Peptide Mediated siRNA Delivery

Current Topics in Medicinal Chemistry, 2009, 9, 000-000

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Peptide Mediated siRNA Delivery Mousa Jafari and P. Chen* Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada, N2L 3G1 Abstract: Applying RNA interference to silence a specific gene has opened a new and promising avenue of gene therapy. But a key bottleneck is the poor stability and inability of naked siRNA to translocate through cell membranes. Among several delivery systems, cationic peptides capable of penetrating cell membranes have drawn attention due to their structural and functional versatility, potential biocompatibility and ability to target cells. In this review, different classes of peptides employed in siRNA delivery are reviewed. In particular, a new class of siRNA delivery peptides with high transfection efficiency and low cytotoxicity is introduced.

Keywords: RNAi, siRNA, peptide, gene therapy, delivery systems 1. INTRODUCTION Thanks to the genome projects, new classes of pharmaceuticals (peptides, proteins and nucleic acid (NA) based therapeutics) are emerging. These novel drugs have shown promising therapeutic potential in the lab. However, they have experienced only limited success in clinical studies. Poor stability and transport through biological barriers, such as the cell membrane, prevent these drugs from reaching their target(s). These new drugs typically cannot be effectively delivered by conventional means. For instance, conventional liposomes, as drug delivery carriers, have been suffering from major limitations including rapid removal from bloodstream, low drug loading capacity, and physical or chemical instability. Furthermore, the efficacy of many conventional pharmaceutical agents may be improved and the side effects reduced if the drug is continuously released in a controlled manner rather than through conventional burst release techniques [1]. Over the past decade, we have witnessed tremendous progress in our understanding of the role of RNA molecules in the regulation of gene expression. The main contribution to this progress was offered by the discovery of RNA interference (RNAi) process. First identified in C. elegans by Fire and Mello [2], RNAi is an evolutionary conserved mechanism that brings about a sequence specific, post transcriptional gene silencing (PTGS) through the use of short RNAs. The basic idea behind RNAi is that a double stranded RNA, termed short interfering RNA or siRNA, complementary to a segment of the target mRNA, can be exogenously synthesized and introduced into the cell. This triggers a process which finally degrades the homologous mRNA and inhibits the production of the corresponding protein. Several types of short RNAs, including short interfering RNA (siRNA), micro RNA (miRNA), tiny noncoding RNA (tncRNA), and short hairpin RNA (shRNA), may be involved in RNAi process [3-5].

*Address correspondence to this author at the Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada, N2L 3G1; E-mail: [email protected]

1568-0266/09 $55.00+.00

Like other newly-emerging NA-based therapeutics, the major limitations for the use of siRNA are the instability of naked siRNA in physiological conditions and the bloodstream, and the inability to cross the cellular membrane to gain access to the intracellular environment. Due to their small size and hydrophilicity, a significant portion of these NA-based drugs are removed from bloodstream through the reticuloendothelial system (RES). Moghimi and Bonnemain described a possible transfer passage through fenestrae in the endothelium of the liver for small particles (less than 100 nm) [6]. It was also reported that highly charged particles can be recognized by the RES more rapidly than neutral or slightly charged particles [7, 8]. Furthermore, the enzymatic degradation of NA based drugs during circulation and within the cell declines their potency, and in some cases an increase of drug dosage is required to compensate these effects. Chemical modifications of siRNA may be applied to improve these characteristics without interfering with its silencing efficiency. Chemical modifications in the sugars, nucleobases, and the phosphate ester backbone of siRNA can significantly increase its nuclease resistance [9-11]. In order to improve cellular uptake, conjugation with hydrophobic functional groups has also been reported [12]. The carrier-mediated delivery system has been recently applied as the main solution to overcome the delivery obstacles and improve the cellular uptake of siRNA therapeutics. The carriers, self-associated or covalently conjugated with siRNA, are designed to enhance cell targeting, prolong drug circulation time, and improve membrane permeation. A safe drug delivery system should cause minimal cytotoxicity and inflammatory response, especially to non-targeted sites. 2. CURRENT SIRNA DELIVERY SYSTEMS Novel drug/gene delivery systems have been constantly sought to overcome various obstacles including premature inactivation and degradation, non-target distribution, zero or minimal celluar uptake of biotherapeutics. Technologies are being developed to minimize drug toxicity or immunogenicity, or to enhance vaccine immunogenicity [1]. A wide spectrum of materials can be engineered and developed to obtain desired capabilities to act as carriers for drug/gene © 2009 Bentham Science Publishers Ltd.

