Self/co-assembling, cell penetrating peptides-a promising siRNAdelivery system

Review Article

Austin J Nanomed Nanotechnol. 2014;2(1): 1008.

Self/co-assembling, cell penetrating peptides-a promising siRNAdelivery system

Xiao Xia Han, Baoling Chen and P. Chen*

Department of Chemical Engineering, Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo, Ontario, N2G 3L1 Canada

*Corresponding author: : P. Chen*, Department of Chemical Engineering, Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo, Ontario, N2G 3L1 Canada

Received: December 13, 2013; Accepted: December 23, 2013; Published: December 27, 2013


SiRNAs (short interfering RNAs) have revolutionized conventional drug therapy for treating many diseases. The clinical applications as anticancer drugsare still facing challenges, such as non-effective delivery systems, offtarget effects, and immune stimulation. Many non-viral carriers have been developed so far to improve drug efficacy and biocompatibility. Among these delivery systems, a class of self/co-assembly, cell penetrating peptides (CPPs) is a most promising class of carriers. Recently, we designed and characterized a series ofself/co-assembly CCPs, C6 family. C6is highlycapable of forming stable and safe nanoparticles with siRNAs and efficiently delivering siRNA complexes into mammalian cells in vitro. In this short review, we will elucidate its physical and chemical characteristics and discuss its in vitro applications.

RNA interference (RNAi) is a cellular regulatory process, in which small, double-stranded RNA molecules that complement a specific gene (typically 21-25 nucleotides) are introduced into biological systems to suppress the expression of that gene [1-3]. Because the RNA molecule only interacts with mRNA to knock down the expression of the targeted gene without interacting with proteins and affecting chromosomal DNA, siRNA techniques greatly reduce possible adverse gene alterations in DNA-based gene therapy and the production of harmful proteins in conventional drug treatment. The effect is often potent: only a few siRNA molecules per cell are required to elicit effective gene silencing [4,5].

Due to RNAi's potency and specificity, siRNAhas revolutionized conventional drug therapy. Itcan also be widely utilized to dissect cell signaling pathways, discover important genes for embryonic development, and elucidate the function of novel genes in various fundamental biological processes, known as functional genomics [3, 6]. In theory, many proteins can be used asgene silencingtargets for treating diseases; thus, ansiRNA-based drug therapy offers versatility for targeting any sequences in mRNAs of interest, regardless of the cellular locations of their translated proteins [7]. For anticancer siRNA gene targeting, there are some popular choices, including anti- Bcl-2, anti-RRM2, anti-VEGF, anti-HER2, anti-Stat3, and survivingtargeted siRNA. Indeed, only a decade after its discovery, siRNA has been employed to treatcancer, HIVand other diseases.

In spite of the advantages of siRNAas a potential new therapeutic, there are still challenges to be overcome forfuture clinical applications, such as off-target effects, immune stimulation, andnon-effective delivery systems. Because they are anionic and hydrophilic, siRNAscannot enter cells by passive diffusion, andthey can berapidlydigested in the circulation and eliminated from the host liver and kidneys.Thus, developing an effective in vivo delivery system is essential.

Development of siRNA delivery systems

In search for effective siRNA delivery systems, the uses of many non-viral carriers have been attempted, including liposomes, polymeric complexes, micro emulsions, and nanoparticles. Among thesesiRNA carriers, liposomes or lipidic compounds arethe most studied. Cationic lipids can encapsulate negatively charged siRNA molecules and deliver them into biological cells. Although some satisfactory results were obtainedin certain in vitro systems, there are still difficulties with the lipidic strategies. First, there is inherent competition between vesicle assembly and intracellular unpacking of their molecular cargo.This phenomenonmarkedlyhinders efficient drug release intothe cytosol or cytoplasmic organelles [8]. Second, the relatively large amount of lipids required for siRNA transfection often induces severe cytotoxicitybecause cationic lipids can interfere with cellsignaling pathways that triggerimmune or anti-inflammatory responses [9]. Hence, these toxic/immunogenic features limit wide utilization of lipidic carriers in vivo. Although new approaches have beeninitiated to addresscytotoxicity and targeting specificity issues, the long-term in vivo safety and efficacy of this strategy remain in question.

Synthetic and natural polymers are another class of carrier systems that are a candidate for use insiRNAtherapeutics. Polymers consist of repeated units of covalently bonded monomers. Their structural and chemical properties are well established.Generally, cationic polymers bind to siRNAs through electrostatic interactions to facilitate crossing the cell membrane. A number of polymers, including poly ethyleneimine (PEI), poly L-lysine (PLL), chitosan, L-lactide-co-glycolide (PLGA), poly dimethylaminoethylmethacrylate (PDMAEMA), gelatin, poly-D, and poly trimethylaminoethylmethacrylate (PTMAEMA), have been studied. Like lipidic carriers, polymeric delivery systems also suffer from cytotoxicity and immunogenicity; thus, their application in vivohas been hampered [10].

