Journal of Controlled Release 161 (2012) 582–591

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Journal of Controlled Release

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Review

Cell entry of cell penetrating peptides: tales of tails wagging dogs

Arwyn T. Jones ⁎, Edward J. SayersCardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, Wales, CF10 3NB, United Kingdom

⁎ Corresponding author. Tel.: +44 2920876431; fax:E-mail address: jonesat@cardiff.ac.uk (A.T. Jones).

0168-3659/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.jconrel.2012.04.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 March 2012Accepted 2 April 2012Available online 10 April 2012

Keywords:Cell penetrating peptides (CPPs)FluorophoresPhotostimulationPlasma membranePeptide therapeutics

Cell penetrating peptides hold considerable potential for academic and pharmaceutical remits with an inter-est in delivering macromolecules to the insides of cells. Hundreds of sequences now fall within the cell pen-etrating peptide classification and HIV-Tat, penetratin, transportan, and octaarginine represent extensivelystudied variants. The process by which membrane translocation is achieved has received significant interestin an aim to exploit new mechanistic knowledge to gain higher efficiency of penetration. There is evidencethat many of the most well studied peptides are able to deliver themselves, relatively small cargo and possi-bly large macromolecular structures directly across the plasma membrane but there is also support for theinvolvement of an endocytic pathway or pathways. This review focuses on recent findings relating to exper-imental protocols and cell penetrating peptide modifications or extensions that yield significant effects onpenetration capability. Relatively small changes in extracellular peptide concentrations, the inclusion or ab-sence of serum from the incubation medium and the in vitro model exemplify variables that significantly in-fluence the capacity of CPPs to penetrate membranes. Attachment of any type of cargo to these entities hasthe potential to affect their interaction with cells. There is increasing evidence to suggest that this is truefor relatively small molecules such as fluorescent probes and hydrophobic adducts such as lipids and shortpeptide sequences designed as peptide therapeutics. Information gained from these findings will improveour knowledge of, and capacity to study the interactions of CPPs with cells, and this will accelerate theirtranslation as efficient vectors from the in vitro setting into the clinical arena.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction and CPP classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5832. Well studied CPPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5833. Methodological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

3.1. Artefacts of fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5843.2. Effective peptide concentration for cell interaction and entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

4. CPP membrane effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5854.1. Peptide concentration and membrane disruption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5854.2. Membrane repair responses and other downstream effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5854.3. Cell surface sugars and CPP interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586

5. Influence of fluorophores and other detection systems on CPP uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5865.1. Fluorophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5865.2. Photostimulation in CPP mediated delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5875.3. Alternative labelling of CPPs for cell uptake studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587

6. CPPs as vectors for peptide therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5876.1. Peptide cargo effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587

7. Hydrophobicity and CPP uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5887.1. Hydrophobic terminals enhancing cell entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5887.2. Influence of tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5887.3. Lipidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588

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583A.T. Jones, E.J. Sayers / Journal of Controlled Release 161 (2012) 582–591

8. Cysteine and disulphide bridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5889. Fusogenic extensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589

10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589

Table 1Peptides described in this review.

Peptides Sequence Ref.

1 PFVYLI PFVYLI [7]2 Kaposi FGF

derivedAAVALLPAVLLALLAP [8]

3 CADY GLWRALWRLLRSLWRLLWRA [9]4 Mastoparan INLKALAALAKKIL [10]5 MAP KLALKLALKALKAALKLA [11]6 TP10 AGYLLGKINLKALAALAKKIL [12]7 Transportan GWTLNSAGYLLGKINLKALAALAKKIL [13]8 GALA WEAALAEALAEALAEHLAEALAEALEALAA [14]9 pHLIP AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT [15]10 KALA WEAKLAKALAKALAKHLAKALAKALKACEA [16]11 Mellittin GIGAVLKVLTTGLPALISWIKRKRQQ [17]12 MPG GALFLGFLGAAGSTMGAWSQPKS [18]13 pVec LLIILRRRIRKQAHAHSK [19]14 Penetratin RQIKIWFQNRRMKWKK [20]15 Pep-1 KETWWETWWTEWSQPKKKRKV [21]16 PasR8 FFLIPKGRRRRRRRR [22]17 R9F2 RRRRRRRRRFFC [23]18 Maurocalcine GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR [24]19 SAP(E) VELPPPVELPPPVELPPP [25]20 R7W RRRRRRRW [26]21 HIV-1 Rev TRQARRNRRRRWRERQR [27]22 HIV-Tat GRKKRRQRRRQ [28]23 R8 RRRRRRRR [29]24 PepFect 14a Stearyl-AGYLLGKLLOOLAAAALOOLLb [30]

CPP-peptidechimaeras

Sequencec

25 IKKγ NBDd DRQIKIWFQNRRMKWKKTALDWSWLQTE [31]26 Tat-HA2 RRRQRRKKRGGDIMGEWGNEIFGAIAGFLG [32]27 NFκB p50d DRQIKIWFQNRRMKWKKVQRKRQKLM [8,33,34]28 R8-PAD RRRRRRRRGGklaklakklaklak [35]29 D-NuBCP-9-r8a,d fsrslhsllG-Ahx-rrrrrrrre [36]

a Peptides not plotted in Fig. 1 due to the presence of non-natural amino acids.b O represents ornithine.c Cargo is underlined in sequence with CPP italicised, lower case denotes D-form

amino acids.d Commercially available.e Ahx represents 6-aminohexanoic acid (aminocaproic acid).

