655-COOH in the cytoplasmic vacuoles of DCs. Twelve endocytic inhibitors demonstrated QD655-COOH endocytosis in DCs, which was recognized by clathrin and scavenger receptors and regulated by F-actin and phospholipase C. In addition, DC maturation with lipopolysaccharide (LPS) caused an increase in QD655-COOH uptake compared with DCs without LPS stimulation. Viability assays, including 96AQ, CCK-8, alamar blue and ApoTox, exhibited minimal toxicity in DCs dosed with QD655-COOH at 24 h. However, glutathione levels showed a significant decrease with 10 nM of QD655-COOH. Finally, QD655-COOH exposure was associated with a decrease in CD80/CD86 expression after LPS stimulation, suggesting suppression with DC maturation.