ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 345 (2005) 55–65

www.elsevier.com/locate/yabio

Cell-penetrating peptides: A comparative membrane toxicity study

Külliki Saar a,¤, Maria Lindgren a, Mats Hansen a, Emelía Eiríksdóttir a, Yang Jiang a, Katri Rosenthal-Aizman a, Meeri Sassian a,b, Ülo Langel a

a Department of Neurochemistry and Neurotoxicology, Stockholm University, SE10691 Stockholm, Swedenb Institute of Organic and Bioorganic Chemistry, University of Tartu, EE51014 Tartu, Estonia

Received 10 February 2005Available online 15 August 2005

Abstract

Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, theabilities of these peptides to translocate through cell membranes can be accompanied by toxic eVects resulting from membrane per-turbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of Wve peptides with well-documented cell-penetrating properties, pAntp(43–58), pTAT(48–60), pVEC(615–632), model amphipathic peptide (MAP), and transportan 10, ontwo human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothe-lial cells. We studied the eVects of these Wve peptides on the leakage of lactate dehydrogenase and on the Xuorescence of plasma mem-brane potentiometric dye bis-oxonol. In all cell lines, pAntp(43–58), pTAT(48–60), and pVEC(615–632) induced either no leakage orlow leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol Xuorescence. MAP and transportan 10 causedsigniWcant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to10 �M of transportan 10 and MAP, accompanied by a signiWcant increase in bis-oxonol Xuorescence. However, none of the CPPstested had a hemolytic eVect on bovine erythrocytes comparable to mastoparan 7. The toxicity proWles presented in the current studyare of importance when selecting CPPs for diVerent applications. 2005 Elsevier Inc. All rights reserved.

Keywords: Cell-penetrating peptides; Membrane perturbation; Lactate dehydrogenase leakage; Hemolytic activity

Cell-penetrating peptides (CPPs),1 also known asprotein translocation domains, membrane transloca-ting sequences, and Trojan peptides, make a new classof transmembrane delivery vectors with high pharma-ceutical potential [1–3]. So far, the most widely usedCPPs are two protein-derived peptides: pAntp(43–58)

* Corresponding author. Fax: +46 8 161371.E-mail address: kyllikis@neurochem.su.se (K. Saar).

1 Abbreviations used: CPP, cell-penetrating peptide; MAP, modelamphipathic peptide; LDH, lactate dehydrogenase; AEC, aortic endo-thelial cell; DiBAC4(3), bis-(1,3-dibutylbarbituric acid)trimethine oxo-nol; FBS, fetal bovine serum; EDTA, ethylenediamine tetraacetic acid;PBS, phosphate-buVered saline; HKR, HEPES-buVered Krebs–Ringersolution; MALDI, matrix-assisted laser desorption/ionization; SEM,standard errors of the mean; ANOVA, analysis of variance.

0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2005.07.033

(also known as penetratin), a fragment of Antennape-dia homeodomain [4], and pTAT(48–60), a fragment ofa transcription factor coded by the human immunode-Wciency virus [5]. Both pAntp(43–58) and pTAT(48–60), and their longer/shorter analogues, have beenshown to translocate anticancer drugs, oligonucleo-tides, peptides, proteins, and viruses into cells (for areview, see Ref. [6]). While the common denominator ofpenetratins, pTAT peptides, and polyarginines is theirhighly basic nature and numerous arginines in theirsequences, model amphipathic peptide (MAP) andtransportans constitute another group of CPPs [7]characterized by amphipathicity and numerous lysinesin their sequences. The sequence of MAP is fully artiW-

cial (e.g., not protein derived), was designed as an

56 Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65

amphipathic model peptide [8,9], and has been shownto translocate oligonucleotides [10,11] and peptides[12]. The sequences of transportans are obtained byengineering galanin and mastoparan fragments [13].Original transportan (galanin(1–12)-Lys-mastoparan)has been shown to translocate oligonucleotides [14–16]and proteins [17].