1. Introduction and CPP classification

“Do not hover always on the surface of things, nor take up sudden-ly, withmere appearances; but penetrate into the depth ofmatters,as far as your time and circumstances allow, especially in thosethings that relate to your profession.” Issac Watts (1674–1748)

As witnessed by literature citation, the profession of an increasingnumber of researchers is in attempting to utilise the membranebreaching properties of cell penetrating peptides (CPPs) to deliver apayload into cells. The term CPP is widely used to describe peptide se-quences, usually less than 30 residues, that have a proven capacity todo this. The alternative terminology of protein transduction domain(PTD) is also used and reflects the fact that early variants were de-rived from proteins. One notable example of this is the HIV-Tat pro-tein whose internalisation into cells, uptake by endocytosis,endosomal escape, translocation into the nucleus and binding toRNA to act as a transcription activator is largely mediated by its trans-duction domain. Quite a feat for a short peptide of less than 20 resi-dues; the protein itself has only 101 residues but over 250identified binding partners [1]. Membrane translocating sequence(MTS) is another nomenclature candidate for these peptides and, asnoted in 2003 [2], this variety in terminology has led to quite some con-fusion, which unfortunately continues today.

The most appropriate definition and nomenclature for these pep-tides will be the subject of debate for time to come and here we sidewith the majority and, unless specified, use the CPP terminology.There is, however, some room for discussing whether sequencessuch as the insect venoms melittin and mastoparan [3,4] and thepHLIP peptide (Table 1), which has membrane spanning features atlow pH, could be classed as CPPs. Antimicrobial peptides also sharetheir physical characteristics and possible mechanism of actionwith CPPs [5,6]. The field of CPPs as a terminological remit is still inits infancy and only celebrated its 20th anniversary in 2008. Studieson antimicrobial peptides and other membrane active peptides,that could equally be classified as CPPs, stretch back much furtherthan this.

Hundreds of different peptide sequences that can be broadlygrouped together as CPPs have now been described and examplesare given in Table 1. All of them have been shown to deliver someform of cargo into cells and these range from small molecules suchas fluorescent probes (b1 kDa) to much larger microparticles [37].In between these are thousands of different bioactive entities suchas peptides, proteins and nucleotides that for therapeutic purposesneed to be delivered into cells or defined subcellular compartments.The CPP may be covalently conjugated to the payload or interactwith it via electrostatic forces and may also be used to aid deliveryof an alternative vector such as a polymer, virus or liposome. Of par-ticular attraction is the fact that the CPP not only aids in initial uptakeinto cells but also has the capacity to promote endosomal escape. Es-cape is important if the payload target, as it most often the case, liesoutside the confines of the endolysosomal system. Thus, here, theCPP acts as a bifunctional entity influencing uptake and facilitatingendosomal escape. It may, however, be the case that CPP payloads ob-served or functioning beyond the endosomal systemmay have arrivedvia direct CPP-mediated translocation across the plasma membrane

without a requirement for endocytosis. A wealth of recent review litera-ture is available on the applications of CPPs for delivering bioactivecargo [38–45] and there is also increasing evidence of their applicationas delivery systems for imaging purposes, especially in the remit of cancerbiology [46]. Their potential for delivery extends beyond the mammalianremit as they have also shown promise in delivering payloads into bacte-ria, plants and nematode worms [47–49].

2. Well studied CPPs

Of the CPPs described only a few have been extensively studiedand these include the HIV-Tat peptide, penetratin (Antp), transportanand its truncated variant TP10, pVec, MAP and also repeating units ofarginine residues, most notably R8 and R9 (Table 1 and referencestherein). Basic residues are prominent in these eight variants andone of the most well studied, octaarginine or R8, is extremely cationic.For oligoarginines optimal cell uptake was noted with lengths be-tween seven and nine residues, however, as the number of residuesincreases or decreases from this number uptake efficiency wasshown to be reduced [50,51]. The importance of the ability of arginine

Fig. 1. Calculated hydrophobicity and absolute charge of selected peptides fromTable 1. The addition of a cargo peptide can significantly alter the properties of CPPs(shown as an arrow). For example, addition of the (klaklak)2 sequence to R8 (23) toform R8-PAD (28) increases the hydrophobicity of the peptide and its absolute charge,whilst the addition of HA2 (26) to Tat (22) reduced its absolute charge while increas-ing its hydrophobicity. In contrast, there is effectively only a change in the absolutecharge with both the IKKγ (25) and NFκB (27) peptides from penetratin (14). A changein peptide properties after the addition of a cargo sequence may produce significantdifferences in the peptides' overall uptake profile (Section 6.1). Properties calculatedusing Bio-Synthesis' peptide property calculator.

584 A.T. Jones, E.J. Sayers / Journal of Controlled Release 161 (2012) 582–591

to form bidendate hydrogen bonds has been described for some timeand recent data with HIV-Tat peptide show that cyclisation of thepeptide to maximise arginine contacts with the plasma membranecan enhance the capacity for translocation [52].

Most of the well studied CPPs have cationic prominence but in CPPsfrom natural systems, rather than synthetic constructs, the cationic res-idues are often separated by hydrophobic residues. In some cases thesealternations between cationic and hydrophobic residues predispose theCPPs to form amphiphilic helices in aqueous solutions. Hydrophobicity(Section 7) is, in many cases, important for cell penetration and indeedsome CPPs are extremely hydrophobic and completely devoid of posi-tive charge. Examples are the PFDYLI peptide [7,53] and a much longersequence derived from Kaposi Fibroblast Growth Factor [8] (Table 1).An anionic CPP termed SAP(E) has also recently been described andshown to deliver fluorophores into a range of cell lines [25].

