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.