The translocation mechanisms of CPPs are beingexplored. However, developments in the Weld have beenhampered by methodological problems. For example,the use of Wxation steps in early Xuorescence microscopystudies has been shown to overestimate membrane pene-tration of pTAT(48–60) and Arg9 [18]. Additional over-estimation may arise from incomplete removal ofmembrane surface-bound peptides by proteases [19].Furthermore, most CPPs have several shorter/longeranalogues that have diVerent translocation eYciencies(for reviews, see Ref. [20]) and perhaps also diVerenttranslocation mechanisms. In addition, the nature of acargo seems to inXuence translocation mechanism andeYciency. For example, CPP-mediated protein translo-cation has been shown to involve endocytosis in thecases of pAntp(43–58) [21,22], pTAT [21–25],pVEC(615–632) [21], transportan [21], and polyarginine[26]. The CPP-mediated Xuorophore translocation hasbeen shown to occur via endocytosis in the cases ofpenetratin [27,28], polyarginines [26,28], and pTAT[18,28] but has been shown to occur independent ofendocytosis in the case of MAP [9]. A combination ofendocytotic and nonendocytotic pathways has beendemonstrated for pTAT and Arg7 analogues [27]. TheCPP-mediated oligomer translocation has been studiedand found to include a nonendocytotic component inthe cases of transportan [29] and MAP [10,11]. More-over, the cellular trajectories of the same CPP cargo maydiVer in diVerent cell lines [30,31], and the peptide-to-cellratio has been reported to inXuence translocationeYciency [32].

Although the number of studies addressing transloca-tion mechanisms of diVerent CPPs is growing rapidly,only a few toxicity studies are available. Instead, the tox-icity of diVerent CPPs is determined by diVerent researchgroups in diVerent cell lines with diVerent methodologies(for a review, see Ref. [33]). To our knowledge, there areonly three comparative studies available. Tréhin andcoworkers compared the lactate dehydrogenase (LDH)leakage from MDCK cells by human calcitonin-derivedCPPs, pTAT(47–57), and pAntp(43–58) [34]. Jones andcoworkers studied the eVect of 7 h exposure of unlabeled,Xuorophore-labeled, and peptide conjugates ofpAntp(43–58), pTAT(48–60), Arg11, and transportan inA549, HeLa, and CHO K1 cells [35]. We have previouslycompared the membrane-perturbing properties of bio-tin-labeled pAntp(43–58), pTAT(48–60), transportan,and MAP in Bowes human melanoma cells [7]. In thecurrent study, we widened the selection of peptides; we

replaced transportan with its shorter analogue transpor-tan 10 [13] and included pVEC(615–632) [36].pVEC(615–632) is derived from the murine sequence ofthe vascular endothelial cadherin and has been shown totranslocate oligonucleotides [36] and proteins [21,36].Transportan 10 (galanin(7–12)-Lys-mastoparan) lacksoriginal transportan’s unwanted eVect on GTPases [13]and has been shown to translocate oligonucleotides [16]and peptides [37]. Instead of Bowes human melanomacells, we used K562 human erythroleukemia cells andMDA-MB-231 human breast cancer cells, both of whichare often used as models in the context of anticancerdrug transport.

In general, cationic amphipathic peptides often pos-sess antiviral [38], antitumoral [39], and antimicrobialactivity, due at least partly to their membrane-disruptingactivity [40]. Because several CPPs are amphipathic, it isinteresting to investigate whether they have such eVectsas well. Indeed, pVEC(615–632) and transportan 10 haveantimicrobial activities [41], and P(�) and P(�) interactwith membranes similar to antimicrobial peptides [42].In the current study, we asked whether the CPPs havehigher membrane toxicity in cancer cells. To investigatethis, we performed membrane toxicity assays in malig-nant cells (K562 and MDA-MB-231) and in immortal-ized human aortic endothelial cells (AECs).

The main barrier to the use of cationic amphipathicpeptides as antimicrobial agents is their hemolytic activ-ity [43]. Numerous structure–activity studies have beenperformed to dissociate the antimicrobial activity fromthe hemolytic activity [43,44]. In the context of delivery,the hemolytic activity of a vector precludes intravenousdelivery and often enhances the toxicity of a conjugatedagent when administered by other routes [45]. Therefore,we decided to assay the hemolytic activities of our selec-tion of CPPs as well.

In summary, in this study, we describe experimentsaddressing the membrane toxicity of Wve CPPs in malig-nant cells and immortalized cells as well as their hemo-lytic activity in bovine erythrocytes.

Materials and methods

The amino acids were obtained from Neosystem(Strasbourg, France) and Bachem (Bubendorf, Switzer-land). The bis-(1,3-dibutylbarbituric acid)trimethineoxonol (DiBAC4(3)) was obtained from MolecularProbes (Eugene, OR, USA). All other chemicals wereobtained from Sigma–Aldrich (Stockholm, Sweden) ifnot stated otherwise. For Xuorescence measurements, adual-scanning microplate spectroXuorometer Spectra-Max Gemini XS (Molecular Devices, Sunnyvale, CA,USA) was used. Mass spectra were collected on a Voy-ager-DE STR Biospectrometry Workstation (AppliedBiosystems, Foster City, CA, USA) and analyzed using

Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65 57

the freeware MoverZ program (http://65.219.84.5/mover-zdl.html).