For those peptides that have been studied in detail there is stillsome controversy regarding almost all aspects of their cellular dy-namics from initial binding to the cell periphery, whether this be tocarbohydrate or lipid, to the mechanism by which they then gain ac-cess to the cell interior, either through an endocytic mechanism or di-rectly across the plasma membrane. Also largely undetermined isexactly what happens to them when they are inside the cell, andhow cells manage them once they reach the cytosol and intracellularorganelles such as the nucleus.

A number of pathogens, and pathogen proteins, first gain access tocells through an endocytic pathway and utilise a feature of endo-somes, such as low pH, to change conformation, fuse with the endoly-sosomal membrane and gain access to the cytosol. Based on this fact itis widely believed that a CPP attached to a large cargo, such as a pro-tein, will do the same. Gaining absolute proof of this mechanism isdifficult, as measuring endosome escape using real-time measure-ments is a major technological challenge. There is clear proof thatCPP-protein variants do gain access to the cytosol and nucleus, andthis is most often based on a readout that is measured several hoursfollowing addition of the CPP-payload to the cellular set up. However,the controversial possibility exists that if a CPP peptide can translo-cate across the endosomal membrane it can also do the same acrossthe plasma membrane. Assays for measuring plasma membrane pen-etration or/versus endosomal release also represent a major techno-logical challenge, especially when considering that only a very tinyfraction of peptide placed on the cell, or even entering the cell via anendocytic mechanism, may actually escape to the cytosol. Whether en-docytosis is necessary in the uptake of CPPs, what endocyticmechanismis favoured and how CPPs can escape from the endosome, if indeed they

do, remain the subjects of debate and are largely unresolved [54–56].The cell biology field is consistently revealing new endocytic uptakeroutes [57,58] but the identification of specific probes for each pathway,andmethods for only attenuating individual pathways, is often a slowerprocess and this currently hinders studies aimed at identifying specificendocytic pathways used by CPPs and other drug delivery vectors.

Generally, it has been difficult to show a clear relationship betweenthe amino acid sequence, the mechanism of action and penetration ca-pacity of CPPs. The literature does, however, highlight that seeminglysmall changes in experimental set up or CPP sequences can have pro-found influences on the cellular dynamics of these entities; aspects ofthese are covered in the following sections.

3. Methodological parameters

3.1. Artefacts of fixation

It is true to say that the CPP field shuddered somewhat with the re-alisation that the abilities of fluorescently labelled peptides to entercells with such high efficiency, and especially enrich in the nucleus tosuch extent, was due to cell fixing being part of the methodology[59,60]. Prior to these findings the literature was heavily inclined to-wards the notion that uptake was occurring through an energy andthus endocytic-independentmechanism. In the aftermath of these find-ings endocytosis gained higher prominence and the peptide that waslocated in the cytosol was gaining initial access through endocytosisand translocation across endocytic membranes. For these experimentsthe cells were not fixed prior to microscopy and flow cytometry andfor the later technique they were washed with heparin and trypsinisedto remove the plasma membrane fraction enabling a more accurate as-sessment on the amount of peptide that actually enters the cell. This isparticularly important for the cationic variants and are now establishedprocedures in this remit. The possibility does, however, exist that pep-tides embedded in the membrane may be resistant to trypsin, whilstcleavage of plasma membrane bound D-form peptides may not occurvia trypsinisation.

3.2. Effective peptide concentration for cell interaction and entry

For fluorescence based uptake studies, labelled CPPs are typicallyplaced on cells at extracellular concentrations ranging from 1 to50 μM. The experiment may be done in the same media that is used toculture the cells; i.e. containing serum. After addition to the experimen-tal setup, cationic variants of CPPs can indiscriminately associate withthe nearest negative charge they encounter, for example, to serum pro-teins such as albumin [61] (Fig. 2). Serum bound CPPs can significantlyreduce cellular uptake as the effective concentration of the free or activepeptide available to interact with the cell is, essentially, reduced. In ad-dition, peptides associated with a protein that is internalised via a re-ceptor mediated endocytic pathway, or via pinocytic fluid phaseuptake, could use this as a mechanism of cell entry.

As the vector, payload or both (e.g. CPP-siRNA) are protease or nu-clease sensitive, uptake assays are often performed in media depletedof serum thus giving the delivery system an extra chance of reachingthe cell surface as an intact entity. CPPs, to different extents, are sensi-tive to degradation [62], thus in vitro experiments can be performed,and often are, in the absence of serum. Rapid degradation of bothMAP and penetratin was observed such that after a 30 min incubationwith cells only 25% of intact peptide remained available for cell entry[63]. Comparison of MAP and penetratin in CHO cells showed bothpeptides to degrade rapidly, with half lives of 10 and 5 min respective-ly. However, in the presence of bovine serum albumin, MAP showedreduced degradation whilst penetratin continued to degrade at thesame rate. CPP uptake levels were also found to be dependent on pep-tide to cell ratios as opposed to concentration alone [64].

Fig. 2. Cell interaction and entry of CPPs. Internalisation of CPPs has been shown tooccur through a variety of mechanisms including macropinocytosis (1) and otherendocytic pathways (2) giving rise to endosomal labelling (3). This may be initiatedby peptide mediated plasma membrane reorganisation either via binding to a plasmamembrane structure or from within the cell itself via an interaction with actin (4).Once inside cells, CPPs have been shown to accumulate in different compartmentssuch as lysosomes or nucleus (5). Cytoplasmic labelling from CPPs may occur throughendosomal escape (6) or direct translocation (7). External factors affecting the uptakeof CPPs include serum proteins (8) and membrane sugars (9).

