Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery Archana Upadhya* ‡ and Preeti C. Sangave ‡ Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condens- ing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide–plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide–plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: cell penetrating peptides; gene delivery; stearylated octa-arginine; noncovalent complexation; membrane translocating sequence; hydrophobic interaction Introduction Delivery of biomolecules especially nucleic acids via non-viral methods is being researched extensively as of today. Among various delivery strategies such as lipoplexes and polyplexes, cell penetrating peptides (CPPs) have evolved as useful tools for intra- cellular delivery of nucleic acids. CPPs are short amphipathic or cationic peptides of less than 30 amino acids which possess the ability to penetrate cellular membranes. Because of their inherent positive charge, CPPs can easily complex nucleic acids and enable their co-transport into the cell [1]. Small oligonucleotides such as siRNA, anti-sense oligonucleotides and splice-correcting oligonu- cleotides have been successfully delivered to cells using cationic CPPs through non-covalent complexation [2]. Delivery for gene expression, however, involves transport of large nucleic acids, such as plasmid DNA to the cell nucleus. Plasmid DNA (size >5 kbp) are cumbersome cargo for CPPs because of their large size and huge number of negative charges. For intracellular transport to a cell which has an inherent negatively charged cell membrane, plasmid DNA charges are neutralized by condensation with the cationic * Correspondence to: Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’s NMIMS University, V.L. Mehta Road, Vile Parle (West), Mumbai 400056, Maharashtra, India. E-mail: archana.upadhya@nmims. edu ‡ Both authors have contributed equally to the publication Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’s NMIMS University, V.L. Mehta Road, Vile Parle (West), Mumbai 400056, Maharashtra, India Abbreviations: CPP(s), (cell penetrating peptide(s)); DNA, (deoxyribonucleic acid); N/P, (peptide nitrogen per nucleic acid ratio); kFGF, (Kaposi fibroblast growth factor); DTT, (dithiothreitol); SVM, (support vector machine); HBS, (Hepes buffered saline); HBG, (Hepes buffered glucose); RBCs, (red blood corpuscles); MTS, (mem- brane translocating sequence); SEAP, (secreted alkaline phosphatase); (PNP), para-Nitrophenol; (pNPP), para-Nitrophenyl Phosphate; SDS, (sodium dodecyl sul- fate); HeLa, (human cervical cancer cell line); NIH/3T3, (mouse embryonic fibro- blast cell line); FBS, (foetal bovine serum); MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide); DPBS, (Dulbecco’s phosphate buffered saline); DMEM, (Dulbecco’s modification of Eagle’s medium); GAGs, (glucosaminoglycans) CO 2 (carbon dioxide); CHAPs, (3-((3-cholamidopropyl)dimethylammonio)-1- propanesulphonate); FGF, (fibroblast growth factor). J. Pept. Sci. 2016; 22: 647–659 Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 14 March 2016 Revised: 27 June 2016 Accepted: 24 August 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/psc.2927 647