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Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 12

delivery. They may include lipids [13-15], polymers [16-20] peptides [21-24], gold particles [25], ceramics [26], and virus vectors [27, 28]. 2.1. Viral Vectors The gene delivery vectors can be generally categorized into viral and nonviral vectors. Typically, viral systems are the most effective carriers for gene delivery. They can selectively target cells and usually possess a high transfection efficiency [29]. However, their isolation from biological sources and their processing can be very costly. Furthermore, the safety risks due to their oncogenic potential and their inflammatory and immunogenic effects have limited the clinical application of the strategies based on viral vector delivery [28, 30]. 2.2. Non-Viral Vectors Although viral vectors possess many of the desired characteristics for efficient NA delivery, nonviral vectors offer several advantages. For a complete overview of nonviral methods for siRNA delivery see Gao and Huang [31]. Due to their lack of immunogenicity, synthetic vector systems are usually safer than viral vectors. In addition, they can be easily modified and produced in large scale. Among the non-viral drug carrier systems, liposomes represent a mature technology for both drug and gene delivery [14, 15]. siRNA molecules are associated with liposomes mainly via electrostatic interaction with the charged head group. However, some issues have led to limited clinical usage of the liposomal systems. The immune system, which tracks foreign materials for destruction, can be a major obstacle to liposomes. Furthermore, the lipid toxicity and lack of long term expression and targeting are problems associated with their use in vivo [6, 32]. Synthetic and natural polymers, made up of repeated units of covalently bonded monomers, are other classes of non-viral macromolecules which have been widely used as carriers for various drug molecules over the past three decades. In particular, such synthetic polymers as polylactic acid and polylactic-co-glycolic acid are very attractive, as compared to biopolymers, since they can be produced in high quantity for relatively low costs. Among the cationic polymers, polyethyleneimine has been widely examined for siRNA delivery [31, 33]. Over the past few years, there have been several drug delivery systems developed that rely on organic polymer technology. Such delivery systems are based on drug entrapment within micelles [34, 35], nanoparticles or the hydrophobic corona formed by block copolymers [17, 36], and they are used to improve solubility and protection of drugs. In particular, polyethylene glycol (PEG) is one of the most frequently used polymers for drug delivery with high water solubility, biocompatibility and chain flexibility [37, 38]. In some cases, it was employed to help protect the siRNA and minimize its interaction with serum proteins. It was also found that covalent attachment of the PEG to the siRNA or its delivery system enhanced stability and efficient delivery to targeted sites [39, 40].