Fig. 3. Endocytosis and direct translocation of octaarginine-Alexa488. After 1 h of ex-tracellular exposure to 2 μM R8-Alexa488, acute myeloid leukaemic KG1a cells inter-nalise the peptide forming punctuate structures typical of endocytic labelling. Athigher concentrations evidence of endocytic uptake remains but this is overshadowedby diffuse cytosolic labelling in some cells suggesting that this fraction has entered thecells independent of endocytosis. DIC, direct interference contrast of the imaged cells,scale bar is 10 μm.

585A.T. Jones, E.J. Sayers / Journal of Controlled Release 161 (2012) 582–591

4. CPP membrane effects

4.1. Peptide concentration and membrane disruption

It is now widely accepted, under certain experimental conditions,that some CPPs can translocate directly across the plasma membraneof cells. CPP mediated toxicity, manifested as a general increase in plas-mamembrane permeability, could explain some of these observed fea-tures. A detailed study with oligoarginines not only showed superiorityin uptake of the D-form over the L-form of the peptide but alsohighlighted a narrow concentration window between effective cellentry of these CPPs and concomitant entry with propidium iodide, anormally membrane impermeable dye [65]. Studies with leukaemiacells showed that at 2 μM extracellular concentration R8-Alexa488was seen to label vesicular structures shown to be endosomes and lyso-somes [66,67]. Upon raising the concentration to 10 μM the peptidewas seen to flood into cells giving, in most cases, uniform labellingthroughout the cytoplasm and nucleus (Fig. 3). However, as shown inthis figure there is some heterogeneity within the cell population assome cells at 10 μM clearly show evidence of both vesicular and cyto-solic labelling. At these higher peptide concentrations propidium iodidedoes not enter the cells [68]. Increasing the peptide concentration fur-ther is required to observe the same phenotype in adherent cells lines,omitting serum from the medium also has the same effect [68].

These findings were supported by others showing predominantlyvesicular labelling of Tat and R9 in adherent HeLa cells at 10 μM con-centration but showing extensive cytosolic labelling at 20 μM [69].Penetratin, however, was still largely confined to vesicles at 20 μM.Together this suggests that the more cationic variants have a highercapacity to translocate across the plasma membrane, as endocytic up-take and subsequent endosomal escape would unlikely result in suchstrong cytosolic labelling at these relatively short (30 min) timepoints. For R9, however, it was suggested that the peptide, at thesehigh concentrations, may cause localised membrane disruption ordamage, as opposed to overall cell leakage, and that this injury or nu-cleation zone serves as a portal for peptide influx that later spreadsthroughout the cytosol [69]. This needs further characterisation as itis not a feature of CPP entry in all cell lines [68]. The possibility exists,therefore, that CPPs under certain conditions and concentrationscould be membrane damaging though not to the same extent as nota-ble membrane disrupting agents such as mastoparan (Table 1).

Retro-inverso forms of CPPs and/or CPP cargo have been widelyinvestigated in an attempt to determine the specificity of uptake orintracellular targeting. Recently significant cell toxicity of someretro-inverso CPPs, including penetratin, was observed over the par-ent peptide highlighting some of the problems of finding suitablecontrol sequences for CPP mediated effects and this is further dis-cussed in Section 6.1. The possible mechanism by which this directtranslocation occurs, has been the focus of much study in cells, artifi-cial membranes and using molecular modelling [70].

4.2. Membrane repair responses and other downstream effects

The possibility that direct translocation accross the plasma mem-brane was a feature of CPPs was clearly evident from earlier studieswith R7, Tat and penetratin [26,71,72]. If the peptides are penetratingvia the generation of plasma membrane pores, it may explain whycells treated with some CPPs induce the services of repair vesiclesfrom the cytoplasm to subsequently plug the damage [73]. This repairmechanism is characterised to occur in response to a rise in intracel-lular calcium as would be expected from membrane damage, thus,some of the effects we see from treating the cells with different CPPentities may well be an attempt at membrane-recovery. The factthat they may also promote other effects, from enhanced internalisa-tion of the TNF receptor [74] to inhibition of proteasome activity [75]also needs further analysis and consideration [76]. Genome wideanalysis and proteomics would likely reveal other unforseen CPP ef-fects that may be looked at in both positive and negative perspectives.

There is a great deal of variation between different cell lines to CPPinduced cytotoxicity and from the limited studies that have looked indetail at this aspect it would appear that non adherent cell lines aremore sensitive [27,77]. This could also explain why some CPPs cantranslocate across their plasma membranes at lower concentrationscompared with adherent cells [68]; possibly a manifestation of thefact that the entire surface of non adherent cells are equally exposedto peptides. These types of cells are also noted for allowing the interna-lisation of CPPs to label the cytosol when experiments are performed onice, thus removing endocytosis form the equation [66,67]. Energy inde-pendent uptake was previously, and has recently been, witnessed inother cell lines with other CPPs [35,72,78].

Fig. 4. Structure and hydrophobicity of fluorophores and CPP additions. (A) Structureand hydrophobicity of parent fluorophores (–R=–H) and when attached via malei-mide (M), succinimidyl ester (SE) and isothiocyanate (ITC) functional groups. (B)Structure and labelling reactions of the three functional groups with an unspecifiedpeptide. (C) Structure and hydrophobicity of other CPP modifications discussed inthis review. LogP and LogD7.4 calculated computationally using Marvin (version 5.8.02012) ChemAxon.