Cell culture

All cells were cultured at 37 °C in 5% CO2. The plasticlabware (Corning) was obtained from Labdesign (Täby,Sweden), and the cell culture reagents (GIBCO) wereobtained from Invitrogen (Lidingö, Sweden) if notstated otherwise.

The K562 human erythroleukemia cells (a kindgift from T. Land, Department of Neurochemistry,Stockholm University) were propagated in suspensionusing an RPMI 1640 medium supplemented withGlutaMAX-I, penicillin (100 U/ml), streptomycin(100 �g/ml), and fetal bovine serum (FBS, heat-inacti-vated, 7.5%). Cell density was kept between 105 and 106

cells/ml.The MDA-MB-231 human breast cancer cells

(ATCC, LGC Promochem, Borås, Sweden) were propa-gated in the RPMI 1640 medium supplemented withGlutaMAX-I, penicillin (100 U/ml), streptomycin(100 �g/ml), and FBS (10%). The cells were detachedonce a week with 0.05% trypsin/ethylenediamine tetra-acetic acid (EDTA) and seeded at a density of 1.2–1.5 £ 106 cells/75-cm2 Xask. The medium was changedonce a week.

The SV40-immortalized AECs (a kind gift from K.Fish, Scripps Research Institute, La Jolla, CA, USA)were propagated in an Endothelial Cell Basal Medium-2supplemented with EGM-2 MV SingleQuots (both fromCambrex Bio Science Walkersville, Walkersville, MD,USA), penicillin (100 U/ml), and streptomycin (100 �g/ml). The cells were detached once a week with 0.25%trypsin/EDTA after extensive washes with phosphate-buVered saline (PBS) and were seeded at a density of1.2–1.5 £ 106 cells/75-cm2 Xask. The medium waschanged once a week.

Peptide synthesis and puriWcation

All peptides used in this study (except MAP pur-chased from AC ScientiWc, Duluth, GA, USA) were syn-thesized essentially as described previously [36] on apeptide synthesizer (Applied Biosystems model 431A,PerkinElmer, Boston, MA, USA) using the tert-butyl-oxycarbonyl strategy of solid-phase peptide synthesis.PuriWcation and analysis of the peptides was also per-formed as described previously [36] except that massspectra were collected on a Voyager-DE STR Biospect-rometry Workstation.

Peptide handling

In all experiments, frozen aliquots of peptides wereused, and the aliquots were prepared as follows.

A suitable amount was weighed, dissolved in water toobtain 1 mM solution, and then stored overnight at 4 °C,aliquoted, and stored at ¡20 °C. Peptide aliquots wereanalyzed regularly by mass spectrometry, and no degra-dation was detected.

Comments on assay design

In general, peptides are notorious for binding to plas-tic surfaces [46] and for being susceptible to proteolysis[47]. In the context of in vitro assay design, this meansthat the eVective peptide concentration can decrease dueto the degradation by serum proteases (in the presenceof the serum in the assay buVer) and due to the peptide’sbinding to plastic. Indeed, at nanomolar concentrations,signiWcant amounts of pAntp(43–58), pTAT(48–60), andall-D-Arg7 and their conjugates have been reported tobind tightly to plastic and glass surfaces [48]. We wantedto avoid the reduction of the eVective peptide concentra-tion; therefore, we did not include serum in the exposuremedium and applied peptides in the micromolar range.Because the aim of our study was to compare Wve CPPsin identical assay conditions, we found it suYcient toperform the LDH and DiBAC4(3) assays at 10-�M pep-tide concentrations. In our preliminary experiments, thiswas the lowest concentration where we could see cleardiVerences among the selected CPPs. In addition, Xuo-rescence microscopy and Xuorescence-activated cellsorter quantiWcation of the translocation of Xuorescein-labeled CPPs are usually performed with 1–10�M of thepeptide [20]. In our hemolysis experiments, 50 �M CPPconcentrations were necessary to see signiWcant eVects. Itshould be pointed out, however, that 50 �M peptide inour hemolysis assay equaled in 2 fmol peptide/cell,whereas 10�M peptide in our LDH and DiBAC4(3)assays equaled 20–30 fmol peptide/cell.

LDH leakage assay

The assay was performed using the CytoTox-ONEHomogeneous Membrane Integrity Assay (Promega,Madison, WI, USA). The Wnal concentrations were asfollows: 10�M for peptides and 0.1% for Triton X-100.

