Cationic cell-penetrating peptides as vehicles for siRNA delivery.

Review

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Therapeutic Delivery

Cationic cell-penetrating peptides as vehicles for siRNA delivery

RNA interference mediated gene silencing has tremendous applicability in fields ranging from basic biological research to clinical therapy. However, delivery of siRNA across the cell membrane into the cytoplasm, where the RNA silencing machinery is located, is a significant hurdle in most primary cells. Cell-penetrating peptides (CPPs), peptides that possess an intrinsic ability to translocate across cell membranes, have been explored as a means to achieve cellular delivery of siRNA. Approaches using CPPs by themselves or through incorporation into other siRNA delivery platforms have been investigated with the intent of improving cytoplasmic delivery. Here, we review the utilization of CPPs for siRNA delivery with a focus on strategies developed to enhance cellular uptake, endosomal escape and cytoplasmic localization of CPP/siRNA complexes.

Sixteen years after the discovery of RNA interference (RNAi) as an intrinsic cellular antiviral defense mechanism [1] , small interfering RNAs (siRNA) constitute an indispensable biological tool and potential therapeutic option for several incurable diseases. siRNAs, synthetic molecules that induce RNAi, are double-stranded RNA molecules, 20–25 base pairs in length that can be synthetically designed to mimic mature endogenous micro RNAs (miRNAs) and small interfering RNAs, the natural mediators of cellular RNAi [2,3] . siRNAs enter the intracellular silencing pathway after bypassing the enzymatic proteins Drosha and Dicer, which are required for processing immature miRNAs [4,5] , and get directly incorporated into the RNA-induced silencing complex (RISC), which mediates cleavage and destruction of complementary messenger RNA (mRNA) [6] . Thus, ‘knockdown’ or ‘silencing’ of gene expression by synthetic siRNAs relies heavily on their being able to access the RISC complex, which is located in the cytoplasm [7] . Since ‘naked’ siRNAs cannot readily penetrate cell membranes (reviewed in [8,9]), a number of methods that transfer siRNA into the cytosol such as viral vectors (reviewed in [10–12]) and

10.4155/TDE.15.2 © 2015 Future Science Ltd

nonviral delivery systems including cationic lipids, dendrimers and polymers (reviewed in [13–15]) are under active investigation. One intensely researched strategy for siRNA delivery has been the use of cell-penetrating peptides (CPPs). The identification of cell-penetrating ability in a protein was first made in 1988 with the observation that the HIV protein Tat could cross cell membranes and enter cells [16,17] . This activity was mapped to a protein transduction domain (PTD) called TAT, comprising an arginine-rich amino acid sequence (GRKKRRQRRRPQ) between positions 48–60 of the protein [18] . Since then, many naturally occurring PTDs have been identified [19–21] . Importantly, many CPPs have also been synthetically generated. Penetratrin (RQIKIWFQNRRMKWKKGG), whose first 16 amino acids are derived from the third helix of the Drosophila Antennapedia protein homeodomain was the first nonviral CPP [22] . Transportan, a chimeric CPP, is a 27 amino acid-long peptide (GWTLNSAGYLLGKINLKALAALAKKIL), containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine [23] . While these

Ther. Deliv. (2015) 6(4), 491–507

Jagadish Beloor‡,1, Skye Zeller‡,1, Chang Seon Choi1,2, Sang-Kyung Lee2 & Priti Kumar*,1 1 Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA 2 Department of Bioengineering, Institute of Nanoscience & Technology, Hanyang University, Seoul, South Korea *Author for Correspondence: Tel: + 1 203 737 3580 Fax: +1 203 737 6179 [email protected] ‡ Authors contributed equally

part of

ISSN 2041-5990

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Review Beloor, Zeller, Choi, Lee & Kumar

Key terms RNA interference: RNA interference is a post-transcriptional pathway in eukaryotes in which short RNA fragments inhibit gene expression by binding to mRNA with a complementary sequence in a highly sequence specific manner and targeting it for destruction. Small interfering RNAs: Small interfering RNAs are a class of double-stranded RNA molecules, 20–25 base pairs in length that can be synthetically designed to bind target mRNA with a high degree of sequence specificity and interfere with its translation to protein by targeting it to destruction. RNA-induced silencing complex: RISC is a ribonucleoprotein complex which is composed of multiple proteins of Argonaute family, siRNA or miRNA and their complementary sequence strands or mRNA. Argonaute2 (AGO2) protein in the RISC complex is the effector molecule that executes degradation of target mRNA. Cell-penetrating peptides: Short peptides that have an intrinsic ability to ‘penetrate’ cells and facilitate cellular uptake of various molecular cargo (from nanosize particles to small chemical molecules and larger nucleic acid molecules).

peptides differ in structure and composition, a common feature that these and many other CPPs possess is a high proportion of positively-charged lysine and arginine amino acid residues that appear critical for cell-penetration. In fact, repeating units of arginine residues, like R8 and R9, are some of the most prominent CPPs studied to date [24] . Indeed, cationic CPPs have found utility in the transport of macromolecules of varying compositions such as proteins and peptides (reviewed in [25–27]), nucleic acids (reviewed in [28–30]) and quantum dots (reviewed in [31,32]) in vitro into a variety of cell types including hard-to-transfect primary cells as well as in vivo in experimental animal models. Mechanisms of cellular entry & trafficking of CPPs The first step of cellular penetration by cationic CPPs is likely initiated by electrostatic interactions of positively charged amino acid residues with the negatively charged lipid and polysaccharide components in the plasma membrane (reviewed in [33]). The critical involvement of positively charged residues in translocating CPPs was demonstrated as early as 1965 when the addition of poly-lysine to media containing albumin-induced protein uptake into tumor cells [34] . Substitution of the arginine residues in TAT with alanine reduced TAT uptake by 70–90% [35] . Substituting arginine with the amino acids histidine, ornithine and lysine reduced TAT penetration into cells and R9 was >20-times efficient than TAT in cell penetration [35,36] . Oligo-arginines are in fact, the only all-cationic residue CPPs that effectively translocate compared with

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other polycationic homopolymers [36] . These findings implicated an important role for the guanidinium headgroup of arginine, which enables formation of bidentate hydrogen bonds with the negatively charged phosphate groups in the plasma membrane in contrast to the single hydrogen bond formed with the ammonium cations in positively charged lysine residues [37] . Additionally, arginine residues have also been implicated in binding to cell-surface heparan sulfate and aid membrane penetration [38,39] . Indeed, of the >100 CPPs characterized in the past 20 years, arginine-rich CPPs (R-CPPs) and oligomers of D and L-arginine (8–15 D/LR), have become a model for mechanistic studies on cellular translocation of macromolecules [40] . However, basic residues may not solely define cell-penetrating activity. In most natural CPPs, cationic residues are often interspersed with hydrophobic residues and hydrophobicity appears to assist in the formation of amphiphilic helices in aqueous solution, which improves penetration of the lipid bilayers [24] . An anionic CPP termed SAP(E) has also been recently described to deliver fluorophores into a range of cell lines [41] . The uptake mechanisms triggered upon CPP attachment to the cell surface are still debatable and may be multifactorial (Figure 1) . Initial studies in 1997 on the entry of TAT revealed energy and temperatureindependent mechanisms and ruled out endocytosis as a means of cellular uptake implying an ability in CPPs to directly translocate across the plasma membrane [18,42,43] . However, further investigations reveal that CPP entry can also be affected by energy and temperature, and that the conclusions of an energy-independent entry mechanism, particularly at low-CPP concentrations, may have been an artifact of the fixation procedure used prior to confocal microscopy [44] . Since this revelation, studies focused on understanding the processes of CPP entry have relied on techniques such as live-cell microscopy, HPLC, flow cytometry, quantitative spectrofluorometry, electron microscopy, MS and functional assays using reporter read-outs such as splice correction and Cre-recombinase activity that do not call for fixation procedures to reduce the risk of artifactual data (reviewed in [45]). A generalized mechanism for CPP entry still remains elusive as the experimental parameters used including the cell line, CPP composition, CPP-associated cargo and CPP concentration markedly influence the route and kinetics of CPP uptake [46–55] . Two major mechanisms are now implicated in the cellular entry of CPPs and their attached cargo: energy-independent direct translocation at CPP concentrations of ≥10 μM [56–58] and endocytosis mechanisms at ≤5 μM [33,48,53,59–61] . Direct penetration is thought to

future science group


Direct penetration Forming of multiamellar vesicle stack

Membrane inversion

Endocytosis

Flattening, thinning and pore formation

?

Review

Macro-pinocytosis

Clathrin-mediated

Caveoline-mediated

? Actin filaments

Dynamin

Lipid bilayer

Dynamin

Clathrin

CPP

Caveolin

Uncoating

Cytoplasm CPPs with NLS ex: Tat peptide

Nucleus

?

Endosome

Caveosome

Endosomal escape ?

Figure 1. Mechanisms of cellular entry and trafficking of cell-penetrating peptides. There are two major pathways implicated in the cellular uptake and entry of CPPs, direct penetration and endocytic entry mechanisms. Direct penetration which occurs mainly at high molar concentrations of CPPs (≥ 5 μM), may involve mechanisms of multilamellar vesicle stack formation at the cell membrane that induces membrane flipping, and flattening and thinning and/or pore formation of the membrane leading to CPP entry through yet undefined mechanisms. Endocytic entry mechanisms are mainly invoked at lower molar concentrations of CPP (≤ 5 μM). Cellular proteins like clathrin and caveolin involved in the formation and pinching off of endocytic vesicles may be involved. Larger CPPs may also be taken in by macropinocytosis. CPPs then traffic to the interior of the cells in within endosomes. How CPPs escape from endosomes is not clear. Some CPPs have a NLS that may lead to the nuclear localization. CPP: Cell-penetrating peptide; NLS: Nuclear localization signal.

occur via energy-independent pathways through different mechanisms including the formation of inverted micelles and membrane pores [22,62,63] or by membrane flattening and thinning [64] ( Figure 1, left half). These mechanisms appear most probable at high CPP concentrations and are initiated by CPP interactions with negatively charged components of the membrane such as heparan sulfate and the phospholipid bilayer, the latter mainly with amphiphilic CPPs [48,65,66] . An interesting energy-independent mechanism reported recently in live-microscopy studies with polyarginine CPPs appears to involve the formation of ‘particle-like’ multivesicular structures on the plasma membrane that promotes interactions with the negatively charged inner membrane inducing temporary topical inversion of the plasma membrane at the site of CPP binding [67] . The presence of hydrophobic residues in the CPP further enhanced the effect of permeation by promoting membrane-CPP interactions.


