Identification of BP16 as a non-toxic cell-penetrating peptide with highly efficient drug delivery properties.

Antimicrobial peptides are an interesting source of non-cytotoxic drug delivery vectors. Herein, we report on the identification of a new cell-penetrating peptide (KKLFKKILKKL-NH2, BP16) from a set of antimicrobial peptides selected from a library of cecropin-melittin hybrids (CECMEL11) previously designed to be used in plant protection. This set of peptides was screened for their cytotoxicity against breast adenocarcinoma MCF-7, pancreas adenocarcinoma CAPAN-1 and mouse embryonic fibroblast 3T3 cell lines. BP16 resulted to be non-toxic against both malignant and non-malignant cells at concentrations up to 200 μM. We demonstrated by flow cytometry and confocal microscopy that BP16 is mainly internalized in the cells through a clathrin dependent endocytosis and that it efficiently accumulates in the cell cytoplasm. We confirmed that the cell-penetrating properties of BP16 are retained after conjugating it to the breast tumor homing peptide CREKA. Furthermore, we assessed the potential of BP16 as a drug delivery vector by conjugating the anticancer drug chlorambucil to BP16 and to a CREKA-BP16 conjugate. The efficacy of the drug increased between 6 and 9 times when conjugated to BP16 and between 2 and 4.5 times when attached to the CREKA-BP16 derivative. The low toxicity and the excellent cell-penetrating properties clearly suggest that BP16 is a suitable vector for the delivery of therapeutic agents into cells.