586 A.T. Jones, E.J. Sayers / Journal of Controlled Release 161 (2012) 582–591

4.3. Cell surface sugars and CPP interaction

Aside from binding to serum proteins, sequestration of CPPs mayalso be mediated by negatively charged carbohydrate such as heparansulphate proteoglycans (HSPGs) located on the plasma membrane(Fig. 2). These may serve as electrostatic traps for the cationic variantsand this could reduce the amount of peptide that can then reach theouter lipid leaflet of the plasma membrane. This has been demon-strated using the Tat peptide incubated with Chinese hamster ovary(CHO) cells or a mutant cell line (CHO pgsB-618) that has no galacto-syltransferase activity and does not synthesise any glycosaminogly-cans [79]. In contrast to these findings, cells with defects in HSPGsynthesis have been shown to have a reduced capacity to internalisecationic CPPs and indeed the full length HIV-Tat protein [80–82]. Itis, however, important to note that pgsB-618 cells or a commonlyused alternative CHO pgsA 745, have not been fully characterised interms of their endocytic properties or the organisation of their plasmamembrane. Any dissimilarity in the endocytic mechanism may influ-ence CPP uptake beyond the most notable difference of cellular ex-pression of surface sugars. A requirement for HSPG for cell uptakesuggests that the peptides can be internalised whilst bound to thesemolecules and/or that binding itself enhances their endocytosis. Sur-face sugars have also been suggested to play a role in the cell uptakeof other CPPs including the Maurocalcine sequence from scorpionvenom [83,84]. This is an unusually long CPP (Table 1) that hasthree disulphide bridges producing a peptide with tertiary structure.

There is evidence to suggest that some CPPs, under specific experi-mental conditions, can induce membrane ruffling and macropinocyto-sis [32,80,85] and this may be achieved through binding to surfacesugars. The HIV-Tat-Cre chimaeric protein induces GFP expression, viarecombination between two loxP sites, if it gains access to its designedregion in the cell nucleus [86]. The capacity of the chimaera to do thiswas the same in control CHO cells and the pgsA 745 variant suggestingthat these surface sugars were not essential for penetration. For pene-tratin entry, the involvement of cell surface carbohydrate was shownto be verymuch a function of peptide concentration [87]. At 1 μMextra-cellular concentration there was no difference in uptake between con-trol cells and variants deficient in expression of glycosaminoglycans(GAGs) and sialic acids.Whilst at 10 μM, cells deficient in GAGs showedreduced accumulation of the peptides but, in contrast, sialic acid nega-tive cells had slightly higher levels of intracellular peptide. Three ofthe most well studied CPPs, R8, HIV-Tat and penetratin also show affin-ity for the cell surface receptor SDC4 (syndecan-4). Over-expression ofsyndecans in chronic myelogenous leukaemia cells provided an in-crease in the uptake of these peptides byflow cytometry [88]. Negative-ly charged proteoglycan heparin has been shown to interfere with theelectrostatic interaction between MPG and its oligonucleotide cargoDNA [89]. It is therefore likely that cell surface sugars have distinctroles to play in both binding and uptake of at least some CPPs butthey could also influence the interaction of CPPs with their cargo.

5. Influence of fluorophores and other detection systems on CPPuptake

5.1. Fluorophores

Most CPP cell uptake studies have relied on covalent linkage of thepeptide to a fluorophore; notable fluorophore examples are shown inFig. 4. As shown, several of these have very different structures andthus physical characteristics. Alexa dyes are gaining increasing popu-larity for a number of applications, including peptide labelling, andthe available variants now span from the UV to the far-red and infra-red range (790 nm). Of note, however, is that despite having the sameAlexa prefix they differ greatly in structure and physical properties(Fig. 4). For example, Alexa568 has a six ring structure that sharessimilarities with Texas Red, whereas Alexa647 has two indole rings

separated by a long alkene chain and is much less hydrophobic. Thepossibility exists that the fluorophores can differentially contributeto CPP uptake either negatively or positively. Fig. 4 highlights valuesrelating to their calculated partition coefficients (LogP, the hydropho-bicity independent of pH, calculated using an non-ionised compound)and diffusion coefficient (LogD7.4, hydrophobicity dependent uponpH, here measured at pH 7.4, taking into account the ionisation statusof the compound). Both these measurements are important parame-ters to take into account when fluorophores are attached to peptidesto investigate membrane dynamics in either artificial membranes, oncells in vitro or for in vivo studies. Fluorophores will also differ in theirexcited-state lifetimes [90], sensitivity to photobleaching and also theirpH sensitivitieswhich can be affected by their immediate aqueous envi-ronment. The later is particularly important for endocytosis studies thatrequire quantification of fluorescence in endocytic compartments (pH6.5–5.0) and lysosomes where pH can be as low as 4.5. The quantumyield of FITC is inhibited by up to 70% at lysosomal pH [91]. Additionaleffects may occur from the linker used to attach a fluorophore to theCPP. Linkers such as succinimidyl esters, provide a short link betweenthe fluorophore and amine groups on the peptide whilst maleimidesprovide a longer link between the fluorophore and peptide cystienes(Fig. 4). Interestingly, maleimides produce a more hydrophobic fluoro-phore than succinimidyl esters but whether this has any effects is yet tobe determined.