For most CPPs at lower, and perhaps physiologically relevant concentrations, it is generally agreed upon that endocytosis is the translocation mechanism. The pathways include macropinocytosis for uptake of large particles and classical endocytosis, dependent on the coat proteins clathrin and/or caveolin [61,68] ( Figure 1, right half). CPP endocytosis is proposed to be a result of hijacking of the natural pathway of glucosaminoglycan recycling that occurs continuously [69,70] or actin remodeling triggered by the clustering of glucosaminoglycans on the cell surface after CPP binding [71,72] . Thus, multiple mechanisms including direct translocation across cell membranes (particularly at high CPP concentrations), macropinocytosis, clathrin- and/or caveolin-mediated endocytosis, operate either independently or in concert to enable CPP entry. It is largely evident that CPPs direct the cellular uptake of attached macromolecules using the same mechanisms [51,73] .

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Review Beloor, Zeller, Choi, Lee & Kumar It is well documented, however, that a major problem is the delivery of CPPs attached to macromolecules to the correct intracellular compartment, which is critical for bioactivity [33,73–76] . In the case of siRNA, as loading onto the RISC complex is necessary for target mRNA knockdown, delivery into the cytoplasm and dissociation of siRNA from the CPP become the rate limiting steps [9,77] . These processes are highly inefficient, and many reports document that endocytosed CPP-siRNA complexes remain entrapped within vesicles for extended periods of time [78–81] . CPP escape from endosomes appears to occur at specific stages of endosomal maturation and not continuously along the route of endosomal trafficking within the cell [82–84] . This stage-specific CPP release appears to depend on the composition of the CPP, for instance, 8R escapes after endosomes mature to the Rab5+ stage, TAT escapes from Rab7+ late endosomes and a unique cationic miniature CPP containing five Arg residues embedded within an α-helix escapes from early Rab5+ endosomes [82] . Computational studies of siRNA release from endocytosed ionizable lipid nanoparticles also suggest that siRNA escape from the endosome is inefficient and restricted to a specific state of endosomal maturation with as little as 1–2% of siRNAs gaining access to the cytoplasm [83] . Vesicles with siRNAs that miss release target siRNA to eventual degradation or may fuse with the Golgi and ER with siRNAs secreted back into extracellular milieu [84] . Thus, for effective siRNA-mediated silencing, strategies that increase siRNA release from the endosome are equally important as those that enhance uptake. Along the lines of such investigation, our own have demonstrated that tagging CPPs to ligands that bind cell-surface receptors can hugely potentiate the process of siRNA delivery to the cytoplasm, using both energy-independent membrane-flipping mechanisms as well as from vesicles involving endosomal protease activity [85–87] . Strategies for CPP-mediated delivery of siRNA CPPs have been used for siRNA delivery broadly in two ways – where the CPP mediates cellular entry as well as transports siRNA, or in combination with another molecule that mediates cellular entry while the CPP functions more as an siRNA carrier and/or an agent for enhancing delivery. As detailed below, siRNAs have been linked covalently to CPPs; however, the complications and poor yields associated with these protocols have made this approach unpopular. The ability of cationic CPPs to noncovalently associate with siRNAs has mostly been exploited for CPP-mediated siRNA delivery. However, this strategy can interfere with the CPP’s ability to interact with cell membranes and also enhance retention of CPP-siRNA complexes

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in endosomal compartments. Therefore, many studies have resorted to modifying CPPs from their native form or using them in multimeric form or in conjunction with another siRNA carrier to permit unhindered uptake and promote endosomal escape. In the more recent years, CPPs have been exploited as siRNA carriers in conjunction with targeting ligands that mediate trafficking and entry into cells after binding cell surface receptors. We detail below, some of the major approaches adopted to elicit siRNA delivery by CPPs and illustrate a few strategic examples. An overview of these approaches is schematically depicted in Figure 2 and representative CPPs used listed in Table 1. Covalent attachment of siRNA to CPP

Covalent conjugation of CPP and siRNA was one of the early approaches explored for CPP-mediated siRNA delivery. Directly conjugating the N-terminal cysteine of TAT through a hetero-bifunctional linker to an amino group engineered at the 3′ terminal end of the antisense strand of duplex siRNA induced RNAi responses in HeLa cells [81] . However, in light of the poorly reproducible efficiencies with this approach, the conjugation strategy was amended to incorporate bioreducible disulfide linkages between the CPP and the sense strand of the linked siRNA [88–91] . This vastly improved siRNA detachment from the CPP particularly within the reducing environment of the cytoplasm and also promoted incorporation of the antisense strand into the RISC complex. Both penetratin and transportan were synthesized with a N-terminal cysteine to enable conjugation with siRNAs modified on the sense strand with a terminal thiol group, which was sensitive to the reducing environment of the cytoplasm [88] . This enhanced silencing of reporter transgene expression in several mammalian cell types. Modification of the N-terminal of penetratin or TAT with a nitropyridine sulfenyl (Npys) group similarly enabled attachment to a 5′thiol-modified sense siRNA strand through a disulfide bond for silencing of target gene expression in primary murine hippocampal neurons with minimal cytotoxicity [89] . However, the complicated multistep protocol for introducing the terminal chemical groups, poor yields of purified CPP-siRNA conjugates and the risk of altering siRNA activity after chemical conjugation preclude large scale use of this approach [92] Electrostatic complexation of siRNA with CPPs

Noncovalent binding of siRNA by cationic CPPs involves simply mixing negatively charged siRNA with positively charged-CPPs at an optimized CPP:siRNA ratio [75] . This produces micro- to nanosized CPPsiRNA complexes wherein the siRNA is fairly



CPP-siRNA covalent conjugate

CPP-siRNA electrostatic complex Hydrophobic moiety

CPP-siRNA

+

+

Cholesterol +

Enhancing endosomal escape

CPP-ss-siRNA ss

Synthetic CPP delivery platform CPP + multimerization CPP-DRBD cell-targeting ss ? fusion ligand ss ss ss + ss ss ss ss ss + ss ss ss

CPP + Lysosomotropic agent/peptide

?

?

?

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ss ss ss ss ssss ss ss ss ss ss ss ss ss ss ss ss ss

?

DsRNA binding domain

?

?

?

Endocytosis Endosome ?

?

? ?

s s

Acidification

H+

Proton sponge effect Osmotic swelling

H+

?

Endosome disruption

H+

RISC

pH dependent 559 peptide ex:GALA (membrane disruptive Calcium peptide) HA2 (fusion peptide)

RISC

Figure 2. Cell-penetrating peptide mediated short interfering RNA delivery. CPP have been exploited for siRNA delivery in many different ways. CPPs have been chemically conjugated to siRNAs and engineering a disulfide bond between the CPP and siRNA enables better cytoplasmic release of the siRNA. Cationic CPPs can electrostatically complex with anionic siRNA. Attachment of hydrophobic groups like cholesterol to CPPs allows better penetration of complexed siRNAs through the lipid-rich cell membrane. CPPs have been tagged with agents that have the ability to disrupt endosomes enabling cytoplasmic delivery. CPP multimers incorporating disulfide bond between the individual CPP molecules facilitate siRNA release in the cytoplasm. DRBD acting as RNA carriers, have been fused to CPPs for siRNA delivery. Incorporation of CPPs into nanoparticle-based siRNA delivery platforms enhances delivery efficacy. Attachment of targeting ligands to CPPs enables delivery to specific cell types and effective endosomal escape after receptor-mediated encocytosis. CPP: Cell-penetrating peptide; DRBD: Double strand RNA binding domain; RISC: RNA-induced silencing complex; siRNA: Short interfering RNA; SS: Disulfide bond.

well-protected from nuclease degradation. Further, CPPs and siRNAs can interact in native form and do not need to be chemically modified to facilitate complex formation, which preserves their intrinsic properties [75] . This has therefore been an approach employed with simple as well as advanced CPPs and CPP-based delivery systems. With simple cationic CPPs, the process, however, most times affects cellular uptake as CPP-membrane interactions are compromised, likely because of the reduction in the net CPP positive charge after siRNA complexation [80] . Hence, for effective siRNA delivery, a huge molar excess of CPP, much larger than required for siRNA complexation is necessitated [80] . Nevertheless, the ease and feasibility of this noncovalent approach has popularized its use in a number of in vitro and in vivo studies [46,73,79,93–95]. Thus siRNA delivered using the homopolymeric arginine CPPs R8 and R9 elicited gene silencing at peptide:siRNA molar ratios of >50:1 in mammalian cell


lines [94,96,97] . As these high concentrations of cationic CPPs induce cytotoxicity in several cell types [93,94] , enhancing CPP hydrophobicity has been explored, which in addition to reducing toxicity, increases cell surface adsorption, membrane translocation and even endosomal escape. Insertion of a stearyl hydrophobic moiety at the N-terminal of 8R (STR-R8) resulted in functional siRNA delivery into primary rat neurons at a 2:1 peptide:siRNA ratio [98] . Stearylation of TP-10, a shorter version of transportan, also improved siRNA transfection efficiencies [47,99,100] . Myristoylated transportan (myr-TP) was twofold more efficient in functional siRNA delivery than the myristoylated versions of TAT or R9 [101] . Similarly, cholesterylation of 9DR (Chol-R9) resulted in a >50% reduction of VEGF gene expression at a 40:1 N/P ratio in the CT-26 colon carcinoma cell line led to tumor regression in mice after intratumoral administration while unmodified R9 was not effective under the same conditions [95] .