A class of self/co-assembling, cell penetrating peptides (CPPs) has been developed recently. In our opinion, they area most promising class of carriers.These CPPs,such as ionic-complementary peptides (ICPs) and amino acid pairing peptides (AAPs), are small in size and typically contain fewer than 30 amino acids.Most CPPs are designed to include both hydrophilic and hydrophobic moieties. Through electrostatic interaction, the hydrophilic side of the peptide interacts with both the hydrophilic heads of the lipid bilayer as well as hydrophilic drugs and siRNAs. Meanwhile, the hydrophobic side is anchored in the hydrophobic core of the bilayer for switching on endocytosis or assisting the direct peptide-cargo translocation to the cytosol [11, 12]. Byalteringthe physical and chemical properties of the peptides, small CPPs can be constructed to promote siRNA cellular uptake and facilitate intracellular complex dissociation to release siRNAsand turn on the RNAi machinery. Furthermore, throughthe targeting ligand valence and the peptide charge,the peptide-cargo complexescan be transported to the desired intracellular compartments to elicit targetspecific RNA delivery and further enhance overall drug deliveryefficiency[13].In comparison, CPPsoffer great advantagesin flexibility and versatility as efficient and safe drug/siRNA delivery vehicles.In fact, peptide-based drug carriers have been designed and employed in researchrecently,and their role as siRNA delivery vehicles has been demonstrated in both in vitro and in vivo systems [14-17].

Design and characterization of a cell penetrating peptide-C6

We have been exclusively working on self/co-assembling CPPs for peptide-assisted anticancer drug/siRNAdelivery systems. The first series of peptides we generated was the C6 family, which is derivatives of C6, an 18-mer amphipathic and amino-acid-pairing peptide (Ac-RLLRLLLRLWRRLLRLLR-NH2) [18]. Strategically, three types of amino acids were incorporated and particularly arranged in our design to achieve (1) ionic interactions between positively charged arginine residues and negatively charged phosphate groups on the siRNA backbone as well asnegatively charged cell surface proteoglycans for initiating cellular uptake [12, 19, 20]; (2)amphiphilic and helix structures for facilitating peptide translocation across the cell membrane through interactions between the hydrophobic tails of the lipid bilayer andleucine residues [11,14]; and (3) an intrinsic fluorescence probe for tracking intracellular siRNAlocalization by inclusion of an aromatic tryptophan residue in the middle of the sequence.

The nanostructure of C6 and C6-siRNA complexes was characterized exclusively with various methods. Initially, C6 formed self-assemblies/aggregates in water. When siRNA was added in the peptide solution, C6 electrostatically formedcoassemblies withsiRNA. Based on Dynamic Light Scattering (DLS), the majority of C6-siRNA co assemblies showed a size of 50 nm.This structure was further confirmed with Atomic Force Microscope (AFM). Since 7 positively charged arginine groups in the peptide C6 and 21 pairs of negatively charged nucleotides in a siRNA molecule, 6 molecules of peptide aretheoretically needed to neutralize the negative charge of a siRNA molecule. But, a molar ratio (MR) of 10:1 was required practically and an excess of peptide molecules (MR=15:1) was needed in order to stabilize the peptide-siRNA complexbased on gel shift assay result [18]. The excess peptide molecules can provide a shield to protect siRNA molecules against degradation and interact with cell membrane to initiate the peptide-siRNA cellular uptake [18]. At a molar ratio of 15:1, C6 completely covered the surface of complex to generate a zeta potential of +30 mV. With increasing C6 concentration at a constant siRNA concentration, the positive value of the surface charge of the complex rose to +60 mV (MR= 40:1).

Cellular uptake and cytoplasmic localization of C6-siRNA

To investigate the capability of C6 facilitating siRNA internalization, Cy3-labeled GAPDH siRNA (50 nM), as an extrinsic fluorescence probe, was complexes with C6 and then transfected into CHO-K1 cells. Cellular uptake of C6-Cy3-siRNA complexes wasmeasuredwith Fluorescence-Activated Cell Sorting (FACS) after 3 hours of incubation. Uptake of C6-Cy3-siRNA correlated well with the molar ratio between C6 and Cy3-siRNA. At a molar ratio of 15:1, C6-Cy3-siRNA uptakeraised to over 90% compared with the non-treated control group. Intracellular fluorescence intensified with increasing molar ratios from 25:1 to 40:1.Moreover, it was noticed under fluorescence microscope that Cy3-siRNA itself was unable to enter the cells due to the negative charge and lack of an appropriate delivery vehicle while C6-Cy3-siRNA was clearly visualized intracellularly. C6-Cy3-siRNA complexes localized to regions in close proximity to the nuclear membrane in the cells. Cy3-siRNA delivered by C6 displayed a nonhomogeneous distribution pattern around the periphery of the nucleus inside the cells, indicating the possibility of endocytosis pathway.

Cytotoxicity of C6-siRNA complexes

While C6 demonstrated high capabilities of protecting siRNAs and delivering them into mammalian cells, cytotoxicity evaluation also showed promising result. In this experiment, CHO-K1 cells were treated either with C6 or with C6-siRNA complexesand cell viability was determined thereafter with a cell counting kit (CCK-8).After 48 hours of treatment; there was no obviouscell death in the treated cells compared with non-treated cells. From molar ratios of 10:1to 40:1, the treated cells all retainedviability over 85%, indicating only a minimum cytotoxicitywith C6-siRNA application. In agreement with this result, similar group of CPPs did not cause significant hemolytic activation and acute immune stimulation in our recent in vitrotests as well as in vivo animal studies.


Among the delivery candidates, small self/co-assembling CPPsareone of the most promising carriers for delivering anticancer siRNAs and drugs. From in vitro studies, these CPPsrevealseveral advantages intargeted gene delivery efficiency and minimalcytotoxicity. While thorough in vitroevaluations on drug efficacy and biocompatibility are still necessary, research on acute and long-term biocompatibilityshould be emphasized. In particular, investigation of cancer treatment efficiency, off-target effects, and long-term immune stimulations/suppressions in animal models should be focused for the future.


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Citation: Han XX, Chen B, Chen P. Self/co-assembling, cell penetrating peptides-a promising siRNAdelivery system. Austin J Nanomed Nanotechnol. 2014;2(1): 1008. ISSN:2381-8956

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