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The influence of thefluorescent probewas directly assessed in humancolon carcinoma Caco-2 cells incubated with the cell penetrating se-quence Dmt-D-R-F-K (Dmt, 2′,6′-dimethyltyrosine) [92] (Table 1). Theterminal lysine residue was replaced with relatively small amino acidbased fluorophores ß-dansyl-L-α,ß-diaminopropionic acid (DNS) or ß-anthraniloyl-L-α,ß-diaminopropionic acid (ATN, Fig. 4). The choice offluorophores was unusual, compared with the majority of CPP studies,but the data highlighted different subcellular localisations of the two pep-tides, with the DNS form causingmitochondrial swelling and cell death. Amore recent study investigated the effects of adding a fluorophore to thesynthetic R6 sequence and then analysing its interactionwith giant unila-mellar vesicles (GUVs) [93]. The peptide alone at specific peptide/lipidmolar ratios was membrane disruptive allowing rapid escape of a mem-brane impermeable dye from inside the GUV. However, under identicalexperimental conditions, addition of FITC to R6 or extending the sequencewith a single tryptophan residue (R6W) changed the peptide–membraneinteraction and the dye was gradually released through the formation ofsmall poreswithout accompanyingmembrane damage. In a different set-ting these pores could presumably accommodate small cargo such asshort peptide sequences attached to these alteredCPPs butwould excludelarger cargo such as whole proteins. As mentioned in Section 4.1, studieswith R8- and Tat-Alexa488 show that direct penetration across the plas-ma membrane is clearly observed in cells but it is still disputed as towhether this is due to pore formation, some other form of direct translo-cation across the entire surface of the plasma membrane or whether nu-cleation zones are required. Amodified 19Fluorine labelled phenylalanine(4-trifluoromethyl-D-Phe) was used to analyse uptake of D-FR8F andD-FITC-GABA-FR8F labelled peptides. Here FITC-GABA increased the up-take of the peptide and this was attributed to its hydrophobic nature [94].

Cationic CPPs can act in synergy with certain fluorophores to influ-ence their interactions with membranes. For example, tetramethylrho-damine (Fig. 4) conjugated to cationic CPPs damages membranesthroughout the cell, as opposed to just those confined to endosomalmembranes. Intriguingly this was confined to CPPs rich in arginines(HIV-Tat and R9) whereas K9 attached to the same fluorophore hadno effect [95]. This was deemed to be due to the production of singletoxygen and, as stated by the authors, this may offer opportunities formore refined disruption without the general cellular damage. It wouldbe interesting to determine whether arginine induced membrane cur-vature has an additional role in thismembrane damage as the bidentatebinding of its guanidinium group to negatively charged phosphate headgroups causes curvature, whereas themonodentate amine groups of ly-sine do not [93].

5.2. Photostimulation in CPP mediated delivery

Illuminating CPPs attached to a photosensitiser, such as fluores-cien, in cells via confocal microscopy was shown to cause extensiveredistribution of fluorescence from vesicular structures into the cyto-sol [96]. This cautions against using similar methods to study endoso-mal escape in general but again this feature of CPP, fluorophore andlight could be exploited for delivering therapeutics into the cytosoland beyond. As an example, a rhodamine labelled Tat-GFP constructwas shown to be able to increase cytosolic levels of the CPP after ex-posure to light via a process known as PCI (photochemical internali-sation). Here, photostimulation of rhodamine generated oxygenspecies damaged the vesicle membrane and released the Tat-GFPinto the cytosol [97]. In another study, a construct containing Tat-Alexa546 was photostimulated to deliver siRNA into cells [98].

5.3. Alternative labelling of CPPs for cell uptake studies

Radiolabelled peptides can also be used to monitor cell uptake,thus eliminating fluorophore artefacts [62], but information regardingthe subcellular location of the peptide is then more difficult to obtain.A procedure based on affinity purification of biotin labelled peptides

followed by avidin based isolation and mass spectroscopy has alsoemerged [99]. Here the peptides are labelled with an N-terminal ex-tension biotin-Gly-Gly-Gly-Gly that allows for post experimental iso-lation with streptavidin beads followed by MALDI MS analysis againstcalibration with a deuterated form of the peptide. This has yielded in-teresting data that often challenges results obtained with fluorescentpeptides [78,87,100]. Biotin is itself a hydrophobic entity (with a hy-drophobicity between Alexa488 and TAMRA) and receptors andtransporters for this molecule have been identified and indeedexploited for drug delivery [101–104]. It may therefore also be thatthis type of CPP modification could also have its drawbacks. Identify-ing a method that is free of a requirement for CPP modification is achallenge. The fact that the main interest in CPPs lies in their capacityto deliver cargo suggests that information with these labelling probeshas value provided that there is not too much emphasis placed on in-dividual data sets with respects to the uniformity of CPP interactionswith cells.

6. CPPs as vectors for peptide therapeutics

Peptides have huge potential as intracellular targeted entities forthe academic and pharmaceutical setting. Often peptide design isbased on an understanding of protein–protein interaction and, withvery high affinities and specificities, peptides can modulate biologicalresponses when introduced into cells. A fundamental problem arisesfrom the fact that the vast majority cannot gain access to intracellulartargets without vector support; herein lies a golden opportunity forCPPs [105–107]. Of the hundreds of manuscripts that have appearedshowing CPP-peptide delivery potential, the translation of this prom-ise to clinical trials has been slow and this may be due to the fact thatit is difficult to tame the CPP to target a defined (cancer) cell withinthe body [108]. There is evidence of promise as some CPP-peptidechimaeras such as KAI-9803 (δ-V1-1) targeting PKCδ to alleviatepain and XG-102 (DJNKI-1) against hearing loss and stroke[43,109–111] have reached clinical trials. For diseases such as cancer,the potential is enormous as more proteins are implicated and theirfunction is now understood at the level of their specific interactionswith other biological entities [105,112]. Peptides are ideal candidatesfor mimicking these specific protein interactions to alter cell behaviour.