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Table 1. Examples of cell-penetrating peptide based delivery systems. CPP-based siRNA delivery system

Sequence/features

Ref.

Covalent CPP: siRNA conjugation TAT(47–57) -siRNA

[81]

Penetratin-SS-siRNA

[88]

Transportan-SS-siRNA

[88]

Electrostatic CPP: siRNA complexes R8

RRRRRRRR:siRNA

[96]

R9

RRRRRRRRR:siRNA

[94]

Stearyl-R8

Stearyl- RRRRRRRR

[98]

Stearyl-TP10

Stearyl-AGYLLGKINLKALAALAKKIL-Cysteamide

[99]

Increased hydrophobicity

cholesteryl oligo-D-arginine

Chol-RRRRRRRRR

Myristoylated Transportan

myr-GWTLNSAGYLLGKINLKALAALAKKI LGGGGKCGNKRTRGC

[101]

MPG

GALFLGFLGAAGSTMGAWSQPKKKRKV

[103]

MPGΔ

NLS

MPG-8

[95]

GALFLGFLGAAGSTMGAWSQPKSKRKV

[103]

βAFLGWLGAWGTMGWSPKKKRK-Cysteamide

[104]

Improved endosomal escape PepFect6/PF6 PepFect14/PF14

[47]

Stearyl- AGYLLGKLLOOLAAAALOOLL-NH2

[100,107]

NickFect51/NF51 Double TAT (dTAT)

[108,109]

RKKRRQRRRHRRKKR

[112]

Membrane-disruptive peptides HA2-penetratin

GLFGAIAGFIENGWEGMIDGRQIKIWFQNRRMKWKK-amide

599

GLFEAIEGFIENGWEGMIDGWYGGGG-RRRRRRRRRK

STR-H16R8

Stearyl-HHHHHHHHHHHHHHHHRRRRRRRR-NH2

STR-H20R8

Stearyl-HHHHHHHHHHHHHHHHHHHHRRRRRRRR-NH2

H5 -S413-PV

HHHHHALWKTLLKKVLKAPKKKRKVC-NH2

[124]

rPOA

(CRRRRRRRRRC)n

[128]

Hph-1-Hph-1

YARVRRRGPRRGHYARVRRRGPRR

[133]

[79,119] [93] [123]

[123]

Multimerization of CPPs

Fusion to RNA-binding agents TAT-dendrimer

PAMAM (G5)-[SMPT linker-S-S-TAT] n

[129]

TAT-poly(CBA-DAH)

(CBA-DAH)] n - C-YGRKKRRQRRR

[138]

TAT-DRDB

MGRKKRRQRRRGHSGRKKRRQRRRGHIYPYDVPVPDYAGDPG RKKRRQRRRGDPAGDLSAG -DRDB- KKAAALEHHHHHH

Hph-1-DRDB

YARVRRRGPRR-RNase III/human dicer dsRBD

TAT-U1A

YGRKKRRQRRR -U1A-Alexa546

[131,132] [133] [80,137]

Synthetic delivery platforms incorporating CPPs R8-MEND

RRRRRRRR-MEND

R8/GALA-MEND

RRRRRRRRWEAALAEALAEALAEHLAEALAEALEALAA-MEND

[147]

ABP

Redox-sensitive polymer

[139]

[56]

CPP sequences are underlined, the functional group is in bold. CPP: Cell-penetrating peptide; siRNA: Short interfering RNA; SS: Disulfide bond.

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Review

Table 1. Examples of cell-penetrating peptide based delivery systems (cont.). CPP-based siRNA delivery system

Sequence/features

PAM-ABP


Ref. [151]

TAT-BMP-PAMAM

Bacterial magnetic nanoparticles based siRNA delivery system driven by magnetic field

[148]

R8/GALA-MEND

RRRRRRRRWEAALAEALAEALAEHLAEALAEALEALAA-MEND

[147]

ABP


[139]

PAM-ABP


[151]

PF6-PF6-NP

PF6/siRNA/lipid core modified with PF6

[149]

F105-P

scFv- HIV gp120 –protaminefusion

[152]

Myr-Tp-Tf

Myristic acid- GWTLNSAGYLLGKINLKALAALAKKIL-GGGGTHRPPMWSPVWP

[153]

TAT-A1

YGRKKRRQRRR - WFLLTM

[156]

RVG-9R

YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG- RRRRRRRRR

scFvCD7–9R

scFvCD7Cys-Cys(Npys)-RRRRRRRRR

[85]

DC3–9R

FYPSYHSTPQRPGGGGSRRRRRRRRR

[155]

ATS-9R

CKGGRAKDRRRRRRRRR

[156]

Improved siRNA release

CPPs with cell-targeting ligands

[86]

CPP sequences are underlined, the functional group is in bold. CPP: Cell-penetrating peptide; siRNA: Short interfering RNA; SS: Disulfide bond.

An interesting set of studies investigating delivery of electrostatically complexed siRNA has revolved around another synthetic hybrid CPP called MPG. MPG has a hydrophobic N-terminal domain derived from the HIV envelope glycoprotein subunit gp41 (GALFLGFLGAAGSTMGAWSQP) and hydrophilic C-terminal domain comprising the nuclear localizing signal (NLS) of the SV40 large T antigen (KKKRKV) [102] . The C-terminal domain not only targets trafficking into the nucleus but also interacts with anionic cargo because of its basic make-up. MPG-siRNA knocked down glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in human foreskin fibroblast HS-68 cells but required a peptide:siRNA molar ratio of 84:1 [103] . To increase cytoplasmic siRNA delivery by MPG, the second lysine residue in the NLS motif was mutated to serine (KSKRKV), which abolished nuclear localization. siRNA delivery with this variant peptide MPGΔNLSresulted in >90% luciferase gene silencing in both HeLa and Cos-7 cells, and ∼80% GAPDH gene silencing in HS-68 cells at peptide:siRNA charge ratio of 10:1. However, intratumoral administration of MPGΔNLScomplexed to siCyclin B1 was poorly effective and attributed to low in vivo stability and precipitation of the CPP-siRNA complexes under the high concentrations used [104] . A shorter version of MPG, called MPG-8 lacking G(1), L(3), S(13), A(17), Q(20) and V(27) and with substitutions of W for the hydrophobic F(7) and A(11) residues, which favored interactions


with both siRNA and the lipid phase of the membrane was developed. In comparison to MPGΔNLS, MPG-8 induced a twofold greater silencing of cyclin B1 with siRNA in HeLa cells. Importantly, intratumoral injection of MPG-8-siCyclin B1 complexes resulted in ∼75% inhibition of tumor growth [104] . Strategies to improve endosomal escape of CPPs

As mentioned above, a well-acknowledged problem associated with CPP:siRNA complexes that are endocytosed is their retention within endosomes resulting in a relatively minute proportion of functional siRNA delivered into the cytosol [33,73–76,99,105] . Fusing CPPs to lysosomotropic agents/peptides has been explored to achieve higher cytoplasmic accumulation of the delivered siRNA. Advances in the design of stearylated-TP10 led to the development of entire family of CPPs termed ‘PepFect or PF’ with better endosomal release properties. PepFect6 (PF6) was derived by linking stearylated TP10 via a succinylated lysine tree to four pH titratable trifluoromethylquinoline moieties, which are chloroquine analogues that facilitate endosomal release [47] . siRNA complexed to PF6-induced gene silencing in several hard-to-transfect cell types including human vein endothelial cells (HUVEC) and Jurkat T-lymphocytic cells as well as in rat cochlear organotypic cultures [106] . This modified CPP also delivered functional siRNA in vivo in mice after systemic injection resulting in ∼50–70% reduction


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Key term Proton-sponge effect: Phenomenon where the extensive buffering capacity of an agent is postulated to increase osmotic pressure within endosomes leading to their swelling, rupture and release of contents into the cytosol.

in hypoxanthine-guanine phosphorybosyltransferase 1 (HPRT1) mRNA levels in lung, liver and kidney and with no toxicity [47] . Replacement of lysines and isoleucines in stearylated TP10 with ornithines and leucines yielded PF14, which along with successfully inducing siRNA-mediated silencing in cell lines, retained activity even upon storage in solid form at elevated temperatures for several weeks [100,107] . Moreover, PF14:siRNA nanoplexes displayed excellent RNAi activity after incubation in simulated gastric fluid that is highly acidic (∼pH 1.2) inducing > 80% reduction of luciferase activity in HEK 293 cells. A recent comparative study with seven different CPPs for siRNA delivery has also revealed superior qualities for PF6 and PF14 [97] Another family of CPPs based on stearylated TP10 engineered with the intention of enhancing endosomal escape is called the NickFects, wherein, reducing the average nanoparticle size was investigated as a means to improve endosomal escape and thus functional efficiency [108] . Replacement of Lys7, which is the linker amino acid between the residues derived from galanin and mastoparan in TP10, with ornithine, a noncoded amino acid that is resistant to protease degradation and coupling it to Gly6 through its δ-NH2 group generated NF51, which enabled the formation of smaller nanoparticles with nucleic acids promoting efficient endosomal escape. NF51 induced excellent gene silencing both under serum free conditions in almost entire cell populations and in presence of serum, given its lysomotropic properties and serum protease resistance [109] . Analogous to the above approach, addition of calcium to CPP-nucleic acid complexes is known to reduce particle size through interaction of calcium with both the amines (polycations) and phosphates (nucleic acids) leading to formation of ‘soft’ cross-links [110,111] . The approach was thus extended to TAT (RKKRRQRRR) and double TAT (comprising two consecutive TAT sequences)-complexed siRNA, resulting in reduction of complex sizes from ∼2200 nm to ∼90 nm which enhanced endosomal escape and induced gene silencing in >90% in A549 cells [112] . In vivo delivery of calcium-condensed TAT-siRNA complexes resulted in pronounced GAPDH gene silencing in lung, muscle and stomach tissues in mice with no evidence of cytotoxicity. An alternative approach is fusing CPPs to pHdependent membrane-disruptive peptides that are inactive at neutral pH but activated under acidic conditions in the endosomes (∼pH 5.5), thus leading to