LDH leakage assay in K562 cellsIn the assay, 15 ml of the cells (6106 cells/ml) was

centrifuged for 10 min at 500g. The cell pellet waswashed twice with 10 ml Hepes-buVered Krebs–Ringersolution (HKR, 5.5 mM HEPES, 138 mM NaCl, 2.7 mMKCl, 2 mM MgCl2, 1.8 mM CaCl2, 5.6 mM glucose, pH7.4). The cells were then suspended to 1 £ 106 cells/ml(counted with a hemacytometer). Then 200 �l of thissuspension was transferred to a 1.5-ml vial that alreadycontained 200 �l of peptide or Triton X-100 solution inHKR. After 10 min of incubation at 37 °C and 300 rpmin a Thermomixer (Eppendorf, Hamburg, Germany),

58 Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65

the vials were centrifuged for 2 min at 500g. Then 100 �lof the supernatant was transferred to a black polypro-pylene FluoroNunc plate (Nunc, Roskilde, Denmark)and 100 �l of the CytoTox-ONE reagent (preparedaccording to the manufacturer’s instructions) wasadded to each well. After 10 min of incubation at roomtemperature, 50 �l of Stop Solution was added to eachwell and the Xuorescence was recorded (�ex 560 nm, �em590 nm).

LDH leakage assay in MDA-MB-231 cells and AECsThe cells were seeded on 24-well plates 1 day before

the experiment, with 0.1 £ 106 cells/well in 0.5 ml of therespective medium. On the day of the experiment, thecells were washed twice with 0.5 ml HKR and thenexposed to the peptide in 300 �l HKR for 10 min at 37 °Cand 300 rpm in a Thermomixer. Then 100 �l of the expo-sure solution was transferred to a black polypropyleneFluoroNunc plate and analyzed as described in theabove section.

Analysis of peptide translocation by mass spectrometry

Two days before the experiment, 0.2 £ 106 AECs/wellwere seeded on six-well plates. On the day of the experi-ment, the cells were washed twice with HKR to removeserum traces. The peptide solutions were diluted to10 �M in HKR, 100 �l aliquots were saved for referencespectra, and 900�l was added to the cells and incubatedfor 45 min at 37 °C and 300 rpm in a Thermomixer. Then200�l of the exposure solution was removed and storedat ¡20 °C until the analysis. The cells were washed oncewith HKR and then treated with trypsin/EDTA solutionfor 2 min at room temperature. Then the trypsin solutionwas carefully aspirated and the cells were washed threetimes with HKR.

To prepare the lysate containing peptide trappedwithin the cells, the cells were treated with 200�l of 0.1%HCl/well for 10 min on ice. Then cell lysates were col-lected and stored at ¡20 °C until the analysis.

Samples were prepared for mass spectrometry usingZipTipC18 devices according to the manufacturer’sinstructions (Millipore, Västra Frölunda, Sweden). TheZipTip technology is widely used for the cleanup andconcentration of peptide mixtures in proteomics [49]. Wehave previously used ZipTipC18 devices when preparingmass spectrometry samples in studies addressing intra-and extracellular stability of pVEC(615–632) [36,50]. Itmust be emphasized here that CPPs accumulate in cellsat high levels; for example, intracellular concentrationsin the 100�M range have been reported for Xuorescein-labeled pVEC(615–632) [36], MAP, and pAntp(43–58)[32]. The combination of a micromolar CPP concentra-tion and the ZipTip sample preparation technologymakes it possible to analyze the fate of a CPP in a com-plex mixture such as a cell lysate.

The matrix (�-cyano-4-hydroxycinnamic acid at10 mg/ml in 50% acetonitrile/0.1% triXuoroacetic acid/water) was spotted before the sample on the sampleplate. As an internal standard, a ProteoMass P14Rmatrix-assisted laser desorption/ionization (MALDI)mass spectrometry standard was used (monoisotopicMW 1533.8582 Da). Data were collected from at leastthree diVerent locations on the spot with intensitygreater than 10,000 and accumulated. The mass spec-trometry analysis was performed on a Voyager-DE STRBiospectrometry Workstation and processed using Mov-erZ freeware.

DiBAC4(3) assay

DiBAC4(3) belongs to a class of anionic slow poten-tial-sensitive dyes [51] and has been shown to respond tomembrane depolarization with an increase in Xuores-cence resulting from the increased intracellular concen-tration and accumulation in intracellular lipid-richcompartments [52]. The DiBAC4(3) Xuorescence-basedassays have been used to estimate viability and/or mem-brane damage of yeasts [53] and bacteria [54,55].

