1. Introduction
RNA interference (RNAi) has been demonstrated as a promising gene therapy approach that regulates the expression of specific genes (Deng et al., 2014; Elbashir et al., 2001; Whitehead et al., 2009). As an RNAi mediator, short-interfering RNA (siRNA) is a double-stranded molecule composed of about 21-23 nucleotides, designed as a sequence complementary to the target mRNA. The exogenously penetrated siRNAs activate RNA-induced silencing complexes (RISC) in the cytoplasm and result in outstanding selective mRNA inhibition with low cytotoxicity. Therefore, gene therapy using siRNA can be a new potent strategy to treat cancer, viral infectious diseases, and local diseases at the genetic level. However, a significant barrier to siRNA delivery is a low uptake efficiency into the cell membranes composed of phospholipid bilayers due to its hydrophilic nature (Sarett et al., 2015; Wang et al., 2010). Moreover, the siRNA is vulnerable to degradation by large amounts of nucleases present in the cytoplasm or interstitial fluid (Lee et al., 2013). Therefore, it is essential to develop an efficient and safe delivery method of siRNA with stability.
Until now, several methods to deliver siRNA have been developed. These techniques could be categorized into physical and chemical methods (Pathak et al., 2009). First, physical methods could deliver siRNAs using the specialized equipment, including microinjector, gene gun, electroporator, sonoporator, laser, and magnetofector (Sinn et al., 2005). Physical methods have limitations for various applications due to the need for special equipment, non-specificity of siRNA delivery, and instability of delivered siRNA. Next, chemical methods could deliver siRNAs using carriers capable of interacting with siRNAs and transferring siRNA into cells. Types of carriers could become lipoplex, polyplex, dendrimer, peptide, and various nanoparticles (Akhtar, 2006; Levine et al., 2013; Yin et al., 2014; Zhou et al., 2017). These carriers could enhance the stability of siRNAs as well as delivery efficiency. On the other hand, chemical methods have shortages of limited delivery efficiency and potential toxicity of chemicals. Therefore, an ideal siRNA delivery method requires enhanced delivery efficiency, biosafety, and siRNA stability.
Recently, peptides have been intensively studied as an attractive siRNA carrier owing to their structural and functional versatility, potential biocompatibility, and targeting ability to cells. Primarily, cell-penetrating peptides (CPPs) have been known to penetrate the cell membrane effectively. The TAT sequence was originated from the Tat protein of human immunodeficiency virus (HIV) (Vives et al., 1997). Positively-charged oligo arginine can assist cellular internalization by forming a hydrogen bond with the sulfate of the cell membrane and phosphate group of the nucleic acid (Koren and Torchilin, 2012; Sarett et al., 2016; Tang et al., 2013; Zeller et al., 2015). The histidine-rich peptide was confirmed using the efficient delivery of siRNA (Langlet-Bertin et al., 2010). Besides, the development of the phage display technique enables us to find new-type cell-penetrating peptides. For example, the skin permeating and cell entering (SPACE) peptide has a superior ability to facilitate the penetration of conjugated cargoes into epidermis and dermis (Hsu and Mitragotri, 2011). However, single peptide showed limited delivery efficiency, and some peptides such as SPACE need the additional conjugation reaction. Therefore, for a facile and useful siRNA carrier, it needs the method with enhanced delivery efficiency without additional reaction.
Herein, we designed novel fusion peptides and investigated their potential as a siRNA delivery carrier. The three fusion peptides were composed of SPACE and cationic oligo arginine (R7, R11, and R15) linked by GCG sequence (Mitchell et al., 2000). The self-assembled complex was identified between each peptide and siRNA without any conjugation. Moreover, each complex was characterized in terms of size, zeta-potential, and siRNA stability. Cellular uptake efficiency of each complex was measured using FACS and fluorescence microscopy. Intracellular co-localization or dissociation of the complex was analyzed using a confocal microscope. The complex-mediated GAPDH knockdown was assessed using mRNA expression level. In addition, the internalization pathway of the siRNA/S-R15 complex was analyzed using FACS and endocytosis inhibitors. Furthermore, the biosafety of each complex was checked using a cytotoxicity test of human dermal fibroblast cells.