Jafari and Chen

3. PEPTIDES Peptides are short sequences of amino acids, usually 30 or less amino acids, covalently linked through an amide or peptide bond. Considering the safety concerns and efficacy issues, peptide-based drug/gene delivery constructs are emerging as alternatives for safe and efficient delivery means since the 1990s. Due to their relatively high polarity and hydrogen bridge forming tendency, it was generally believed that peptides would be unable to translocate through cell membrane. However, the complex nature of the cell membrane was neglected. The rationale for peptide mediated NA delivery initially evolved from the biochemical knowledge that the active sites of enzymes, receptor ligands and antibodies involve about 5 to 20 amino acids. Thus, it should be possible to design small synthetic peptides to mimic the active sites of proteins, especially the sites which are responsible for cell penetration, and formulate synthetic peptide based drug/gene delivery systems that may be as efficient as viruses without their limitations. Peptide-based delivery systems have the potential to deliver therapeutic proteins, bioactive peptides, small molecules, and any size nucleic acids [41-43]. Several peptides investigated as NA carriers are of biological origin which makes them biodegradable and likely to be biocompatible. For example, cell penetrating peptides (CPPs) [41], fusogenic peptides [44], and receptor-based targeting peptides [45] are derived from existing cellular or viral proteins. The main attraction of the peptide carriers is their versatility, through the use of the 20 naturally occurring amino acids, each with different hydrophobicity, size, and other solution properties. This versatility in peptide design also allows the synthesis of multifunctional transfer reagents that can enhance transfection, stability, and targeting ability [41]. The secondary structure of peptides seems to play an important role in the cell membrane translocation. Depending on the sequence and the solvent, a peptide can attain such secondary structures as an -helix or -pleated sheet. The importance of -helical [46] and -sheet [47] structures to membrane translocation has been discussed previously. However, some peptides, such as oligoarginine, can also deliver drugs across the cell membrane in the random coil conformation, indicating that the secondary structure is not the only factor that determines cellular uptake of drug molecules [48]. In the next sections, different classes of peptides employed as gene delivery carriers including protein-derived CPPs, cationic peptides, and model amphipathic peptides will be discussed. 3.1. Protein-Derived Cell Penetrating Peptides A cell penetrating peptide, by definition, is a relatively short peptide, 5-40 amino acids, with the ability to gain access to the cell interior by means of different mechanisms and with the capacity to promote the intracellular delivery of covalently or noncovalently conjugated bioactive cargoes. The mechanism(s) by which peptides enter the cell and

Peptide mediated siRNA delivery


mediate the entry of cargo molecules inside the cell are still not understood in any detail. In the early 1990s, the discovery of CPPs led to proposals of direct entry mechanism [41]. In general, cellular uptake can either be energy dependent or independent. The energy dependent pathways for cells generally include macropinocytosis, clathrinmediated endocytosis, and caveolin-mediated endocytosis [49]. Endocytotic mechanisms were almost ruled out in the present cases because the translocation could be observed at low temperature. The most discussed energy independent cellular uptake mechanism is inverted micelle based [50]. In this model, the peptide first associates with the bilayer surface through electrostatic interaction. The lipid bilayer reorganizes the peptide-cargo complex, and minimize the exposure of the complex to the solvent, which eventually leads to the formation of an inverted micelle in the bilayer and is later released to the cytosol. Table 1 shows a number of CPPs derived from some viral proteins. These peptides are the shortest peptide sequences responsible for cell penetration in the corresponding viruses. Among these peptides, penetratin and Tat are the most studied peptides. Penetratin is the third -helix of AntennaTable 1.

pedia, a membrane transduction protein [51]. It is internalized by energy-independent mechanism at both 4 and 37°C, and has access to the cytoplasm and nucleus. The presence of three lysine residues confers to the peptide an isoelectric point above 12. It has - helical structure in a hydrophobic environment but is poorly structured in aqueous solution. Experiments have shown that the basic amino acids and the tryptophan residue at position 6 of the peptide (48 of Antp) are essential to cellular uptake of penetratin [52]. Moreover, the -helical structure is not essential to membrane translocation since disturbing the secondary structure of the peptide by point mutation with proline did not prevent its internalization [53]. Discovered in 1988 by two independent groups, transactivating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) can be efficiently taken up from the surrounding media by numerous cell types in culture [22, 75]. The Tat protein has 86 amino acids but only the cluster of basic amino acids, RKKRRQRRR, residues 49 to 57, is responsible for the cell penetrating property of the Tat peptide. Due to charge repulsion resulting from six arginine and two lysine residues, the Tat peptide undergoes a random coil configuration in solution. The Tat peptide has

Sequences of Protein-Derived CPPs

Peptide

Origin

Sequence

Reference

Penetratin (43-58)

Antennapedia

RQIKIWFQNRRMKWKK

54

Tat (48-60)