The assay was performed in suspension in the cases ofall cell lines. This means that the MDA-MB-231 cellsand the AECs were Wrst detached in the routine cell-splitting procedure (e.g., Wrst detached with trypsin/EDTA, which was then neutralized by the addition ofcomplete growth medium). Cells (5–10 £ 106) were cen-trifuged for 10 min at 300g. The pellet was washed twicewith HKR. After the second wash, the cells were sus-pended in 0.5 �M DiBAC4(3) (in HKR), counted, anddiluted to obtain a suspension at a density of0.5 £ 106 cells/ml (K562) or 0.25 £ 106 cells/ml (AEC andMDA-MB-231). Cell suspension (400 �l) was pipettedinto each well of a black polypropylene FluoroNuncplate. After 15 min of incubation at 37 °C and 300 rpm ina Thermomixer, Xuorescence was recorded (�ex 494 nm,�em 518 nm). Then 4�l of 1 mM peptide stock solutionwas added to the respective wells. After 30 min of incu-bation at 37 °C and 300 rpm in a Thermomixer, Xuores-cence was recorded again. Analogous recordings werealso made in the absence of cells (400 �l of 0.5 �MDiBAC4(3) + 4 �l peptide stock solution).

Hemolysis assay

Bovine blood (supplemented with citrate) was pur-chased from the National Veterinary Institute of Sweden(Uppsala, Sweden), stored at 4 °C, and used within 10days after the production date. Blood (10 ml) was centri-fuged for 10 min at 1000g, and the pellet was washedonce with PBS and once with HKR and was then sus-pended in HKR to obtain a 4% suspension. Then 50 �l ofthis suspension was transferred to a 1.5-ml vial thatalready contained 950 �l of peptide or Triton X-100

Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65 59

solution in HKR. Final concentrations were as follows:50 �M for peptides and 0.1% for Triton X-100. After30 min of incubation at 37 °C and 300 rpm in a Ther-momixer, the vials were centrifuged for 2 min at 1000g.Then 300 �l of the supernatant was transferred to a 96-well plate and the absorbance was recorded at 540 nm ona Digiscan Microplate Reader (ASYS Hitech, Eugen-dorf, Austria).

Statistical analysis

Data were expressed as the means and standarderrors of the mean (SEM) for the various statisticalgroups (two to four experiments in duplicate). To Wndout whether the two mean values of interest were signiW-cantly diVerent, an unpaired t test was performed usingGraphPad Prism version 4.00 for Windows (GraphPadSoftware, San Diego, CA, USA). When comparing morethan two values, a one-way analysis of variance(ANOVA) test was performed using GraphPad Prism.

Results and discussion

The aim of this project was to study systematicallyone eVect of the CPPs: their toxicity due to membraneperturbation. We restricted ourselves to “naked” CPPs,that is, CPPs without any Xuorophore or cargo. Thesequences of the CPPs tested are listed in Table 1. Ourselection included CPPs with following properties: (i)protein derived (pAntp(43–58), pVEC(615–632), and

pTAT(48–60)) as well as engineered (MAP and trans-portan 10) and (ii) amphipathic (pVEC(615–632), MAP,and transportan 10) as well as nonamphipathic(pAntp(43–58) and pTAT(48–60)).

We chose three cell lines to work with: K562 humanerythroleukemia cells, MDA-MB-231 human breast can-cer cells, and S40-transformed human AECs. In otherwords, we used one immortalized cell line (an in vitroculture model for normal endothelial cells) and twomalignant cell lines (K562 and MDA-MB-231). Bothmalignant cell lines are useful models in the context of(anticancer) drug transport. That is, the MDA-MB-231cells are inherently methotrexate resistant due toimpaired antifolate transport [56], are highly invasive,and are often used in animal models to produce tumors(for a review, see Ref. [57]). The K562 cells are widelyused in diVerentiation studies and are often selected withanticancer drugs to establish drug-resistant sublines [58].

During the preliminary experiments, we comparedfour membrane disturbance assays: the LDH leakageassay, the 2-deoxy-D-[1-H3]glucose-6-phosphate leakageassay, the calcein AM leakage assay, and the ethidiumhomodimer-1 entry assay. In our hands, the latter twoassays were not suitable for quantitative measurementsdue mainly to the unstable baseline. Then we comparedthe LDH leakage assay (the commercially availableCytoTox-ONE Homogeneous Membrane IntegrityAssay) and the 2-deoxy-D-[1-H3]glucose-6-phosphateleakage assay [59]. Previously, it was reasoned that LDHis a far too large molecule for the detection of earlyevents in membrane integrity [33]. However, in our

Table 1Peptides used in the current study

Note. Basic amino acids are underlined. Amphipathic moments were calculated using WinPep [77].

