Cell-Penetrating Peptides, PepFects, Show No Evidence of Toxicity and Immunogenicity In Vitro and In Vivo Julia Suhorutsenko,* ,§,† Nikita Oskolkov, § Piret Arukuusk, § Kaido Kurrikoff, § Elo Eriste, § Dana-Maria Copolovici, § and U ̈ lo Langel §,† § Institute of Technology, Tartu University, 50411 Tartu, Estonia † Department of Neurochemistry, Stockholm University, 10692 Stockholm, Sweden * S Supporting Information ABSTRACT: Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR) 4 peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1β, IL-18, and TNF-α cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 μM and 5 μM, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions. ■ INTRODUCTION Efficient internalization of therapeutic agents into target cells is very important to gain the desired therapeutic effect. However, since the plasma membrane limits the internalization of high molecular weight molecules into the cell, therapeutic applications of nucleic acids and proteins have been restricted. 1 Several techniques have been used to promote efficient drug delivery, for instance, electroporation, encapsulation in polymers or liposomes, viral delivery systems, and so forth. However, these approaches have several serious drawbacks, including complex manipulation, limited efficiencies in in vitro and in vivo systems, cytotoxicity, and undesired immunogenic effects. 2,3 Cell-penetrating peptides (CPPs) have proven their ability to deliver various drug molecules, DNA, RNA, and proteins in cultured cells. 4 Synthetic CPPs were designed with predeter- mined structures and they maintain the parent peptide’s functions. This enables different chemical modifications of the CPPs as well as expression with specific proteins or application together with other delivery vectors, which gives an advantage for the construction of novel, more efficient therapeutic agents. 5 Several modifications of CPPs have been investigated in order to improve gene delivery. In particular, stearic acid modification of different membrane-permeable relatively short peptides, such as TAT, 6 transportan, 7,8 octa-arginine (R 8 ), 9 and (RxR) 4 , was shown to substantially increase their transfection efficiency. 6,8,10 It has been shown that stearic acid modification enhances CPP- mediated plasmid DNA delivery, promoting more efficient endosomal escape, 10 enhanced cellular association, as well as higher nuclear delivery. 9 Lysosomotropic agents such as chloroquine and methylamine are also capable of improving the release of therapeutic molecules from acidic endosomal compartments, raising the pH, and performing proteolytic cleavage; 11,12 however, their application in vivo for therapeutic use is limited, due to their toxicity. 13,14 Although therapeutic protein agents and peptides used in clinical settings are generally considered nontoxic, antither- apeutic protein antibodies can develop during the treatment. Antitherapeutic protein antibodies or antidrug antibodies might neutralize or otherwise compromise the clinical effect of therapeutics and can also be associated with serious adverse events related to cross-reactivity with autologous proteins of the host organism. 15 Immune response to therapeutic proteins is categorized by activation of the immune system by foreign Received: June 6, 2011 Revised: September 12, 2011 Published: October 5, 2011 Article pubs.acs.org/bc © 2011 American Chemical Society 2255 dx.doi.org/10.1021/bc200293d | Bioconjugate Chem. 2011, 22, 2255-2262