HIV-1

GRKKKRRQRRRPPQ

55

Transportan

Galanin-wasp venom

GWTLNSAGYLLGKINLKALAALAKKIL

56

LLIILRRRIRKQAHAHSK

57

pVEC peptide mu

Adeno virus

MRRAHHRRRRASHRRMRGG

58

E5

Influenza virus

GLFEAIAEFIEGGWEGLIEG

59

E5CA

Influenza virus

GLFEAIAEFIEGGWEGLIEGCA

60

E5WYG

Influenza virus

GLFEAIAEFIEGGWEGLIEGWYG

61

gp41 fusion

Influenza virus

GALFLGWLGAAGSTMGA

62

H5WYG

Influenza virus

GLFHAIAAHFIHGGWHGLIHGWYG

63

HA

Influenza virus

GLFEAIAGFIENGWEGMIDG

64

HBV

PLSSIFSRIGDP

65

hCT

LGTYTQDFNKFHTFPQTAIGVGAP

66

Integrin

VTVLALGALAGVGVG

67

INF-1

Influenza virus

GLFEAIAGFIENGWEGMIDGGGC

68

INF-7

Influenza virus

GLFEAIEGFIENGWEGMIDGWYG

69

K5

Influenza virus

GLFKAIAKFIKGGWKGLIKG

70

Melittin

venom of Apis

GIGAVLKVLTTGLPALISWIKRKRQQ

71

MPM

K-FGF

AAVALLPAVLLALLAP

72

RHIKIWFQNRRMKWKK

73

RGGRLSYSRRRFSTSTGR

74

PDX-1 SynB1

Protegrins

3

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been successfully used to deliver oligonucleotides [76], proteins [77], and fluorophores [78] in both in vitro and in vivo models. 3.2. Cationic Peptides Basic amino acids, such as lysine and arginine, are positively charged in physiological pH; thus, they can interact with negatively charged drug molecules, such as siRNAs, and cell membranes through columbic interactions. Fuchs et al. reported that the oligomers of the four cationic amino acids, arginine, lysine, histidine, and ornithine can cross the cell membrane and be localized in the cytosol and the nucleus [79]. Due to the removal of highly charged oligolysine by the RES, the transfection efficiency of therapeutic materials delivered by oligolysine is generally low [7, 8]. Various methods have been employed to increase its efficiency. It was found that incorporation of cysteine in a lysine-rich peptide allows the formation of disulfide bond between peptide molecules, resulting in smaller complexes and lower opsonisation rate compared with uncross-linked complexes [80]. Also, the disulfide bond reduction in the cytosol triggers the release of NAs, resulting in higher in vitro transfection efficiency [81]. Bhadra et al. have also reported that conjugation of oligolysine with PEG can increase transfection efficiency and protect it from serum attacks [38]. 3.3. Designed Amphiphilic Cell Penetrating Peptides Since the lipid bilayer of a cell membrane is amphiphilic, it seems reasonable to employ an amphiphilic peptide to carry a drug across the cell membrane. In this delivery model, the hydrophilic section of the peptide first interacts with the membrane surface with subsequent translocation to the cytosol assisted by the hydrophobic section of the peptide. There are two major types of amphiphilic cell penetrating peptides, namely primary and secondary amphiphilic peptides. Primary amphiphilic peptides have specific hydrophobic and hydrophilic domains joined by a linker in the primary sequence. The hydrophobic region can interact with hydrophobic drugs and anchor itself in the cell membrane. The hydrophilic region, on the other hand, interacts with hydrophilic drugs and the cell membrane surface through electrostatic interactions. In general, the primary amphiphilic peptides adopt a random coil structure at neutral pH but a defined secondary structure upon a change in pH or interaction with the cell membrane [47, 82]. The high efficiency of primary amphiphilic peptides may be attributed to the change in secondary structure at low pH, which can induce leakage of the endosomal membrane and facilitate endosomal escape of carrier-drug complexes. In secondary amphiphilic peptides, the amphiphilic nature of the molecule is originated from its secondary structure, i.e., -helix or -sheet structures. Many peptide delivery carriers are designed based on -helix amphiphilicity [46, 83] while investigations based on amphiphilic -sheet peptides are relatively limited [47, 84]. Secondary amphiphilic peptides can be embedded in the cell membrane so that the hydrophobic side is anchored in the hydrophobic core of the bilayer and the hydrophilic side interacts with the hydrophilic heads of the lipid bilayer. Alternatively, the