6.1. Peptide cargo effects

As fluorophores affect CPP dynamics within cells then the poten-tial also exists that any peptide sequence attached to a CPP couldhave an influence. This was clearly highlighted when the PKI peptide(TYADFIASGRTGRRNAI), that inhibits protein kinase A, was linked towell studied CPPs, penetratin, transportan, oligoarginine (R11) andHIV-Tat and their effects were observed in a range of cancer celllines. Addition of the targeting sequence to R11 significantly reducedthe capacity of the CPP to enter cells but the reverse was observedwhen the same cargo was attached to transportan [113]. The studyalso highlighted significant non-specific toxicity when CPPs in the ab-sence of cargo were incubated with cells. In another study, the shortnine residue sequence derived from the Bcl-2 interacting portion ofNur77 was shown, conjugated to D-R8, (Table 1) to change the be-haviour of Bcl-2 from being a guardian of the cell and protectorfrom apoptosis to being a cell killer [36]. Mutating the first and lastresidue of D-NuBCP-9-r8 (or BCl-2 converted peptide) cargo to an al-anine led to loss of activity providing a useful control peptide. Laterexperiments, however, demonstrated that loss of the N-terminal phe-nylalanine on the cargo had dramatic effect on the initial interactionof this peptide with the plasma membrane, its uptake into cells andpromotion of Bcl-2 independent cell death [114].

Sequence (KLAKLAK)2, also known as the proapoptotic domain(PAD) peptide, was initially identified as an antimicrobial peptidewith an MIC of 6 μM for Gram positive and negative bacteria [115].

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It lacked toxicity against mammalian cells at >500 μM but was latershown to induce cell death when conjugated to tumour homing pep-tides [116]. Its mechanism of action was proposed to be via mitochon-drial damage but there was little information available regarding themechanism by which it reached the cytosol of targeted cells. To im-prove cell entry (KLAKLAK)2 has been conjugated to CPPs includingPTD-5, penetratin, R6W3 and r7/r8, giving cell toxicity in the low mi-cromolar range [35,117–119]. Analysing cells immediately followingexposure to r8-(KLAKLAK)2 highlighted that this peptide caused ex-tensive plasma membrane reorganisation within 10 min and thatthis was closely followed by increased permeability to propidium io-dide [53]. Co-addition of r8 and (KLAKLAK)2 as single entities wasnon-toxic. Interestingly, the inclusion of a glycosylated peptide be-tween CPP and PAD significantly reduced the capacity of the CPP todeliver the cargo and induce cell death [117].

There are now a number of commercial CPP-peptide chimaeras forintracellular targeting and examples of two that are designed to in-hibit the NFκB pathway are shown in Table 1. A simple glance at thecargo sequences suggests that they could influence the penetrationcapacity of the CPP vector, in this case penetratin. Stringent controlmeasures are therefore required with these and other CPP-peptidechimaeras to assess specificity and this is unlikely to be provided byusing the CPP sequence alone or even scrambled cargo sequences.Mutations in the cargo are likely to provide answers with respect tobinding in cell free assays but as highlighted a small modification insequence could also have profound effects on cellular interaction.Retro or retro-inverso forms are alternatives but, as described earlier,data with these forms also need to be interpreted with caution. It maybe that the control setting is better placed, if possible, at the cellularlevel via, for example, modification of expression or activity of thetarget protein or pathway.

7. Hydrophobicity and CPP uptake

7.1. Hydrophobic terminals enhancing cell entry

A number of studies have shown that placing hydrophobic residues,such as a phenylalanine, at either N- or C-terminus of CPPs can enhancemembrane interaction. The effect of the inclusion of an F residue toD-NuBCP-9-r8 was highlighted in Section 6.1. In line with thesestudies are those showing that addition of FF to the C terminus of R9has significant effects on its delivery capacity [120,121]. Elongation ofthe N-terminus of R8 with a so-called penetrating accelerating se-quence (FFLIPKG) to form PasR8 (Table 1) increased the uptake ofAlexa488 and of note was the fact that cytosolic and nuclear labellingwas extremely prominent suggesting that it was penetrating directlyacross the plasma membrane [22]. Whether the full seven residuesare required for this enhanced translocation remains to be determined.The CPP pVEC (Table 1) has an extremely hydrophobic N-terminus(LLIIL) and single residue mutation of any of these to an alanine led toa significant decrease in cell uptake with the largest effects seen witha leucine to alanine change at the first amino acid [122]. Whether addi-tion of single or multiple hydrophobic residues to other CPPs enhancemembrane translocation remains to be seen.

7.2. Influence of tryptophan

In the plasma membrane, a lipophilic interior sandwiched be-tween hydrophilic, negatively charged phosphate head groups pro-vides a complex chemical surface with which peptides and smallmolecules can interact. Tryptophan has been extensively studied be-cause of its role within the membrane and its prevalence at theedge of transmembrane regions in proteins [123]. Many CPPs containeither a tryptophan or a tyrosine residue and this is especially trueamong the amphiphilic peptides such as TP10. These often contain

at least one tryptophan residue at one end of the helix, (Table 1 andFig. 1).