Abbreviation Sequence Mr Amphipathic moment

pAntp(43–58) RQIKIWFQNRRMKWKK amide 2246 0.01pTAT(48–60) GRKKRRQRRRPPQ amide 1719 0.04pVEC(615–632) LLIILRRRIRKQAHAHSK amide 2209 0.08MAP KLALKLALKALKAALKLA amide 1876 0.16Transportan 10 AGYLLGKINLKALAALAKKIL amide 2181 0.07, residues 6–21: 0.16

Table 2Summary of toxic eVects of studied peptides in diVerent cells (percentages)

Note. See text and Figs. 1, 3, and 4 for details. The LDH leakage (F590) and the changes in DiBAC4(3) Xuorescence (�F518/F518) were determined at a10-�M peptide concentration. The hemoglobin leakage (A540) was determined at a 50-�M peptide concentration. The leakage induced by 0.1% TritonX-100 was deWned as 100%. “No peptide” indicates the background Xuorescence or absorbance value from the wells containing the cells and HKR.“nd” indicates not determined.

K562 MDA-MB-231 AEC Bovine erythrocytes

LDH F590

DiBAC4(3) �F518/F518

LDH F590

DiBAC4(3)�F518/F518

LDH F590

DiBAC4(3) �F518/F518

Hemoglobin A540

No peptide 16.4 § 3.2 11.0 § 1.9 12.0 § 1.9 12.1 § 0.7pAntp(43–58) 16.0 § 2.0 ¡2.1 § 5.2 13.6 § 2.0 ¡6.0 § 2.3 10.4 § 1.1 4.9 § 2.2 20.0 § 1.8pVEC(615–632) 16.0 § 2.0 ¡0.7 § 2.9 10.2 § 0.1 ¡6.4 § 2.5 11.6 § 0.03 ¡1.7 § 4.0 18.1 § 0.3pTAT(48–60) 17.7 § 2.2 3.2 § 2.7 9.7 § 0.05 2.7 § 3.6 9.6 § 0.6 2.5 § 3.1 11.2 § 1.5MAP 49.9 § 5.6 33.4 § 3.6 44.3 § 5.9 12.5 § 3.2 17.5 § 1.7 10.4 § 1.0 20.9 § 1.8Transportan 10 50.3 § 3.4 nd 44.7 § 1.5 nd 21.0 § 1.5 nd 29.0 § 4.9

60 Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65

hands, the LDH leakage assay and the 2-deoxy-D-[1-H3]glucose-6-phosphate leakage assay gave identicalresults (data not shown) in spite of the 1000-fold diVer-ence in the size of the marker molecules (LDHMW » 132 kDa, 2-deoxy-D-[1-H3]glucose-6-phosphateMW » 221 Da). The 2-deoxy-D-[1-H3]glucose-6-phos-phate leakage assay is a more cumbersome methodbecause the marker molecule precursor (e.g., 2-deoxy-D-[1-H3]-glucose) is radioactive and must be preloaded tothe cells. Moreover, some cell lines take up this com-pound at insuYcient levels. In addition, LDH leakagecorrelated well with the cytotoxic histamine leakage inRBL-2H3 rat basophilic leukemia cells (data notshown). In summary, we found that the LDH assay wasthe most suitable assay for screening of membrane toxic-ity. The particular commercial kit we chose (CytoTox-ONE) has been used previously by several diVerentresearch groups [60–64].

Results from the LDH leakage assay are presentedin Table 2 and illustrated in Fig. 1. pAntp(43–58),pVEC(615–632), and pTAT(48–60) did not cause anysigniWcant LHD leakage in any of the cell lines. TheeVects of MAP and transportan 10 were signiWcant inthe K562 and MDA-MB-231 cells. Strikingly, theeVects of MAP and transportan 10 were nearly identi-cal within and between these cells. When the back-ground Xuorescence (“no peptide”) was taken as 0%and the Triton X-100-induced signal was taken as100%, 40–41% of the total cellular LDH leaked outfrom the K562 cells and 38–39% leaked out from theMDA-MB-231 cells when exposed to one of those pep-tides. Intriguingly, the AECs seemed to be less sensitiveto the membrane-perturbing eVects of MAP and trans-portan 10 than did the K562 and MDA-MB-231 cells.Only a small leakage was recorded, but this was notstatistically signiWcant when compared with the back-ground signal.