Jafari and Chen

peptides can first form micelles or aggregates to minimize the exposure of hydrophobic residues to the solvent, and then associate with the cell membrane. One example of this class of peptides is model amphiphilic peptide (MAP) with a sequence of KLALKLALKALKAALKLA [75]. Table 2 shows some sequences of amphiphilic cell penetrating peptides and their types of amphiphilicity. MPG was the first peptide used to introduce siRNA into cells. It consists of a hydrophobic domain derived from a HIV gp41 fusion sequence GALFLGFLGAAGSTMGA and a nuclear localization sequence (NLS) KSKRKV, joined through a linker WSQP [47]. The linker domain contains a proline residue, which improves the flexibility and integrity of both hydrophobic and hydrophilic domains. The sequence of MPG has been modified by a single mutation of a lysine residue in NLS to a serine residue in order to limit its nuclear translocation and rapid release of the cargo in the cytoplasm. The resulting peptide is MPG-NLS. MPG family peptides exhibit high affinity to siRNA and large plasmid DNA. The positively charged NLS domain mediates the interaction of the peptide with NAs. On the other hand, the hydrophobic domain mediates the peptide-peptide interactions, resulting in a peptide cage around the NA molecules. The resulting nanoparticles significantly improve the stability of siRNA inside the cell and protect it against enzymatic degradation. MPG-mediated delivery of siRNA has brought about significant gene silencing in several cell lines including Hela [95, 96], SW620 [97], MEF [98], HEK [95, 99], C2C12 [97], HS-68 [97], CEM/macrophage [100], and HEpG2 [97]. MPG has been also successfully applied for siRNA delivery in vivo through intravenous or intratumoral injections [41]. 3.4. PEPTIDE-SIRNA CONJUGATES/COMPLEXES In order to enhance the cellular uptake and gene silencing efficiency, siRNA should properly bind or co-assemble with its carrier molecules. Two different strategies are mainly applied to form peptide-siRNA conjugates: either peptides are covalently attached to siRNAs, or they interact through electrostatic interactions to form non-covalent complexes. 3.4.1. Covalent Peptide-siRNA Conjugates Covalent attachment of peptides to siRNA molecules offers a potential strategy for peptide-mediated siRNA delivery. It can be of several advantages for in vivo applications, including reproducibility of the procedure, and control of the stoichiometry of peptide/siRNA ratios. Also, less peptide is required in this method as compared to non-covalent strategies. This is especially important if the peptide shows toxicity in high concentrations. Several options are available for covalent conjugation of peptides to NAs, including the use of cross-linking agents, triple helix- forming oligonucleotides, and chemical attachment to the end of linear NAs 41, 101. The main method applied for the peptide-siRNA conjugation is disulfide linkage, which is cleaved in the cytosol due to its reducing environment. There are limited papers (Table 3), reporting significant gene silencing through covalent attachment of peptides and siRNAs as it is suspected to alter the biological activity of siRNA molecules [24].


Table 2.


Amphiphilic cell Penetrating Peptides Names

Sequence

Amphiphilicity

Reference

MPG

GALFLGFLGAAGSTMGAWSQPKKKRKV

Primary

47

MPG-NLS

GALFLGFLGAAGSTMGAWSQPKSKRKV

Primary

47, 85

Pep-1

KETWWETWWTEWSQPKKKRKV

Primary

86

Pep-2

KETWFETWFTEWSQPKKKRKV

Primary

86

GALA

WEAALAEALAEALAEHLAEALAEAEALEALAA

Secondary

87

KALA

WEAKLAKALAKALAKHLAKALAKALKACEA

Secondary

88

MAP

KLALKLALKALKAALKLA

Secondary

89

SP

MGLGLHLLVLAAALQGAWSQPKKKRKV

Primary

90, 91

SP-NLS

MGLGLHLLLAAALQGAKKKRKV

Primary

90, 92

SPM

MGLGLWLLVLAAALQGAKKKRKV

Primary

93

Transportan

GWTLNSAGYLLGKINLKALAALAKKIL

Primary

56

Hel 9-9

KLLKKLLKLWKKLLKKLK

Secondary

83

Hel 11-7

KLLKLLLKLWKKLLKLLK

Secondary

83

Hel 13-5

KLLKLLLKLWLKLLKLLL

Secondary

83

[Pa]