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the disruption of only the endosomal membrane but not the plasma membrane [77,113,114] . The 23 aminoacid long HA2 fusion peptide derived from hemagglutinin HA2 subunit is able to fuse and disrupt lipid bilayers upon protonation of its glutamate and aspartate residues under acidic conditions [115,116] . CPPs are normally linked to the C-terminal end of HA2 as the N-terminal G residue in HA2 is required for its membrane fusion activity [117,118] . Attachment of HA2 to penetratin, improved siRNA delivery [79,119] . A recent study similarly used a synthetic peptide named 599, composed of an influenza virus-derived endosome disruptive peptide IFN-7 fused to R9 (GLFEAIEGFIENGWEGMIDGW YGGGGRRRRRRRRRK), to induce knockdown of cancerous inhibitor of protein phosphatase 2A (CIP2A) mRNA levels in CAL 27 and SCC-25 oral cancer cell lines [93] . The ‘proton sponge’ effect, in which the extensive buffering capacity of an agent is postulated to increase osmotic pressure within endosomes leading to their swelling, rupture and release of contents into the cytosol [120,121] , has been investigated as another means to enhance endosomal release of CPP-siRNA complexes. The pK a of the imidazole group in histidine matches the pH within endosomes [122] and coupling histidine to the R8 CPP such that the ratio of histidine to arginine is >1.5 (e.g., STR-HnR8 where n = 16 and 20), induced pronounced gene silencing due to the proton-sponge effect [123] . Modification of the S413-PV CPP, derived from the dermaseptin peptide fused to NLS of SV40 large T antigen through the addition of a five-histidine tail to its N-terminus elicited superior biological activity when complexed to siRNAs targeting survivin in HT1080 cells [124] . Similarly, a 7000-fold improvement in gene transfection efficiency was observed with a modified Tat peptide covalently fused with ten histidine residues (Tat-10H) [125] . Multimerization of CPPs

CPP multimerization can concentrate CPPs in a localized manner on the cell surface leading to improved membrane penetration. Multimerization can be achieved by polymerizing CPP monomers through amino-carboxyl or disulfide linkages between terminal cysteine residues [126–128] . Dimethylsulfoxide oxidation of Cys-(9DR)-Cys monomers yielded a polymeric CPP, reducible poly (oligo-D-arginine) or rPOA extending in size upto 96 kDa [128] . The interpeptide redox-sensitive disulfide bonds aided dissemination of the large polymer into monomeric units of 9DR in the cytoplasm enhancing biocompatibility and enabling rapid siRNA release both in cell lines and in a murine subcutaneous tumor model where siRNA targeting VEGF-induced tumor regression.



Fusion of CPPs to RNA-binding agents

The approaches described this far relied on a CPP for mediating cellular binding/entry as well as actively transporting siRNA into cells. Coupling a CPP to another siRNA carrier, on the other hand, prevents masking of CPP activity due to siRNA binding leaving the CPP free to mediate translocation across cell membranes. One way this has been achieved is by attaching CPPs to dendrimers that can bind nucleic acids. Attachment of TAT to poly(amidoamine) (PAMAM) dendrimers induced RNAi-mediated gene silencing in NIH3T3 cells [129] A second approach has utilized biological dsRNA binding-domain containing proteins (DRBD or dsRBD) as the siRNA carrier. DRBDs constitute a family of eukaryotic, prokaryotic and viral-encoded products with an evolutionarily conserved motif that facilitates interaction with dsRNA [130] . Fusing three TAT peptide repeats with the N-terminal of an ∼65 residue dsRNA binding domain (PTD-DRBD) allowed delivery of functional siRNA into a variety of cell types resistant to conventional transfection reagents such as H1299 lung adenocarcinoma cells, Jurkat T cells, primary human fibroblasts, keratinocytes, macrophages, melanoma and glioma cells [131] . Intranasal inoculation of ROSA26 mice expressing luciferase in the nasal and tracheal passages resulted in strong knockdown in luciferase expression within 24 h demonstrating in vivo delivery of functional siRNA. To further investigate the utility of this strategy, recently, three different CPPs (TAT, PTD4 and Hph-1) were individually fused with distinct dsRBD proteins including the human protein kinase R (PKR), human dicer and Escherichia coli ribonuclease III. The Hph-1 CPP fusion with the ribonuclease III or human dicer dsRBDs appeared superior inducing ∼70% target gene silencing in HeLa cells [132] . In fact, fusing dimerized Hph1 with the dsRBD enabled siRNA transfer into a whole mouse heart graft in an in vivo heart transplantation model [133] . Although, the studies did not rule out a role for the CPP in siRNA binding, they nevertheless demonstrated that the inclusion of another carrier for siRNA enhances functional siRNA delivery by the CPP [134] One other study utilizing TAT with an RNA binding protein also incorporated an additional feature to promote endosomal escape of CPP-siRNA complexes. The approach was based on the use of photo-sensitive fluorescent dyes whose exposure to light results in the generation of short-lived reactive oxygen molecules which can destabilize lipid membranes [135,136] . Fusing TAT to the 98 amino acid long RNA binding domain from the human U1 small nuclear ribonucleoprotein A (TatU1A) enabled binding to siRNA engineered with a 5′ U1A specific sequence (GGGCAUUGCA-


Review

CUCCGCCC) [80,137] . TAT-mediated transduction resulted in excellent uptake of the siRNA complexes, but cytoplasmic release was poor due to endosomal retention. Covalent attachment of the alexa fluor 546 to the C-terminus of the TatU1A and photo stimulation at 540 nm triggered cytosolic delivery and siRNA mediated gene silencing in CHO and A431cells Incorporation of CPPs into synthetic siRNA delivery platforms

CPPs have been included as components of synthetic siRNA delivery platforms comprised liposomes, dendrimers and polymers in a bid to increase cellular internalization [129,138–140] . This also orients CPPs in close proximity akin to multimerization, while minimizing the risk of immunogenicity that sometimes results with multimeric branched CPPs due to their large size and structural resemblance to multiple antigenic peptides [141,142] . One of the early approaches with a CPP-modified liposome used TAT linked to the surface of a phosphatidylcholine, cholesterol and Rh-PE based liposome [143] . Although siRNA delivery was not investigated, the approach demonstrated efficient uptake of the formulation into human ovarian carcinoma [144] , tumor and dendritic cells [145] . Incorporation of stearylated-R8 at a relatively low concentration (5 mole%) into a lipid-based mixture was found to vastly improve cellular uptake of DNA [146] . This formulation, termed the ‘multifunctional envelope-type nanodevice’ or MEND was adapted for siRNA delivery by condensing siRNA with stearyl-R8 to form a condensed core of 80% silencing of target gene expression in HeLa cells 24 h after transfection. The R8-MEND system was further evolved to include a pH sensitive fusogenic peptide GALA, promoting endosomal escape. The GALA peptide, a 30 amino acid (WEAALAEALAEALAEHLAEALAEALEALAA) amphipathic peptide with a glutamic acid-alanine-leucine-alanine repeat sequence, undergoes a pH-induced transition that exposes the hydrophobic face of the peptide and allows interaction with lipid membranes [50] . Effective knockdown of SOCS-1 gene expression with this system in primary dendritic cells induced anti-tumor responses in C57BL/6 mice [147] . Another siRNA delivery system made of bacterial magnetic nanoparticles (BMPs) purified from magnetotactic bacteria was conjugated with PAMAM dendrimers and TAT (TAT-BMP-PAMAM) and enabled trans-membrane delivery of siRNA under the influence of a magnetic gradient for gene therapy of sub-cutaneously implanted tumors in mice [148]


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Review Beloor, Zeller, Choi, Lee & Kumar Enhancing siRNA release within the cytoplasm

Some of the more evolved CPP-integrating siRNA delivery platforms incorporate features that accelerate siRNA release from the carrier in the cytoplasm after endosomal escape, which further enhances gene knockdown. In contrast to R8, the conventional homopolymeric cationic CPP, pH sensitive polycations such as octahistidine (H8) acquire a positive charge at a lower pH but are uncharged at the cytoplasmic pH (7.4), which aids in easy dissociation from siRNA. Thus, incorporation of stearylated-H8 in the abovedescribed R8/GALA-MEND, resulted in an ∼55-fold increase in cytoplasmic siRNA release, which translated >95% gene silencing in HeLa-GL3 cells [149] The previously described PF6 contains bulky chloroquine analogs that leads to premature decondensation of siRNA in the endosome. Therefore, the PF6/ siRNA core was coated with lipid membranes (PF6NP) that were again modified with PF6 for endosomal escape [150] . The PF6/PF6-NP thus generated hugely enhanced siRNA distribution within the cytoplasm, which induced a potent RNAi effect resulting from a combination of features that resulted in high cellular uptake, efficient endosomal escape and cytoplasmic decondensation of the polyplexes Similarly, ABP, a polymer, poly(cystaminebisacrylamide-diaminohexane) grafted with 9–11 residues of the amino acid, arginine in each repeating subunit, has several redox-sensitive internal disulfide bonds that get reduced in the glutathione-rich reducing environment of the cytoplasm. In addition to eliciting effective release of complexed nucleic acids, the biodegradability significantly reduced cytotoxicity [139] . PAM-ABP, a high molecular weight polyplex generated by incorporation of ABP onto a PAMAM dendrimer [151] was able to elicit excellent gene silencing by promoting cytoplasmic release of complexed siRNA in cell lines [139] and effectively regressed tumors in a mouse model with siRNAs targeting the VEGF, Bcl-2 and c-Myc mRNAs [140] . Use of CPPs with cell-targeting ligands