The low toxicity of pAntp(43–58), pVEC(615–632),and pTAT(48–60) compared with that of MAP is inaccordance with the general picture emerging from pre-vious studies (for a review, see Ref. [33]). The diVerencein membrane activity, or induction of LDH leakage,may indicate a diVerence in mode of uptake, therebygrouping the Arg-rich peptides (pAntp(43–58),pVEC(615–632), and pTAT(48–60)) as milder comparedwith the Lys-rich peptides (MAP and transportan 10)with a higher degree of membrane activity.

To conWrm the cellular translocation ability of theselected peptides, a qualitative mass spectrometry assaywas performed. This assay was developed to be able todetect unlabeled CPPs in cell lysate preparations fromtissue culture. In Fig. 2, only the non-leakage-inducingCPPs are shown: pAntp(43–58), pVEC(615–632), andpTAT(48–60). However, pTAT(48–60) was not detect-able in either the extracellular fraction or lysate, perhapsreXecting its rapid degradation or low intracellular levels

of the CPP. In addition, MAP and transportan 10 weredetected in high amounts in the intracellular fraction(data not shown). It has also been shown previously byour group that Xuorophore-labeled pAntp(43–58) andpVEC(615–632) translocate eYciently into AECs [36].

Because the membrane-perturbing eVects of MAPand transportan 10 were less pronounced in AECs thanin malignant cells, we were eager to investigate the possi-

Fig. 1. LDH leakage after 10 min exposure to 10 �M peptide in HKRat 37 °C. LDH was determined using CytoTox-ONE, and the readoutwas the Xuorescence value at 590 nm (F590). The signal induced by0.1% Triton X-100 was deWned as 100%. “No peptide” indicates thebackground Xuorescence value from the wells containing the cells andHKR. The experiments were performed twice in duplicate, and thedata are presented as means § SEM. *P < 0.05, **P < 0.001.

Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65 61

Fig. 2. Qualitative mass spectrometry analysis of pAntp(43–58) (A), pVEC(615–632) (B), and pTAT(48–60) (C) translocation into AECs. “Extracel-lular” denotes a sample taken from the exposure medium after 45 min incubation with the 10-�M peptide in HKR at 37 °C. After brief trypsination,the cells were lysed with 0.1% HCl and the obtained sample was denoted as the “lysate”. All of the collected samples were puriWed with ZipTipC18

devices and mixed on a plate 1:1 with 10 mg/ml �-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% triXuoroacetic acid/water matrix.

pAntp(43-58) Mr 2246

lysate

extracellular

stock solutionm/z1400 2400

Inte

nsity

100

0

1533.9

2245.0

pVEC(615-632) Mr 2209

lysate

extracellular

stock solution

m/z1500 2400

Inte

nsity

100

0

2207.61533.3

pTAT(48-60) Mr 1719

lysate

extracellular

stock solution

m/z1550 1700

Inte

nsity

30

0

1533.1

1717.2

A

B

C

62 Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65

bility that these peptides could possess selectivity towardmalignant cell membranes. Namely, inherent selectivetoxicity for the membranes of malignant cells has beenreported for several naturally occurring cationic amphi-pathic peptides (e.g., cecropins, magainins, dermaseptins)(for a review, see Ref. [39]) as well as for engineered pep-tides [65,66]. Moreover, some cationic amphipathicpeptides have been shown to be equally lytic for drug-sensitive and multidrug-resistant cancer cells and toenhance the action of anticancer drugs [67].

The mechanism of the anticancer activity of cationicamphipathic peptides is not fully understood. Becausetheir antimicrobial activity is due to the membrane dis-ruptive action and/or binding to intracellular targets[40], these mechanisms most likely also account for theiranticancer activity. Indeed, bovine lactoferricin causesleukemia and carcinoma cells to lose their membraneintegrity and triggers the mitochondrial pathway ofapoptosis [68]. It is not known which factors provide thespeciWcity of cationic peptides for the malignant cellmembranes. However, the certain features of the malig-nant cell membranes (e.g., diVerent membrane composi-tion and Xuidity, more negative charge, highertransmembrane potential, increased level of acidic com-ponents on the surface) have been pointed out as possi-ble reasons [39,65].