The placing of tryptophan residues in different positions in thehelix of a model (KAAKKAA)3 peptide has been shown to have alarge influence on its cytotoxic effects [124]. The most cytotoxic pep-tides were found when tryptophan sits on the interface between thehydrophilic lysines and hydrophobic alanines when the peptide is ina helical formation. For the CPP Pep-1 (Table 1), tryptophan residueswere used as part of a hydrophobic domain created to interact withmacromolecules [21]. When in a helical formation, the tryptophanswere arranged on one side of the helix and were shown to be embed-ded within the membrane. This caused the peptide to line up perpen-dicular to the membrane and form a pore, possibly aiding intranslocation. Another peptide, CADY (Table 1), also forms a helixwith tryptophan residues again present on only one side [9]; the pep-tide was shown to successfully deliver siRNA into cells. This studysuggests that tryptophan plays a dual role within this peptide byinteracting with the membrane, to aid translocation, and formingelectrostatic interactions with the end of the siRNA, reducing nucleo-tide degradation. In the non-amphiphilic peptide, R7, addition of atryptophan to the C-terminal end of the peptide was shown to in-crease uptake and lower toxicity at 37 °C and 4 °C when comparedto R7 alone [26] and as previously described a single tryptophan res-idue has significant influence on the way R6 interacts with GUVs [93].

7.3. Lipidation

Lipid tails have also been added to CPPs in order to enhance theirmembrane binding and penetration. The addition of a C14 myristoyl(Fig. 4) to R7-FITC significantly enhanced its association with cellsand subsequent internalisation [125]. However, there was little evi-dence of increased cytosolic labelling suggesting that the lipopeptidewas merely associating with the membrane rather than translocatingacross it. Similar findings using myristoylated oligoarginine peptides,in three different cells lines were observed [126] and both studiesdemonstrated that the extent of myristoyl mediated cell associationis dependent on the length of the arginine chain. The possibility existsthat the lipid aids initial membrane interaction but the comfort of thelipid tail in the bilayer may hinder the penetrating capacity of thepeptide. There was, however, evidence that a myristoylated R11 pep-tide was also gaining access to the cytosol [126]. Myristoylation isknown to affect the interaction of peptides with artificial membranesand cells and indeed it is suggested that otherwise impermeable pep-tides may gain access to cells via attachment to this lipid group[127,128]. The true extent of cytosolic labelling versus membrane as-sociation needs to be determined to confirm whether this is a usefulstrategy for aiding CPP-mediated delivery. The C16 stearyl group(Fig. 4) was initially found to increase the ability of CPPs R8 andHIV-1 Rev to transfect cells with plasmid DNA; addition of cholesterolor lauryl group was also advantageous [129]. Following on from thesestudies, the stearyl group has been attached to other CPPs, such asPepFect 14, for enhancing cell uptake and bioactivity of payloads in-cluding proteins and siRNA [130,131]. More information regardingthe influence of hydrophobicity and amphiphilicity on CPP dynamicsis available in this review [132].

8. Cysteine and disulphide bridges

A number of CPP delivering strategies are based on linking thepeptide to cargo via a disulphide bridge. This may be to attach a bio-active cargo or a label such as a fluorophore. For the former it then of-fers the possibility that the reducing environment of the cell allowsrelease of the vector from the payload. However, reduction may alsooccur before cell entry with the possibility that the new active thiolcould react with the plasma membrane and be internalised, withother components of the delivery system, via plasma membrane

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turnover [100]. More recently the addition of a single cysteine residueto CPPs penetratin and PenArg (RQIRIWFQNRRMRWRR) was found toimprove gene delivery in vitro [133]. The fact that this was largely de-pendent on addition of the weak base chloroquine to the cell systemwas evidence that this was mediated via some form of endocytic up-take and endosomal escape.

9. Fusogenic extensions

Penetration and delivery capacity of CPPs may also be enhancedby elongation with a fusogenic sequence; an example of this is theHA2 peptide from the influenza virus [32,134]. Tat-HA2 fusogenicpeptides have been investigated with a number of drug delivery sys-tems as a means of reducing and increasing endolysosomal sequestra-tion and escape respectively [32,134]. These effects may be mediatedby loss of membrane integrity as the addition of a HA2 derived se-quence to CPPs was not only shown to enhance cell internalisationbut also cause cytotoxicity [135]. The pH sensitive fusogenic amphi-philic peptide GALA (Table 1) has been extensively studied as a vectorfor drug delivery and has been modified to enhance its binding capac-ity to nucleotides and improve its cell entry [136]. Using CPPs in con-junction with GALA has also been shown to enhance endosomalescape [137]. Replacing the glycine residues of GALA to Lys to giveKALA (Table 1 and reference therein) produces a more CPP-like se-quence, but its capacity to escape from endosomes is reduced.

10. Conclusions

CPPs most certainly “Do not hover on the surface of things” andclearly interact with cells and penetrate into their depths. There is a re-quirement for CPP researchers to do the same to gain a better under-standing of the mechanism of action of these fascinating entities. It isclear that enhanced penetration of CPPs can be achieved by modifyingthe experimental systems and also supplementing the peptides withrelatively small appendages. This may be to enhance uptake and endo-somal escape or to direct penetration across the plasma membrane;both have their advantages and shortfalls as mechanisms for cellulardelivery of therapeutic macromolecules. It is hoped that the knowledgegained from all these studies will eventually allow formore rational de-sign of CPP sequences with enhanced penetration, and capacity to de-liver payloads.

Acknowledgement

Structures in Fig. 4, unless referenced in the text, were obtainedfrom Molecular Probes, Life Technologies and Sigma-Aldrich.

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