Cavaco, MarcoValle, JavierSilva, RúbenCorreia, João D. G.Castanho, Miguel A. R. B.Andreu, DavidNeves, Vera2023-04-212023-04-212020Current Pharmaceutical Design, 2020, 26, 1495-15061381-6128http://hdl.handle.net/10451/57223© 2020 Bentham Science PublishersBackground: The use of peptides as drug carriers across the blood-brain barrier (BBB) has increased significantly during the last decades. PepH3, a seven residue sequence (AGILKRW) derived from the α-helical domain of the dengue virus type-2 capsid protein, translocates across the BBB with very low toxicity. Somehow predictably from its size and sequence, PepH3 is degraded in serum relatively fast. Among strategies to increase peptide half-life (t1/2), the use of the enantiomer (wholly made of D-amino acid residues) can be quite successful if the peptide interacts with a target in non-stereospecific fashion. Methods: The goal of this work was the development of a more proteolytic-resistant peptide, while keeping the translocation properties. The serum stability, cytotoxicity, in vitro BBB translocation, and internalization mechanism of DPepH3 was assessed and compared to the native peptide. Results: DPepH3 demonstrates a much longer t1/2 compared to PepH3. We also confirm that BBB translocation is receptor-independent, which fully validates the enantiomer strategy chosen. In fact, we demonstrate that internalization occurs trough macropinocytosis. In addition, the enantiomer demonstrates to be non-cytotoxic towards endothelial cells as PepH3. Conclusion: DPepH3 shows excellent translocation and internalization properties, safety, and improved stability. Taken together, our results place DPepH3 at the forefront of the second generation of BBB shuttles.engAdsorption-mediated transcytosisBlood-brain barrierD-amino acidsMacropinocytosisPepH3Peptide shuttlesStabilityjournal article10.2174/13816128266662002130945561873-4286