To verify the results obtained in the LDH leakageassay, we decided to study their eVect on DiBAC4(3)Xuorescence. This dye belongs to a class of anionic slowpotential-sensitive dyes [51] and has been used previ-ously to estimate viability and/or membrane damage ofyeasts [53] and bacteria [54,55]. As seen in Fig. 3 andTable 2, the largest increase in DiBAC4(3) Xuorescencewas caused by MAP and the eVect was most pronouncedin the K562 cells. In this cell line, the pAntp(43–58),pVEC(615–632), and pTAT(48–60) were clearly distin-guishable from MAP. In the MDA-MB-231 cells, theMAP-induced change in DiBAC4(3) Xuorescence wassigniWcantly diVerent from that induced by pAntp(43–58) and pVEC(615–632) but not from that induced bypTAT(48–60). In the AECs, no statistical diVerence wasfound among the eVects of four tested peptides. In sum-mary, the selectivity of MAP for malignant cells in thisassay was less evident than in the LDH leakage assay.Unfortunately, measurements with transportan 10 werecomplicated because this peptide induced an increase inDiBAC4(3) Xuorescence in the absence of cells; there-fore, the data are not presented. The eVect of cationiccompounds on DiBAC4(3) Xuorescence has beenreported previously (e.g., valinomycin [69]).

We also investigated the hemolytic activities of theCPPs. As pointed out in the introductory paragraphs, anideal delivery vector should not exert any hemolyticactivity because this activity precludes the intravenousdelivery route. Moreover, in the context of cationicamphipathic antimicrobial peptides, erythrocytes are

used as a model for normal eukaryotic cells. As a controlpeptide, we used a representative of the family of mast-oparans, known for their hemolytic and mast celldegranulating activities [70]: mastoparan 7, a potentpore maker [71,72] and G protein activator [73,74].

Fig. 3. Changes in DiBAC4(3) Xuorescence after 30 min exposure to10 �M peptides in HKR at 37 °C. The readout was the Xuorescencevalue at 518 nm (F518). The peptide-induced changes (�F518) areexpressed as percentages of the respective initial Xuorescence values(F518) (i.e., before the addition of peptide). Experiments were per-formed twice in duplicate, and the data are presented asmeans § SEM. *P < 0.05.

Cell-penetrating peptides / K. Saar et al. / Anal. Biochem. 345 (2005) 55–65 63

Results from the hemolysis assay are presented in thelast column of Table 2 and illustrated in Fig. 4. Asexpected, mastoparan 7 was most hemolytic, causing thecomplete lysis of erythrocytes at a 50-�M concentration(and even at 25 �M [data not shown]). The hemolyticeVects of 50 �M pAntp(43–58), pVEC(615–632), MAP,and transportan 10 were much less pronounced,although the low leakage detected was statistically sig-niWcant when compared with the control value (e.g., “nopeptide”). There was no signiWcant diVerence among theeVects of pAntp(43–58), pVEC(615–632), MAP, andtransportan 10. The eVect of pTAT(48–60) was not sig-niWcant when compared with the background absor-bance value (“no peptide”). In our hands, MAP was lesshemolytic, whereas transportan 10 and pVEC(615–632)were more hemolytic, than reported previously [41,75].Such discrepancies could be caused by species diVerences(bovine vs. rat [41] vs. human [75]). Unfortunately, noneof those studies included a peptide with known hemo-lytic properties as a reference compound. The ability ofCPPs to translocate into erythrocytes and lymphocyteshas been demonstrated previously [76].

Interestingly, the amphipathic nature of CPPs doesnot seem to be the only criterion for membrane pertur-bation. That is, pVEC(615–632) possesses clearly seg-mental amphipathicity (hydrophobic N terminus andhydrophilic C terminus) yet expresses low membranetoxicity comparable to nonamphipathic CPPs(pAntp(43–58) and pTAT(48–60)) (Table 2).

In summary, the experiments described in this studyindicate the higher membrane toxicity of MAP andtransportan 10 when compared with pAntp(43–58),pVEC(615–632), and pTAT(48–60). This property wasmost pronounced in the K562 leukemia cells and leastevident in erythrocytes. Consequently, MAP and trans-portan 10 could be the vectors of choice when enhancing

Fig. 4. Peptide-induced hemoglobin leakage from bovine erythrocytesafter 30 min exposure to 50 �M peptides in HKR at 37 °C. The read-out was the absorbance at 540 nm (A540). The signal induced by 0.1%Triton X-100 was deWned as 100%. “No peptide” indicates the back-ground absorbance value from the wells containing the cells andHKR. Experiments were performed in duplicate two times (four timesin the case of transportan 10), and the data are presented asmeans § SEM. *P < 0.05, **P < 0.001, ***P < 0.0001.

the action of anticancer drugs. Furthermore, the overalllow hemolytic activity of tested CPPs should encouragethe development of CPPs as drug delivery vectors.

Acknowledgments

This work is funded by grants from the EC Frame-work 5 (QLK3-CT-2002-01989) and the SwedishResearch Council (VR-M and VR-NT) and was sup-ported by CePeP and Orexo. We are grateful for helpfuladvice on the DiBAC4(3) assay from Margus Pooga.

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