The more recent, and perhaps, the most evolved strategies utilizing CPPs for siRNA delivery have exploited cell-targeting ligands, which has the significant benefit of selectively delivering siRNA to a specific cell types increasing efficiencies in vivo. Herein, the CPP functions as a siRNA carrier and enhances siRNA delivery while binding and uptake of the CPP-siRNA complex to cells is initiated by ligand-receptor interactions One of the first ligand-guided approaches investigated the targeted delivery of anti-viral siRNAs to HIV-infected cells that express the viral envelope glycoprotein gp120 on their surface [152] . A single

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chain antibody recognizing gp120 was conjugated to a siRNA carrier comprised an arginine-rich peptide sequence derived from protamine. siRNA transduction was specific to cells surface-expressing the gp120 envelope and siRNAs targeting a HIV-gag gene sequence inhibited HIV replication in infected cells expressing the HIV envelope protein. Further, systemic injection in mice implanted with B16 melanoma cells expressing gp120 resulted in targeted siRNA delivery to the tumor. In an analogous approach for achieving neuronal delivery of siRNA, myristoylated transportan (myrTP) was used as a chimera with a transferrin-receptor targeting peptide (Tf) for functional delivery of siRNA into mouse primary neurons and astrocytes [153] . TAT has also been attached to a vascular endothelial growth factor receptor 1 (VEGFR1)-binding peptide A1 (WFLLTM) mediating effective knockdown of target gene expression in tumor cells expressing the VEGFR [154] . We, in our work, have utilized the R9 CPP in conjunction with several targeting ligands for effective transfection of complexed siRNA into multiple hardto-transfect primary cells in vivo. A short 32 aminoacid long peptide derived from the rabies virus glycoprotein conjugated to R9 (RVG9R) targeted siRNA delivery into neuronal cells [86] . Intravenous administration resulted in transvascular delivery of RVG-9RsiRNA complexes across the blood–brain barrier and elicited virus gene silencing in brain tissue protecting mice against fatal challenge with the Japanese encephalitis flavivirus. A single chain antibody targeting the pan-T cell receptor CD7 fused to 9DR for targeted siRNA delivery into human T cells [85] . The strategy proved therapeutic for HIV infection in humanized mice when a combination of siRNAs targeting HIV RNA sequences was administered systemically and helped control plasma viral RNA levels. A dendritic cell-targeting 12-mer peptide conjugated to R9 (DC3–9R) delivered antiviral siRNA for inhibited dengue viral replication in multiple subsets of human DCs [155] . Intravenous treatment of humanized mice with siRNA targeting TNF-α complexed to DC3–9R also suppressed poly(I:C)-induced TNF-α production in humanized mice. In another recent study, an adipocyte-targeting peptide fused to R9 (ATS–9R) selectively transfected mature adipocytes by binding to prohibitin [156] . Injection of ATS–9R complexed to short-hairpin RNA (shRNA) for silencing fatty-acidbinding protein 4 (shFABP4), induced metabolic recovery and body-weight reduction. Our investigations into the mechanism of siRNA delivery by the ligand-9R system revealed that the major mechanism of cytoplasmic entry involved ligand-tethered aggregation of siRNA complexes on the plasma membrane



after receptor binding followed by CPP-induced topical membrane inversion translocating the entire ligand-9R–siRNA complex into the cytosol [87] . A second pathway of cytosolic entry from endosomes after ligand-internalization also occurred, this however contributed to extending the dynamics of gene silencing and required endosomal proteolytic activity More complex siRNA delivery platforms incorporating CPPs have also been engineered with targeting ligands to enable cell-specific delivery in vivo. A gene delivery system assembled from a pDNA core (comprised short biodegradable polyamines for complexing plasmid DNA encoding shRNA sequences) and a pPAC core (comprised PEG-tris-acridine core conjugated to multiple peptides - TAT for enhancing delivery, a NLS to guide the pDNA to the nucleus for transcription and apolipoprotein E receptor-specific peptide or a novel brain homing peptide for targeting) delivered pDNA and enabled shRNA expression in mouse cerebral cortex cells after intravenous administration [157] . Incorporation of TAT in the previously described poly(CBA-DAH) formulation along with a cardiomyocyte-specific peptide (PCM), not only increased transduction efficiency but also enabled specific targeting of cardiomyocytes [138,158,159] . The approach was effective in preventing hypoxia-induced cardiomyocyte apoptosis when PCM-Tat-CBA-DAH complexed siRNA targeting SHP-1 or Fas was used as treatment. Taken together, these studies demonstrate that the CPPs can find application in two ways for siRNA delivery. CPPs can perform as general transfection reagents enabling siRNA delivery to a broad variety of cell types by initiating diverse mechanisms of CPP uptake. When used with a cell targeting ligand, CPPs can aid targeted delivery of siRNA into specific cell types utilizing CPP entry mechanisms as well as ligand-uptake pathways. Conclusion Problems of poor bioavailability and ineffective delivery have prevented siRNAs from being broadly useful therapeutically. siRNA therapeutics that have demonstrated promise in the clinic utilize sophisticated delivery platforms that enhance bioavailability but focus largely on liver-related diseases, exploiting the natural absorptive function of hepatocytes [160] . The rate-limiting problem is the delivery of siRNAs into the majority of cells and tissues that do not readily take up the RNAs by endocytosis, particularly when using the systemic route for delivery. The ability of CPPs to enter cells in an autonomous and receptor-independent manner with low cytotoxicity and immunogenicity presents an avenue for the exploitation of these simple peptides as vehicles of siRNA delivery. The simple formulation where CPP:siRNA


Review

complexes are formed within a matter of minutes after mixing is clearly an advantage compared with other in vivo siRNA delivery systems that use complex procedures for siRNA encapsulation. CPPs also offer advantages of enhancing dynamics and kinetics of siRNA delivery, enhancing physiological stability and bioavailability of complexed siRNA. In this review, we have discussed several approaches developed with cationic CPPs for siRNA delivery. However, given the fact that CPPs still need to be used with siRNAs at concentrations above the therapeutic threshold for eliciting physiological effects, there is considerable scope for improving CPPs as platforms for siRNA delivery. Carefully dissecting CPP trafficking pathways can enable refinement in CPP design to ultimately yield an approach that results in cytoplasmic localization of CPP and their siRNA cargo, which is key for siRNA bioactivity. A focus on other aspects such as siRNA stability, increased circulation times of CPP-siRNA, cell-specific delivery by attachment of targeting ligands and immunogenicity, is also required for enhancing in vivo potency. Approaches with CPPs aimed at cell-specific RNAi through the use of cell-binding ligands could enable targeted pharmacology and make an impact on siRNAs as medicine Future perspective The cell translocation property of CPPs has immeasurable value in the delivery of biomolecules. Several studies have demonstrated the utility of CPPs in delivering siRNA and eliciting biological effects in animal models. Given the recent discovery of more advanced nuclease-based genome editing systems, CPPs offer a potential platform for introducing permanent changes into the genetic make-up of a cell as well. Key to success is the design of a CPP-incorporating multimodal delivery platform with individual modules performing specific functions such as cell binding, entry, cytoplasmic release and detachment of the biomolecule effectors, that can be temporospatially controlled and disassembled after activity. Combining advanced technologies from the fields of polymer and lipid chemistry with CPPs and the use of cell-targeting ligands has a high probability of achieve this and advancing CPP-based delivery systems several steps closer to the clinic Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties No writing assistance was utilized in the production of this manuscript


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Executive summary Cell-penetrating peptides for siRNA delivery • Cell-penetrating peptides (CPPs) can direct the cellular uptake of attached macromolecules on account of their intrinsic property of translocation into multiple cell types. • Delivery of short interfering RNA (siRNA) to the cytoplasmic compartment and dissociation of siRNA from the CPP are rate-limiting steps for CPP-mediated delivery of associated siRNA. • Strategies for attaching CPPs to siRNA: –– Covalent conjugation of CPPs to siRNA, directly or through bioreducible disulfide linkages, has been explored as a means to deliver siRNA. –– Non-covalent binding of siRNA to cationic CPPs by simply mixing the two components is a more commonly investigated strategy for siRNA delivery. • Strategies to improve cytoplasmic availability of CPP-attached siRNA: –– Enhancing CPP hydrophobicity can increase adsorption on the cell surface, membrane translocation and endosomal escape of CPPs and attached siRNAs. –– CPP multimerization can concentrate CPPs in a localized manner on the cell surface leading to improved membrane penetration. –– Fusing CPPs to lysosomotropic agents/peptides can induce better release of endocytosed CPP-siRNA complexes and higher cytoplasmic accumulation of the delivered siRNA. –– Coupling CPPs to another siRNA carrier, like a double stranded RNA binding domain, reduces masking of CPP activity and enables better membrane-CPP interactions improving translocation into cells. –– CPPs have been included as cocomponents of synthetic siRNA delivery platforms comprised liposomes, dendrimers and polymers to enhance cellular internalization. –– Attachment of cell-targeting ligands to CPPs provides the significant added benefit of selectively delivering siRNA to a specific cell types while increasing efficiency.

References 1

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature 391(6669), 806–811 (1998).

2

Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl Acad. Sci. USA 98 (17), 9742–9747 (2001).

