Protein Transduction in Human Cells Mediated by Arginine-Rich Cell-Penetrating Peptides in Mixed Covalent and Noncovalent Manners

Cell-penetrating peptides (CPPs) are small peptides with a highcontent of basic amino acid residues. They possess the ability to translocate through the plasma membrane and facilitate exogenous cargo delivery into living cells. In this chapter, we demonstrate that arginine rich CPPs are able to not only traverse cellular membranes by themselves,but also carry macromolecules into human A549 lung carcinoma cells in mixed covalent and noncovalent manners. This special macromolecular delivery system was named as mixed covalent and noncovalent protein transductions (CNPT). We found that cells treated with nona-arginine(R9)-red fluorescent protein (RFP) fusion protein mixed with green fluorescent protein (GFP), referred to as R9-RFP/GFP complexes, exhibit both red and green fluorescent images. Cells treated with R9-GFP fusion protein mixed with RFP, denoted as R9-GFP/RFP complexes, emitted green and red fluorescence, vice versa. Furthermore, mechanistic studies revealed that the cellular uptake mechanism of CNPT may involve a combination of multiple internalization pathways. Therefore, applications of this binary CNPT system may provide an efficient tool for delivery of multiple proteins in bioscience and clinical research.