GALFLAFLAAALSLMGLWSQPKKKRKV

Primary

47

[Pb]

GALFLGFLGAAGSTMGAWSQPKKKRKV

Primary

47

gp41 fusion

GALFLGWLGAAGSTMGA

Primary

94

Table 3.

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siRNA Delivery by Covalent Peptide-siRNA Conjugates Peptide

Cell line

Target gene

Reference

Tat

NIH-3T3 MDR

MDR1

102

Tat

Hela

P38

103

Penetratin

Neuron

SOD1

104

Penetratin

CHO

Luciferase

105

Transportan

CHO

Luciferase

105

3.4.2. Non-Covalent Peptide-siRNA Complexes As an alternative to covalent strategies, non-covalent interactions of carrier peptides and cargos have recently been developed. Positively charged peptides can interact with the negatively charged backbone of siRNA through non-specific electrostatic interactions, providing cell permeability for siRNA molecules by covering the siRNA surface with positive charges of the peptides. This is a very simple and effective strategy for carrier mediated siRNA delivery without any need to chemical modification of siRNA. However, if high peptide/siRNA molar ratios are applied, the high concentration of positively charged peptides may induce some side-effects through interactions with anionic molecules in the cell. The molar excess of the peptide as

compared to siRNA can also yield complexes of varing sizes. This could be considered as a disadvantage of this strategy as a certain size of complexes is required in most therapeutic applications. One example of peptides employing this strategy is MPG as we discussed before. Table 4 shows some reports of using non-covalent peptide-siRNA complexes for RNAi. 4. NEW CLASS OF SIRNA DELIVERY PEPTIDES: AMINO ACID PAIRING (APP) PEPTIDES Amino acid pairing peptides are a new class of peptides recently developed for siRNA delivery [111]. It consists of two main domains: amino acid pairing domain which is responsible for peptide self assembly and cell permeation domain which is a functional group for cell penetration or a

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Table 4.