3

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836), 494–498 (2001).

4

Sliva K, Schnierle BS. Selective gene silencing by viral delivery of short hairpin RNA. Virol. J. 7, 248 (2010).

11

Maetzig T, Baum C, Schambach A. Retroviral protein transfer: falling apart to make an impact. Curr. Gene Ther. 12(5), 389–409 (2012).

12

Liu YP, Berkhout B. miRNA cassettes in viral vectors: problems and solutions. Biochim. Biophys. Acta 1809(11–12), 732–745 (2011).

13

Vicentini FT, Borgheti-Cardoso LN, Depieri LV et al. Delivery systems and local administration routes for therapeutic siRNA. Pharm. Res. 30(4), 915–931 (2013).

14

Gao Y, Liu XL, Li XR. Research progress on siRNA delivery with nonviral carriers. Int. J. Nanomedicine 6, 1017–1025 (2011).

15

Zhang S, Zhi D, Huang L. Lipid-based vectors for siRNA delivery. J. Drug Target 20(9), 724–735 (2012).

5

Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409(6818), 363–366 (2001).

16

Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55(6), 1189–1193 (1988).

6

Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: doublestranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101(1), 25–33 (2000).

17

Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55(6), 1179–1188 (1988).

7

Sen GL, Blau HM. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat. Cell Biol. 7(6), 633–636 (2005).

18

8

Behlke MA. Progress towards in vivo use of siRNAs. Mol. Ther. 13(4), 644–670 (2006).

Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272(25), 16010–16017 (1997).

19

Marinova Z, Vukojevic V, Surcheva S et al. Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission. J. Biol. Chem. 280(28), 26360–26370 (2005).

9

502

Lee Y, Ahn C, Han J et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425(6956), 415–419 (2003).

10

Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8(2), 129–138 (2009).

Ther. Deliv. (2015) 6(4)



20

Magzoub M, Sandgren S, Lundberg P et al. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis. Biochem. Biophys. Res. Commun. 348(2), 379–385 (2006).

37

Lattig-Tunnemann G, Prinz M, Hoffmann D et al. Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cellpenetrating peptides. Nat. Commun. 2, 453 (2011).

21

Sandgren S, Wittrup A, Cheng F et al. The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis. J. Biol. Chem. 279(17), 17951–17956 (2004).

38

Ma DX, Shi NQ, Qi XR. Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells. Int. J. Pharm. 419(1–2), 200–208 (2011).

39

Richard JP, Melikov K, Brooks H, Prevot P, Lebleu B, Chernomordik LV. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280(15), 15300–15306 (2005).

22

Derossi D, Joliot AH, Chassaing G, Prochiantz A. The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269(14), 10444–10450 (1994).

40

23

Pooga M, Hallbrink M, Zorko M, Langel U. Cell penetration by transportan. FASEB J. 12(1), 67–77 (1998).

Lindgren M, Langel U. Classes and prediction of cellpenetrating peptides. Methods Mol. Biol. 683, 3–19 (2011).

41

24

Jones AT, Sayers EJ. Cell entry of cell penetrating peptides: tales of tails wagging dogs. J. Control. Release 161(2), 582–591 (2012).

Martin I, Teixido M, Giralt E. Design, synthesis and characterization of a new anionic cell-penetrating peptide: SAP(E). Chembiochem 12(6), 896–903 (2011).

42

25

Grdisa M. The delivery of biologically active (therapeutic) peptides and proteins into cells. Curr. Med. Chem. 18(9), 1373–1379 (2011).

Futaki S, Suzuki T, Ohashi W et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276(8), 5836–5840 (2001).

26

Van Den Berg A, Dowdy SF. Protein transduction domain delivery of therapeutic macromolecules. Curr. Opin. Biotechnol. 22(6), 888–893 (2011).

43

27

Du J, Jin J, Yan M, Lu Y. Synthetic nanocarriers for intracellular protein delivery. Curr. Drug Metab. 13(1), 82–92 (2012).

Suzuki T, Futaki S, Niwa M, Tanaka S, Ueda K, Sugiura Y. Possible existence of common internalization mechanisms among arginine-rich peptides. J. Biol. Chem. 277(4), 2437–2443 (2002).

44

Richard JP, Melikov K, Vives E et al. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278(1), 585–590 (2003).

45

Holm T, Andaloussi SE, Langel U. Comparison of CPP uptake methods. Methods Mol. Biol. 683, 207–217 (2011).

46

Al Soraj M, He L, Peynshaert K et al. siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat. J. Control. Release 161(1), 132–141 (2012).

47

Andaloussi SE, Lehto T, Mager I et al. Design of a peptidebased vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo. Nucleic Acids Res. 39(9), 3972–3987 (2011).

48

Duchardt F, Fotin-Mleczek M, Schwarz H, Fischer R, Brock R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8(7), 848–866 (2007).

49

Fretz MM, Penning NA, Al-Taei S et al. Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochem. J. 403(2), 335–342 (2007).

50

Subbarao NK, Parente RA, Szoka FC, Jr, Nadasdi L, Pongracz K. pH-dependent bilayer destabilization by an amphipathic peptide. Biochemistry 26(11), 2964–2972 (1987).

51

Tunnemann G, Martin RM, Haupt S, Patsch C, Edenhofer F, Cardoso MC. Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J. 20(11), 1775–1784 (2006).

28 29

Hoyer J, Neundorf I. Peptide vectors for the nonviral delivery of nucleic acids. Acc. Chem. Res. 45(7), 1048–1056 (2012). Lehto T, Kurrikoff K, Langel U. Cell-penetrating peptides for the delivery of nucleic acids. Expert Opin. Drug Deliv. 9(7), 823–836 (2012).

30

Margus H, Padari K, Pooga M. Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery. Mol. Ther. 20(3), 525–533 (2012).

31

Suzuki Y. Exploring transduction mechanisms of protein transduction domains (PTDs) in living cells utilizing singlequantum dot tracking (SQT) technology. Sensors (Basel) 12(1), 549–572 (2012).

32

Liu BR, Huang YW, Chiang HJ, Lee HJ. Cell-penetrating peptide-functionalized quantum dots for intracellular delivery. J. Nanosci. Nanotechnol. 10(12), 7897–7905 (2010).

33

Madani F, Lindberg S, Langel U, Futaki S, Graslund A. Mechanisms of cellular uptake of cell-penetrating peptides. J. Biophys. 2011, 414729 (2011).

34

Ryser HJ, Hancock R. Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture. Science 150(3695), 501–503 (1965).

35

Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl Acad. Sci. USA 97(24), 13003–13008 (2000).

36

Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB. Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res. 56(5), 318–325 (2000).



Review

503

Review Beloor, Zeller, Choi, Lee & Kumar 52

Tunnemann G, Ter-Avetisyan G, Martin RM, Stockl M, Herrmann A, Cardoso MC. Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J. Pept. Sci. 14(4), 469–476 (2008).

67

Hirose H, Takeuchi T, Osakada H et al. Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells. Mol. Ther. 20(5), 984–993 (2012).

53

Verdurmen WP, Bovee-Geurts PH, Wadhwani P et al. Preferential uptake of L- versus D-amino acid cellpenetrating peptides in a cell type-dependent manner. Chem. Biol. 18(8), 1000–1010 (2011).

68

Nakase I, Tadokoro A, Kawabata N et al. Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis. Biochemistry 46(2), 492–501 (2007).

54

Walrant A, Correia I, Jiao CY et al. Different membrane behaviour and cellular uptake of three basic arginine-rich peptides. Biochim. Biophys. Acta 1808(1), 382–393 (2011).

69

Belting M. Heparan sulfate proteoglycan as a plasma membrane carrier. Trends Biochem. Sci. 28(3), 145–151 (2003).

55

Zaro JL, Vekich JE, Tran T, Shen WC. Nuclear localization of cell-penetrating peptides is dependent on endocytosis rather than cytosolic delivery in CHO cells. Mol. Pharm. 6 (2), 337–344 (2009).

70

Yanagishita M. Metabolism of plasma membrane-associated heparan sulfate proteoglycans. Adv. Exp. Med. Biol. 313, 113–120 (1992).

71

56

Nakamura Y, Kogure K, Futaki S, Harashima H. Octaarginine-modified multifunctional envelope-type nano device for siRNA. J. Control. Release 119(3), 360–367 (2007).

Jiao CY, Delaroche D, Burlina F, Alves ID, Chassaing G, Sagan S. Translocation and endocytosis for cell-penetrating peptide internalization. J. Biol. Chem. 284(49), 33957–33965 (2009).

72

57

Rothbard JB, Jessop TC, Wender PA. Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells. Adv. Drug Deliv. Rev. 57 (4), 495–504 (2005).

Poon GM, Gariepy J. Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells. Biochem. Soc. Trans. 35(Pt 4), 788–793 (2007).

73

El-Andaloussi S, Jarver P, Johansson HJ, Langel U. Cargodependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study. Biochem. J. 407(2), 285–292 (2007).

74

Mager I, Eiriksdottir E, Langel K, El Andaloussi S, Langel U. Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay. Biochim. Biophys. Acta 1798(3), 338–343 (2010).

58

59

Fischer R, Kohler K, Fotin-Mleczek M, Brock R. A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. J. Biol. Chem. 279(13), 12625–12635 (2004).

75

60

Payne CK, Jones SA, Chen C, Zhuang X. Internalization and trafficking of cell surface proteoglycans and proteoglycan-binding ligands. Traffic 8(4), 389–401 (2007).

Chao TY, Raines RT. Mechanism of ribonuclease A endocytosis: analogies to cell-penetrating peptides. Biochemistry 50(39), 8374–8382 (2011).

76

61

Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10(3), 310–315 (2004).

Erazo-Oliveras A, Muthukrishnan N, Baker R, Wang TY, Pellois JP. Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. Pharmaceuticals (Basel) 5(11), 1177–1209 (2012).

