Evaluating The Characteristics Of An Efficient Cell-penetrating Peptide And Its Application

Cell-penetrating peptides(CPPs),a class of peptides consisting of 5-30 amino acids,are able to carry plasmid DNA,small interfering RNA,proteins,nucleic acids and other biomacromolecules into cells.CPPs have been widely studied due to the high transduction efficiency.The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles.Previous studies from our laboratory identified a heparin binding domain(HBD)as a novel human-originated CPP which could efficiently deliver trichosanthin or MAP30 protein into tumor cells resulting in enhanced anti-tumor effects.In order to obtain a human-originated CPP with higher transduction efficiency than HBD,several reported heparin binding domains were investigated in this study.The protein transduction efficiency of four heparin binding domains was evaluated by the fluorescence microscopy and flow cytometry analysis.One of these heparin binding domains(termed HBP)derived from human heparin-binding epidermal growth factor-like growth factor(HB-EGF)showed stronger protein transport capacity than HBD.The study of endocytic pathway of HBP suggested that HBP-driven endocytosis process was an energy-dependent direct translocation and likely occurred through lipid raft-,caveolae-and calthrin-mediated pathways,as well as macropinocytosis.The result also indicated that the translocation progress of HBP was involved the interaction with the heparin proteoglycans or heparin sulfate proteoglycans on cell surface.In order to further evaluate the application potential of HBP,HBP was fused with MAP30(Momordica anti-HIV protein of 30 kDa)which was often obstructed by barriers in the cell membrane.The pharmacological activity of MAP30-HBP on tumor cells was assessed.MAP30,a representative type I ribosome inactivating protein with antitumor activity,was isolated from bitter melon.The data indicated that the intrinsic bioactivities of MAP30-HBP fusion protein were well preserved compared to those of free MAP30 and the internalization efficiency of MAP30-HBP was significantly improved.The results of MTT assay confirmed that the introduction of HBP significantly enhanced the cytotoxic of MAP30 to six kinds of tumor cells in a time-and concentration-dependent manner.MAP30-HBP achieved more than 100-fold improvement in cytotoxicity against 95D,B16,MGC803 and HepG2 cells.Apoptosis pathway analysis showed that MAP30-HBP increased the levels of activated caspase-8,caspase-9,caspase-3 and PARP compared to free MAP30.More obvious upregulation of cleaved caspase-3,cleaved PARP and the ratio of Bax/Bcl-2 were also detected after treatment with MAP30-HBP.These results revealed that HBP enhanced MAP30-induced apoptosis in HeLa cells through the activation of the mitochondrial-and death receptor-mediated signaling pathways.In addition,the MAP30-HBP fusion protein could cause more HeLa cells to become arrested in S phase compared to MAP30 alone.In this research,we demonstrated that HBP,a CPP with high transduction efficiency,was a new potential vector for antitumor drug delivery.The application of HBP for the highly efficient intracellular delivery of MAP30 made this recombinant protein a promising drug candidate for cancer therapy.