Targeted Delivery of a Ligand-Drug Conjugate via Formyl Peptide Receptor 1 through Cholesterol-Dependent Endocytosis.

G protein-coupled receptors (GPCRs) undergo ligand-induced internalization that carries the cognate ligands into intracellular compartments. The present study explores this property for the use of formyl peptide receptor 1 (FPR1), a class A GPCR that binds formylated peptides, as a potential target for drug delivery. A pH-sensitive peptide-drug conjugate consisting of doxorubicin (DOX), N-ε-maleimidocaproic acid hydrazide (EMCH), and the formyl peptide fMet-Leu-Phe-Cys (abbreviated as DEF) was prepared. DEF retained pharmacological activities of formyl peptides in binding to FPR1 and mobilization of Ca2+ from intracellular stores. However, the conjugated DOX was no longer cell membrane-permeable and relied on FPR1 for cellular entry. DOX was released from DEF into acidic compartments labeled with fluorescent trackers for endosomes. Treatment of cells with pharmacological inhibitors that block clathrin- or caveolae-mediated endocytosis did not abrogate FPR1-dependent DEF internalization, nor did inhibition of macropinocytosis and phagocytosis. In contrast, cholesterol depletion abrogated DEF internalization through FPR1, suggesting characteristics of cholesterol-dependent uptake mediated by a cell surface receptor. These results demonstrate the possibility of using FPR1 for targeted drug delivery.