77

62

Lee MT, Hung WC, Chen FY, Huang HW. Many-body effect of antimicrobial peptides: on the correlation between lipid’s spontaneous curvature and pore formation. Biophys. J. 89(6), 4006–4016 (2005).

El-Sayed A, Futaki S, Harashima H. Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment. AAPS J. 11(1), 13–22 (2009).

78

63

Matsuzaki K, Yoneyama S, Murase O, Miyajima K. Transbilayer transport of ions and lipids coupled with mastoparan X translocation. Biochemistry 35(25), 8450– 8456 (1996).

Mae M, Andaloussi SE, Lehto T, Langel U. Chemically modified cell-penetrating peptides for the delivery of nucleic acids. Expert Opin. Drug Deliv. 6(11), 1195–1205 (2009).

79

Pouny Y, Rapaport D, Mor A, Nicolas P, Shai Y. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry 31(49), 12416–12423 (1992).

Lundberg P, El-Andaloussi S, Sutlu T, Johansson H, Langel U. Delivery of short interfering RNA using endosomolytic cellpenetrating peptides. FASEB J. 21(11), 2664–2671 (2007).

80

Deshayes S, Morris MC, Divita G, Heitz F. Interactions of amphipathic CPPs with model membranes. Biochim. Biophys. Acta 1758(3), 328–335 (2006).

Endoh T, Sisido M, Ohtsuki T. Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference. Bioconjug. Chem. 19(5), 1017–1024 (2008).

81

Chiu YL, Ali A, Chu CY, Cao H, Rana TM. Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells. Chem. Biol. 11(8), 1165–1175 (2004).

82

Appelbaum JS, Larochelle JR, Smith BA, Balkin DM, Holub JM, Schepartz A. Arginine topology controls escape of minimally cationic proteins from early endosomes to the cytoplasm. Chem. Biol. 19(7), 819–830 (2012).

64

65

66

504

Rydstrom A, Deshayes S, Konate K et al. Direct translocation as major cellular uptake for CADY selfassembling peptide-based nanoparticles. PLoS ONE 6(10), e25924 (2011).

Kosuge M, Takeuchi T, Nakase I, Jones AT, Futaki S. Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans. Bioconjug. Chem. 19(3), 656–664 (2008).

Ther. Deliv. (2015) 6(4)



83

Gilleron J, Querbes W, Zeigerer A et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31(7), 638–646 (2013).

98

Tonges L, Lingor P, Egle R, Dietz GP, Fahr A, Bahr M. Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons. RNA 12(7), 1431–1438 (2006).

84

Sahay G, Querbes W, Alabi C et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 31(7), 653–658 (2013).

99

85

Kumar P, Ban HS, Kim SS et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 134(4), 577–586 (2008).

Mae M, El Andaloussi S, Lundin P et al. A stearylated CPP for delivery of splice correcting oligonucleotides using a noncovalent co-incubation strategy. J. Control. Release 134(3), 221–227 (2009).

86

Kumar P, Wu H, Mcbride JL et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature 448(7149), 39–43 (2007).

87

Zeller S, Choi C, Uchil PD et al. Attachment of cellbinding ligands to arginine -rich cell penetrating peptides enables cytosolic translocation of complexed siRNA. Chem. Biol. 22(1), 50–62 (2014).

88

Muratovska A, Eccles MR. Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells. FEBS Lett. 558(1–3), 63–68 (2004).

89

Davidson TJ, Harel S, Arboleda VA et al. Highly efficient small interfering RNA delivery to primary mammalian neurons induces MicroRNA-like effects before mRNA degradation. J. Neurosci. 24(45), 10040–10046 (2004).

90

Turner JJ, Jones S, Fabani MM, Ivanova G, Arzumanov AA, Gait MJ. RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA. Blood Cells Mol. Dis. 38(1), 1–7 (2007).

91

92

93

Moschos SA, Jones SW, Perry MM et al. Lung delivery studies using siRNA conjugated to TAT(48–60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug. Chem. 18(5), 1450–1459 (2007).

101 Ren Y, Hauert S, Lo JH, Bhatia SN. Identification and

characterization of receptor-specific peptides for siRNA delivery. ACS Nano 6(10), 8620–8631 (2012). 102 Morris MC, Vidal P, Chaloin L, Heitz F, Divita G. A new

peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res. 25(14), 2730–2736 (1997). 103 Simeoni F, Morris MC, Heitz F, Divita G. Insight into the

mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res. 31(11), 2717–2724 (2003). 104 Crombez L, Morris MC, Dufort S et al. Targeting cyclin B1

through peptide-based delivery of siRNA prevents tumour growth. Nucleic Acids Res. 37(14), 4559–4569 (2009). 105 Lehto T, Simonson OE, Mager I et al. A peptide-based vector

for efficient gene transfer in vitro and in vivo. Mol. Ther. 19(8), 1457–1467 (2011). 106 Dash-Wagh S, Jacob S, Lindberg S, Fridberger A, Langel

U, Ulfendahl M. Intracellular Delivery of Short Interfering RNA in Rat Organ of Corti Using a Cell-penetrating Peptide PepFect6. Mol. Ther. Nucleic Acids 1, e61 (2012).

Cantini L, Attaway CC, Butler B, Andino LM, Sokolosky ML, Jakymiw A. Fusogenic-oligoarginine peptidemediated delivery of siRNAs targeting the CIP2A oncogene into oral cancer cells. PLoS ONE 8(9), e73348 (2013).

108 Arukuusk P, Parnaste L, Oskolkov N et al. New generation

95

Kim WJ, Christensen LV, Jo S et al. Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma. Mol. Ther. 14(3), 343–350 (2006).

97

novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids Res. 39(12), 5284–5298 (2011).

107 Ezzat K, Zaghloul EM, El Andaloussi S et al. Solid

Wang YH, Hou YW, Lee HJ. An intracellular delivery method for siRNA by an arginine-rich peptide. J. Biochem. Biophys. Methods 70(4), 579–586 (2007).

96

100 Ezzat K, Andaloussi SE, Zaghloul EM et al. PepFect 14, a

Juliano R, Alam MR, Dixit V, Kang H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 36(12), 4158–4171 (2008).

94

Zhang Y, Kollmer M, Buhrman JS, Tang MY, Gemeinhart R A. Arginine-rich, cell penetrating peptide-antimicroRNA complexes decrease glioblastoma migration potential. Peptides 58, 83–90 (2014). Van Asbeck AH, Beyerle A, Mcneill H et al. Molecular parameters of siRNA–cell penetrating peptide nanocomplexes for efficient cellular delivery. ACS Nano 7(5), 3797–3807 (2013).


Review

formulation of cell-penetrating peptide nanocomplexes with siRNA and their stability in simulated gastric conditions. J. Control. Release 162(1), 1–8 (2012). of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids. Biochim. Biophys. Acta 1828(5), 1365–1373 (2013). 109 Arukuusk P, Parnaste L, Margus H et al. Differential

endosomal pathways for radically modified peptide vectors. Bioconjug. Chem. 24(10), 1721–1732 (2013). 110 Baoum A, Xie SX, Fakhari A, Berkland C. “Soft” calcium

crosslinks enable highly efficient gene transfection using TAT peptide. Pharm. Res. 26(12), 2619–2629 (2009). 111 Baoum AA, Berkland C. Calcium condensation of DNA

complexed with cell-penetrating peptides offers efficient, noncytotoxic gene delivery. J. Pharm. Sci. 100(5), 1637–1642 (2011). 112 Baoum A, Ovcharenko D, Berkland C. Calcium condensed

cell penetrating peptide complexes offer highly efficient, low toxicity gene silencing. Int. J. Pharm. 427(1), 134–142 (2012). 113 Schmid S, Fuchs R, Kielian M, Helenius A, Mellman I.

Acidification of endosome subpopulations in wild-type Chinese


505

Review Beloor, Zeller, Choi, Lee & Kumar hamster ovary cells and temperature-sensitive acidificationdefective mutants. J. Cell Biol. 108(4), 1291–1300 (1989). 114 Serresi M, Bizzarri R, Cardarelli F, Beltram F. Real-time

measurement of endosomal acidification by a novel genetically encoded biosensor. Anal. Bioanal. Chem. 393(4), 1123–1133 (2009). 115 Wharton SA, Martin SR, Ruigrok RW, Skehel JJ, Wiley DC.