Jafari and Chen

siRNA Delivery by Non-Covalent Peptide-siRNA Complexes Peptide

Cell line

Target gene

Reference

MPG, MPG-NLS

HS-68

GAPDH

96

MPG, MPG-NLS

Hela

Luciferase

96, 106

MPG

ECV304

Luciferase

107

H2A-Penetratin

Hela

Luciferase

108

Bprp

Hela

Luciferase

108

Tat

CHO

EGFP

109

R9

GC

EGFP

110

EB1

Hela

Luciferase

108

group of cationic amino acids. In their patent [111], Chen et al. described different mechanisms including electrostatic interaction, hydrogen bonding, hydrophobic, and - stacking interactions incorporated in peptide assembly. The peptides were designed to be sequence complementary, and possess geometric matches between basic amino acid residues and the phosphate backbone of siRNA. Among the screened peptides, the peptide C1 [112] showed promising results. It consists of an AAP domain and a cationic domain. The AAP domain is capable of hydrogen bonding and hydrophobic interactions. The hydrophobic residues of two adjacent peptide molecules can interact with each other through hydrophobic interactions while hydrogen donor and acceptor residues form hydrogen bonding between two adjacent peptide molecules. The cationic domain is responsible for cellur internalization while a histidine contain portion provides endosomal disruption. 4.1. Silencing Efficiency of C1-siRNA Formulation in C166-GFP Cell Line [112] The flow cytometry technique was applied to evaluate the GFP-encoding gene silencing. For effective eGFP silencing, the eGFP fluorescence intensity is expected to decrease after siRNA transfection, since the mRNA encoding for the eGFP is degraded. Upon reaching the cytosol, the successfully delivered siRNA would perform RNAi, which prevents the downstream production of eGFP until the siRNA is eventually degraded by endonucleases. However, GFP that is already present in the cytosol prior to siRNA delivery would still give fluorescence before it is degraded by intracellular proteases. Therefore, the effect of silencing was monitored over a sufficiently long time (24 hours and 48 hours). The base line of eGFP fluorescence was obtained from the fluorescence of untreated cells, one of the controls. The normal and positive controls were cells transfected with naked siRNA and siRNA-Lipofectaminee 2000 complexes, respectively [112]. Figure 1-a shows the flow cytometry results, indicating fluorescence intensity distributions for the cells treated by the peptide C1. Fluorescence intensities of the untreated cells (red line) and the cells treated by Lipofectamine 2000 (green line) are also shown. It can be seen that there is no

significant difference between the transfection efficacies of the positive control Lipofectamine 2000 and the peptide C1. The percentage of silencing in the cells treated by Lipofectamine/siRNA and the C1/siRNA complexes is also shown in Figure 1-b, for easy comparison. 4.2. MTT Assay To evaluate the viability of the cells treated by complexes, the MTT assay was applied. As it can be seen from Figure 2-a, the viability of the cells treated by the C1/siRNA complex is considerably higher than that of the cells treated by the Lipofectamine/siRNA complex. This indicates higher biocompatibility of the peptide. It is also observed that the complexation did not have significant additional effect on the viability of the cells. 4.3. Serum Effect on the Stability and Transfection Efficacy of the Complex The next screen experiments included serum plus and serum free groups, to investigate the serum effect on the transfection efficacy of the complexes. The transfection experiments were conducted in the serum-free medium to avoid any effect of serum on the degradation or destabilization of the complex. However, in in vivo transfection, serum is inherently present, so the transfection reagent should be able to preserve and deliver the drug/gene in a serum-plus environment. For this purpose, the complexes of the peptide C1 with siRNA were prepared in DMEM with 10% FBS. From Figure 2-b, it can be seen that the presence of serum in the treatment did not lead to a pronounced reduction in silencing in C166-GFP cells, indicating that the C1/siRNA complex was serum stable. CONCLUSIONS Several delivery systems have been developed to enhance cellular uptake of therapeutic siRNA and protect it against pre-mature degradation. Peptide-mediated gene delivery is a comparatively new area as compared to some well-developed delivery systems. Different types of peptides, including protein-derived cell penetrating peptides, model amphiphilic peptides, and cationic peptides have been applied for siRNA delivery with moderate success as



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A B Fig. (1). a) Flow cytometry results for the untreated cells (—), cells treated by C1/siRNA (—) and Lipofectamine/siRNA (—) in 48 hours. b) Percentage of silencing Cell line: C166-eGFP, siRNA concentration: 100nM, C1 concentration: 4 M [112].

A B Fig. (2). a) MTT cytotoxicity results, b) Effect of serum on the transfection efficiency Cell line: C166-eGFP, siRNA concentration: 100nM, C1 concentration: 4 M. Each bar shows the average measurement based on three replicates of the experiment. The error bars represent the standard deviation of the measurements [112].

compared to viral vectors. Endosomal entrapment of siRNA is one of the problems associated with peptide mediated siRNA delivery. Researchers have addressed this problem by incorporating endosomal disruptive residues in the peptide sequence. One example is the MPG peptide which has shown promising performance both in vitro and in vivo. Recently, a new class of siRNA delivery peptides, termed amino acid pairing peptides, was discovered, which offers high transfection efficiency, serum stability, and low toxicity in vitro.

ACKNOWLEDGMENTS The authors are grateful for the financially support from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Foundation for Innovation (CFI), and the Canadian Research Chairs (CRC) program. REFERENCES [1] [2]

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Received July 21, 2009

Accepted August 6, 2009

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