Kiss and Run: Promoting Effective and Targeted Cellular Uptake of a Drug Delivery Vehicle Composed of an Integrin-Targeting Diketopiperazine Peptidomimetic and a Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) have emerged as powerful tools in terms of drug delivery. Those short, often cationic peptides are characterized by their usually low toxicity and their ability to transport diverse cargos inside almost any kinds of cells. Still, one major drawback is their nonselective uptake making their application in targeted cancer therapies questionable. In this work, we aimed to combine the power of a CPP (sC18) with an integrin-targeting unit (c[DKP-f3-RGD]). The latter is composed of the Arg-Gly-Asp peptide sequence cyclized via a diketopiperazine scaffold and is characterized by its high selectivity toward integrin ??. The two parts were linked via copper-catalyzed alkyne-azide click reaction (CuAAC), while the CPP was additionally functionalized with either a fluorescent dye or the anticancer drug daunorubicin. Both functionalities allowed a careful biological evaluation of these novel peptide-conjugates regarding their cellular uptake mechanism, as well as cytotoxicity in ?? integrin receptor expressing cells versus cells that do not express ??. Our results show that the uptake follows a "kiss-and-run"-like model, in which the conjugates first target and recognize the receptor, but translocate mainly by CPP mediation. Thereby, we observed significantly more pronounced toxic effects in ?? expressing U87 cells compared to HT-29 and MCF-7 cells, when the cells were exposed to the substances with only very short contact times (15 min). All in all, we present new concepts for the design of cancer selective peptide-drug conjugates.