Membrane fusion by peptide analogues of influenza virus haemagglutinin. J. Gen. Virol. 69(Pt 8), 1847–1857 (1988). 116 Tamm LK, Han X. Viral fusion peptides: a tool set to disrupt

and connect biological membranes. Biosci. Rep. 20(6), 501–518 (2000). 117 Steinhauer DA, Wharton SA, Skehel JJ, Wiley DC. Studies

of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin. J. Virol. 69(11), 6643–6651 (1995). 118 Qiao H, Armstrong RT, Melikyan GB, Cohen FS, White

JM. A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype. Mol. Biol. Cell 10(8), 2759–2769 (1999). 119 Meade BR, Dowdy SF. Enhancing the cellular uptake of

siRNA duplexes following noncovalent packaging with protein transduction domain peptides. Adv. Drug Deliv. Rev. 60(4–5), 530–536 (2008). 120 Shete HK, Prabhu RH, Patravale VB. Endosomal escape: a

bottleneck in intracellular delivery. J. Nanosci. Nanotechnol. 14(1), 460–474 (2014). 121 Benjaminsen RV, Mattebjerg MA, Henriksen JR, Moghimi

SM, Andresen TL. The possible “proton sponge “ effect of polyethylenimine (PEI) does not include change in lysosomal pH. Mol. Ther. 21(1), 149–157 (2013). 122 Midoux P, Monsigny M. Efficient gene transfer by histidylated

polylysine/pDNA complexes. Bioconjug. Chem. 10(3), 406–411 (1999). 123 Chu D, Xu W, Pan R, Ding Y, Sui W, Chen P. Rational

modification of oligoarginine for highly efficient siRNA delivery: structure-activity relationship and mechanism of intracellular trafficking of siRNA. Nanomedicine doi:10.1016/j. nano.2014.08.007 (2014) (Epub ahead of print). 124 Cardoso AM, Trabulo S, Cardoso AL et al. Comparison of the

efficiency of complexes based on S4(13)-PV cell-penetrating peptides in plasmid DNA and siRNA delivery. Mol. Pharm. 10(7), 2653–2666 (2013). 125 Lo SL, Wang S. An endosomolytic Tat peptide produced by

incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials 29(15), 2408–2414 (2008). 126 Lee SJ, Yoon SH, Doh KO. Enhancement of gene delivery

using novel homodimeric tat peptide formed by disulfide bond. J. Microbiol. Biotechnol. 21(8), 802–807 (2011). 127 Won YW, Kim HA, Lee M, Kim YH. Reducible poly(oligo-

D-arginine) for enhanced gene expression in mouse lung by intratracheal injection. Mol. Ther. 18(4), 734–742 (2010). 128 Won YW, Yoon SM, Lee KM, Kim YH. Poly(oligo-D-

arginine) with internal disulfide linkages as a cytoplasmsensitive carrier for siRNA delivery. Mol. Ther. 19(2), 372–380 (2011).

506

Ther. Deliv. (2015) 6(4)

129 Kang H, Delong R, Fisher MH, Juliano RL. Tat-conjugated

PAMAM dendrimers as delivery agents for antisense and siRNA oligonucleotides. Pharm. Res. 22(12), 2099–2106 (2005). 130 Saunders LR, Barber GN. The dsRNA binding protein

family: critical roles, diverse cellular functions. FASEB J. 17(9), 961–983 (2003). 131 Eguchi A, Meade BR, Chang YC et al. Efficient siRNA

delivery into primary cells by a peptide transduction domaindsRNA binding domain fusion protein. Nat. Biotechnol. 27(6), 567–571 (2009). 132 Li H, Tsui T. Six-cell penetrating peptide-based fusion

proteins for siRNA delivery. Drug Deliv. (2014) (Epub Ahead Print). 133 Li H, Zheng X, Koren V, Vashist YK, Tsui TY. Highly

efficient delivery of siRNA to a heart transplant model by a novel cell penetrating peptide-dsRNA binding domain. Int. J. Pharm. 469(1), 206–213 (2014). 134 Geoghegan JC, Gilmore BL, Davidson BL. Gene Silencing

mediated by sirna-binding fusion proteins is attenuated by double-stranded RNA-binding domain structure. Mol. Ther. Nucleic Acids 1, e53 (2012). 135 Hogset A, Prasmickaite L, Selbo PK et al. Photochemical

internalisation in drug and gene delivery. Adv. Drug Deliv. Rev. 56(1), 95–115 (2004). 136 Matsushita M, Noguchi H, Lu YF et al. Photo-acceleration

of protein release from endosome in the protein transduction system. FEBS Lett. 572(1–3), 221–226 (2004). 137 Endoh T, Sisido M, Ohtsuki T. Spatial regulation of specific

gene expression through photoactivation of RNAi. J. Control. Release 137(3), 241–245 (2009). 138 Nam HY, Kim J, Kim S, Yockman JW, Kim SW, Bull DA.

Cell penetrating peptide conjugated bioreducible polymer for siRNA delivery. Biomaterials 32(22), 5213–5222 (2011). 139 Kim TI, Ou M, Lee M, Kim SW. Arginine-grafted

bioreducible poly(disulfide amine) for gene delivery systems. Biomaterials 30(4), 658–664 (2009). 140 Beloor J, Choi CS, Nam HY et al. Arginine-engrafted

biodegradable polymer for the systemic delivery of therapeutic siRNA. Biomaterials 33(5), 1640–1650 (2012). 141 Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo

protein transduction: delivery of a biologically active protein into the mouse. Science 285(5433), 1569–1572 (1999). 142 Tam JP. Synthetic peptide vaccine design: synthesis and

properties of a high-density multiple antigenic peptide system. Proc. Natl Acad. Sci. USA 85(15), 5409–5413 (1988). 143 Torchilin VP, Rammohan R, Weissig V, Levchenko TS. TAT

peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc. Natl Acad. Sci. USA 98(15), 8786–8791 (2001). 144 Fretz MM, Koning GA, Mastrobattista E, Jiskoot W, Storm

G. OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis. Biochim. Biophys. Acta 1665(1–2), 48–56 (2004). 145 Marty C, Meylan C, Schott H, Ballmer-Hofer K,

Schwendener RA. Enhanced heparan sulfate proteoglycan-



mediated uptake of cell-penetrating peptide-modified liposomes. Cell Mol. Life Sci. 61(14), 1785–1794 (2004). 146 Khalil IA, Kogure K, Futaki S, Harashima H. Octaarginine-

modified liposomes: enhanced cellular uptake and controlled intracellular trafficking. Int. J. Pharm. 354(1–2), 39–48 (2008). 147 Akita H, Kogure K, Moriguchi R et al. Nanoparticles for ex

vivo siRNA delivery to dendritic cells for cancer vaccines: programmed endosomal escape and dissociation. J. Control. Release 143(3), 311–317 (2010). 148 Han L, Zhang A, Wang H et al. Tat-BMPs-PAMAM

conjugates enhance therapeutic effect of small interference RNA on U251 glioma cells in vitro and in vivo. Hum. Gene Ther. 21(4), 417–426 (2010). 149 Toriyabe N, Hayashi Y, Harashima H. The transfection

activity of R8-modified nanoparticles and siRNA condensation using pH sensitive stearylated-octahistidine. Biomaterials 34(4), 1337–1343 (2013). 150 Mitsueda A, Shimatani Y, Ito M et al. Development

of a novel nanoparticle by dual modification with the pluripotential cell-penetrating peptide PepFect6 for cellular uptake, endosomal escape, and decondensation of an siRNA core complex. Biopolymers 100(6), 698–704 (2013). 151 Nam HY, Nam K, Lee M, Kim SW, Bull DA. Dendrimer

type bio-reducible polymer for efficient gene delivery. J. Control. Release 160(3), 592–600 (2012). 152 Song E, Zhu P, Lee SK et al. Antibody mediated in vivo

delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23 (6), 709–717 (2005).


Review

153 Youn P, Chen Y, Furgeson DY. A myristoylated cell-

penetrating peptide bearing a transferrin receptor-targeting sequence for neuro-targeted siRNA delivery. Mol. Pharm. 11(2), 486–495 (2014). 154 Fang B, Jiang L, Zhang M, Ren FZ. A novel cell-penetrating

peptide TAT-A1 delivers siRNA into tumor cells selectively. Biochimie 95(2), 251–257 (2013). 155 Subramanya S, Kim SS, Abraham S et al. Targeted delivery

of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production. J. Virol. 84(5), 2490–2501 (2010). 156 Won YW, Adhikary PP, Lim KS, Kim HJ, Kim JK, Kim YH.

Oligopeptide complex for targeted non-viral gene delivery to adipocytes. Nat. Mater. 13(12), 1157–1164 (2014). 157 Zhang H, Gerson T, Varney ML, Singh RK, Vinogradov

SV. Multifunctional peptide-PEG intercalating conjugates: programmatic of gene delivery to the blood-brain barrier. Pharm. Res. 27(12), 2528–2543 (2010). 158 Kim SH, Jeong JH, Ou M, Yockman JW, Kim SW, Bull DA.

Cardiomyocyte-targeted siRNA delivery by prostaglandin E(2)-Fas siRNA polyplexes formulated with reducible poly(amido amine) for preventing cardiomyocyte apoptosis. Biomaterials 29(33), 4439–4446 (2008). 159 Nam HY, Mcginn A, Kim PH, Kim SW, Bull DA. Primary

cardiomyocyte-targeted bioreducible polymer for efficient gene delivery to the myocardium. Biomaterials 31(31), 8081–8087 (2010). 160 Bender E. The second coming of RNAi. Scientist 28(9),

52–57 (2014).


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Protease-triggered siRNA delivery vehicles.

Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas.

Cationic lipid nanodisks as an siRNA delivery vehicle.

Amylose-Based Cationic Star Polymers for siRNA Delivery.

Design and development of effective siRNA delivery vehicles.

Glycol chitosan nanoparticles as specialized cancer therapeutic vehicles: sequential delivery of doxorubicin and Bcl-2 siRNA.

Cells as delivery vehicles for cancer therapeutics.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Aptamers as drug delivery vehicles.

DNA-carbon dots function as fluorescent vehicles for drug delivery.

Functional Delivery of siRNA by Disulfide-Constrained Cyclic Amphipathic Peptides.

Molecular imprinted polymers as drug delivery vehicles.

Exosomes as drug delivery vehicles for Parkinson's disease therapy.

Lignin nanotubes as vehicles for gene delivery into human cells.

pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery.

Redox-responsive, reversibly-crosslinked thiolated cationic helical polypeptides for efficient siRNA encapsulation and delivery.

Polyene-based cationic lipids as visually traceable siRNA transfer reagents.

Cationic Polymer Modified Mesoporous Silica Nanoparticles for Targeted SiRNA Delivery to HER2+ Breast Cancer.

Delivery materials for siRNA therapeutics.

Polymer-based vehicles for therapeutic peptide delivery.

Combined delivery of BCNU and VEGF siRNA using amphiphilic peptides for glioblastoma.

A role for peptides in overcoming endosomal entrapment in siRNA delivery - A focus on melittin.

pectin nanocomplexes as potential oral delivery vehicles.

Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome.