Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide

Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden, and University of Ta...
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Bioconjugate Chem. 2010, 21, 1662–1672

Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates ¨ lo Langel†,‡ Emelı´a Eirı´ksdo´ttir,§,† Imre Ma¨ger,*,§,†,‡ Taavi Lehto,‡ Samir El Andaloussi,¶,†,‡ and U Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden, and University of Tartu, Institute of Technology, Tartu, Estonia. Received April 9, 2010; Revised Manuscript Received June 22, 2010

Cell-penetrating peptides (CPPs) belong to a class of delivery vectors that have been extensively used for the cellular delivery of various, otherwise impermeable, macromolecules. However, results on the cellular internalization efficacy of CPPs obtained from various laboratories are sometimes challenging to compare because of differences in the experimental setups. Here, for the first time, the cellular uptake kinetics of eight well-established CPPs is compared in HeLa pLuc 705 cells using a recently published releasable luciferin assay. Using this assay, the kinetic behavior of cytosolic entry of these luciferin-CPP conjugates are registered in real time. Our data reveal that the uptake rate of CPPs reaches its maximum either in seconds or in tens of minutes, depending on the CPP used. Tat and higher concentrations of MAP and TP10 display fast internalization profiles that resemble the kinetic profile of membrane-permeable free luciferin. The uptake of the other peptides, pVec, penetratin, M918, and EB1, is much slower and is consistent with the reported observations of endocytosis being the predominant internalization mechanism. Additionally, to some extent, the latter CPPs can be clustered into subgroups which are based on time points when the most pronounced uptake rates are observed. This may indicate once more involvement of various (concentration dependent) mechanisms in the uptake of CPPs. In summary, the variances in the internalization profiles for the CPPs demonstrate the importance of measuring kinetics instead of only relying on simple end-point studies, and with the luciferin-CPP assay, more lucid information can be retrieved when studying the internalization mechanisms of CPPs.

INTRODUCTION Ever since the first cell-penetrating peptides (CPPs)1were introduced in mid-1990s (1, 2), research on these cargo delivery vehicles has been highly active. The origin of CPPs is manifold, ranging from protein-derived sequences (3, 4) with some having inherent biological activity (5-7) to fully artificial peptides, which are designed based on physical and chemical properties required for specific interactions with cargo molecules and/or cell-membrane components (8-10). Also, several successful attempts have been made to modify well-established CPPs in order to increase their potency (11-16). These polycationic and/ or amphipathic peptides have shown great promise in cargo delivery, both in vivo and in vitro (17-20). However, it is important to keep in mind that CPPs might have disparate translocation capabilities and their potency often depends on the type of a cargo molecule used and whether the cargo is attached to the CPP using covalent or noncovalent strategy. There are several methods available to assess the delivery efficacy of CPPs that are mostly based on the use of fluorescent labels or functional assays, which generate readouts that arise from a biological response. Each method has its advantages and limitations. While fluorometric assays are relatively fast and * Corresponding author. Tel.: +372 737 4871; fax: +372 737 4900; E-mail address: [email protected]. § These authors contributed equally to this work. † Stockholm University. ‡ University of Tartu. ¶ Present address: Department of Laboratory Medicine, Karolinska Institute, 141 86, Stockholm, Sweden. 1 Abbreviations: CPP, cell-penetrating peptide; TP10, Transportan 10; MAP, model amphipathic peptide; HKRg, HEPES-buffered Krebs Ringer containing 1 mg/mL glucose; PBSg, phosphate buffered saline containing 1 mg/mL glucose; LDH, lactate dehydrogenase.

Figure 1. Structure of the luciferin-CPPs. The luciferin-CPP conjugates are composed of luciferin, a reducable/releasable linker, and a cysteine-containing CPP, as previously described (23).

straightforward to implement, these methods might not provide biologically relevant information because the readout might be biased by fluorescence originating from membrane-bound and/ or endosomally entrapped CPP-cargo constructs, which are not biologically available (21). Therefore, in order to avoid misleading results by overestimation of internalization efficacy, it is more relevant to take advantage of biological assays, such as the splice correction assay (22). However, the readout depends on whether the cargo molecule is delivered into the correct cellular compartment, e.g., the nucleus in case of the splice correction assay. This might, on the other hand, not reflect the delivery efficacy into other parts of a cell, such as the cytoplasm, which is also of great interest from a pharmacological point of view (when, for example, RNAi effects are studied). Both the high background in fluorescence assays and the lack of biological readout call for novel assays to measure the delivery efficacy of CPPs. A releasable luciferin-transporter system (Figure 1) is a recently published assay, which allows real-time analysis of cellular uptake and cytosolic release of the transporter molecule (23). This assay relies on the luciferin-luciferase reaction which is known for its high

10.1021/bc100174y  2010 American Chemical Society Published on Web 08/04/2010

Uptake Kinetics of Luciferin-CPP Conjugates

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to monitor the kinetics of the uptake process since the total uptake depends on the treatment duration. Furthermore, inhibition of a certain internalization pathway in a mechanistic study might in some cases only change the kinetics but not the total uptake or vice versa (26). Therefore, one time point for the readout does not reveal all aspects of a certain peptide carrier. Here, the releasable luciferin-CPP conjugate system is used for the first time to compare the cargo delivery efficacy of eight CPPs into cells: the protein derived peptides penetratin (1), EB1 (27), Tat (2), M918 (28), pVec (29), and TP10 (30) (with cargo attached to the N-terminus or to Lys7), and a designed peptide MAP (31). We demonstrate that this assay is sensitive enough to efficiently detect differences of the CPPs in terms of their uptake kinetics. We also show how the CPPs under this study could be clustered into subgroups regarding to their uptake kinetics profile and kinetic parameters.

EXPERIMENTAL PROCEDURES

Figure 2. Scheme of the cellular fate of the luciferin-CPP conjugates. Upon cytosolic entry via an endocytic or direct pathway, (1) the disulfide bridge is reduced by cytosolic glutathione, (2) the linker between the luciferin and the CPP undergoes cyclization and free luciferin is released, (3) the luciferase enzyme converts the luciferin to oxyluciferin and a photon, and (4) the photon is detected by a luminometer.

quantum yield and minimal background (24). In short, after the cytosolic entry, the disulfide bond between the luciferin and the transporter (a CPP in our study) is reduced, free luciferin is released, and in turn, it is converted by luciferase into oxyluciferin and a photon (Figure 2). The produced light thereby reflects the amount of the corresponding CPP from the luciferin-CPP conjugate. This approach has previously been used to quantify polyarginine uptake and cargo release in luciferase expressing transgenic mice by whole body imaging technique (25). In order to correctly assess and compare uptake efficacy of CPP-cargo constructs with in vitro model systems, it is important

Peptide Synthesis. The peptides (Table 1) were synthesized in a stepwise manner on an automated peptide synthesizer (Applied Biosystems model 433A, USA) by tert-butoxycarbonyl (t-Boc) chemistry. t-Boc amino acids (Neosystem, France; Iris Biotech, Germany; Bachem AG, Switzerland) were coupled as hydroxybenzotriazole (HOBt) esters (Iris Biotech, Germany) in the presence of N,N′-dicyclohexylcarbodiimide (DCC; Iris Biotech, Germany) and N,N′-diisopropylethylamine (DIEA; Iris Biotech, Germany) on 4-methylbenzhydrylamine resin (Iris Biotech, Germany) to generate C-terminally amidated peptides. Manual coupling of the Cys residue to the ε-amino group on Lys7 of TP10 was carried out with HOBt, 2-(1H-benzotriazole1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and DIEA activation. Peptides containing His(Dnp) or Trp(For) were deprotected with 20% thiophenol in N,N′-dimethylformamide (DMF) for 1 h or 20% piperidine in DMF for 1 h, respectively, and all peptides were then cleaved from the resin under anhydrous hydrogen fluoride/p-cresol/p-thiocresol (90/5/5) for 1 h at 4 °C and collected with ether precipitation. Finally, the peptides were purified by semipreparative reverse-phase HPLC column (DiscoveryBIO Wide Pore C-18, Supelco, Sigma-Aldrich, Sweden), and their purity was analyzed by RP-HPLC analytical column (Discovery C-18, Supelco). The correct molecular weight was verified by MALDI-TOF (Perkin-Elmer prOTOF 2000, PerkinElmer, Sweden) mass spectrometry. Synthesis of Luciferin-CPP Conjugates. Luciferin linker was synthesized as previously reported (see Scheme 1 in ref 32). Luciferin linker and Cys-CPPs were mixed in 1:1 molar ratio at 0.88 mM concentration in DMF/acetic acid buffer (pH 5, 50 mM) for 1 h under nitrogen. Luciferin-CPP conjugates (Figure 1) were purified by semipreparative RP-HPLC column, and their purity (>99%) was analyzed by RP-HPLC analytical column. The correct molecular weight was verfied by MALDITOF mass spectrometry (Table 1). Cell Culture. HeLa pLuc 705 cells, kindly provided by Ryszard Kole, were grown in DMEM (Dulbecco’s modified Eagle’s medium) with glutamax, supplemented with 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 10% FBS (fetal bovine serum), 100 U/mL penicillin, and 100 mg/mL streptomycin (hereafter referred to as complete medium). Cells were grown at 37 °C in a humidified 5% CO2 incubator. All cell culture media and supplements were purchased from Invitrogen, Sweden. Uptake Kinetics. 106 cells were seeded onto a 6 cm cell culture dish 96 h prior to experiments. 48 h prior to experiments, the cells were transfected with 4 µg luciferase encoding pGL3 plasmid (Promega, Sweden) using 10 µL Lipofectamine 2000 (Invitrogen, Sweden) in 6 mL complete medium for 4 h. The

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Table 1. Sequences of the Used Cell-Penetratin Peptides (CPPs) with Physicochemical Characteristicsa calcd

exp

CPP

Cys-CPP sequence

ref

Lys

number of Arg

[M+H+]

[M+H+]

〈H〉b

〈µ〉c

MAP TP10 TP10(Cys) EB1 Penetratin pVec Tat M918

C-KLALKLALKALKAALKLA-amide C-AGYLLGKINLKALAALAKKIL-amide AGYLLGK(C)dINLKALAALAKKIL-amide C-LIRLWSHLIHIWFQNRRLKWKKK-amide C-RQIKIWFQNRRMKWKK-amide C-LLIILRRRIRKQAHAHSK-amide C-GRKKRRQRRRPPQ-amide C-MVTVLFRRLRIRRASGPPRVRV-amide

31 30 30 27 1 29 2 28

5 4 4 4 4 2 2 0

0 0 0 3 3 4 6 7

2375.32 2680.42 2680.42 3597.86 2745.32 2707.42 2217.08 3133.62

2375.32 2680.42 2680.43 3597.88 2745.29 2707.42 2217.07 3133.61

0.99 0.93 0.93 -0.63 -1.73 -0.44 -3.49 -0.05

0.16 0.07 0.07 0.04 0.01 0.08 0.04 0.01

a

Luciferin is conjugated to the N-terminal cysteine residue, unless stated otherwise. Amino acids with a positive charge at physiological pH are underlined. b Mean hydrophathy (GRAVY score) was calculated using WinPep with Kyle and Doolittle scale (73). c Mean amphipathy moment was calculated using WinPep (calculations assume R-helical conformation of the peptides). d Luciferin was coupled to the cysteine at the ε-amino group on Lys7.

transfection medium was replaced with complete medium and the cells grown for further 20 h. 9 × 103 cells were seeded in a white 96-well clear-bottom plate (Greiner Bio-One, Germany) 24 h prior to luciferin-CPP treatment. On the day of the experiment, the cells were washed once with 100 µL cell culture media or HEPES-buffered Krebs Ringer buffer containing 1 mg/mL glucose (HKRg), which was then replaced with various concentrations of 120 µL luciferin-CPP solution in complete medium or HKRg, respectively. All experiments were carried out at RT (25 °C) for either 2 h (in complete medium) or 1 h (in HKRg) in atmospheric CO2. Luminescence was measured by GLOMAX 96 microplate luminometer (Promega, Sweden) where data points were recorded in 93 s time interval. In order to exclude overestimation of peptide cytosolic delivery and overrule possibilities of false-positive signals due to extracellular cleavage of the conjugates by potential leak of glutathione, we monitored changes in extracellular free thiol concentration. The cells were seeded into 96-well tissue culture plates one day prior experiment, as described above. On the day of the experiment, the cells were washed with phosphate buffered saline (PBS) and incubated with 5 µM luciferin-CPP conjugates in a solution of 250 µM Ellman’s reagent (5,5′dithiobis-(2-nitrobenzoic acid) or DTNB) in PBS supplemented with 1 mg/mL glucose (PBSg). Ellman’s reagent reacts with free thiols, releasing the yellow dianion 2-nitro-5-thiobenzoate (TNB2-). By monitoring absorption at 412 nm (the absorption peak of TNB2-), change in the free thiol concentration in the extracellular media can be determined. The absorption was measured over time in triplicate using a Tecan Sunrise microplate absorbance reader (Tecan Group Ltd., Switzerland). As a positive control, we measured the absorbance of lysed cells which were incubated in a solution of luciferin-CPP and Ellman’s reagent. Lactate Dehydrogenase (LDH) Leakage. Membrane integrity was measured using the Promega CytoTox-ONE assay (Promega, USA). 9 × 103 cells were seeded in a clear 96-well plate (Greiner Bio-One, Germany) 24 h prior to treatment. Cells were washed twice with 120 µL complete medium prior to 35 min or 2 h incubation with 120 µL luciferin-CPP conjugates at four different concentrations (1, 2.5, 5, and 10 µM) in complete medium at RT (25 °C). Finally, 80 µL of extracellular medium was transferred to a black 96-well plate (Labdesign, Sweden) containing 80 µL of CytoTox-ONE reagent, incubated for 10 min at RT, and fluorescence measured at 560/590 nm. LDH release from cells lysed with 0.2% Triton X-100 in HKRg was defined as 100% leakage and LDH release from untreated cells as 0% leakage. Statistical Analysis. All data were processed in GraphPad Prism v 5 for Windows (GraphPad Software Inc., USA) with results displayed as mean ( SEM. The uptake measurements in complete medium are three independent experiments per-

Figure 3. (A) An increase in extracellular free thiol content after 30-60 min treatment with 5 µM CPP in PBSg buffer. (B) Cellular toxicity. LDH leakage was measured after 35 min or 2 h treatment with 2.5-10 µM CPP in complete medium. LDH release from cells lysed with 0.2% Triton X-100 in HKRg was defined as 100% leakage, and LDH release from untreated cells as 0%. The values display the means of two independent experiments performed in HeLa pLuc 705 cells in triplicates (mean ( SEM, n ) 2).

formed in triplicate while the uptake experiments in HKRg were carried out twice in triplicate. The cytotoxicity measurements include two independent experiments performed in triplicate. Data Fitting and Comparison of Uptake Kinetics Parameters. The uptake kinetics raw data of replicate samples were averaged for each experiment, and a modified logistic model was used to fit the averaged data. The fitting procedure was carried out in Prism 5 (GraphPad Software Inc., USA) according to the formula

Uptake Kinetics of Luciferin-CPP Conjugates

S(t) )

P2 + P5·t + P1 1 + exp[-P4·(t - P3)]

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(1)

where S(t)is the signal intensity at time t, P1 approximates the baseline signal intensity, P2 approximates the amplitude of the plateau above the baseline, P3 (s) correlates with the time point at which maximal change of the signal occurs, P4 (s-1) correlates with the maximum slope of the signal, and P5 (s-1) describes the terminal slope of the uptake curve (33). Thereafter, the maximum slope (maximum first derivate) of the uptake curve and the time at which the maximum slope occurs (i.e., the inflection point of a sigmoidal curve) were determined from the fitted curve for each experiment. Finally, average of these parameters along with standard error of mean (SEM) were calculated.

RESULTS Uptake Measurements in Complete Medium; Exponential Kinetic Profiles versus Sigmoidal. Here, we have used the luminescence of the samples in each time point to produce kinetic curves of the uptake that correlate with turnover of the luciferin-luciferase reaction in these data points (in units of RLU per second). By calculating the area under the kinetic curve (Figure 4), the total emitted light from the luciferin-luciferase reaction, and thereby the total cytosolic release of luciferinCPP, could be evaluated. This integration can be carried out for any chosen time point from a single experiment, and we have chosen 15, 30, and 60 min time points (Figure 5B-D) since the majority of CPP end point uptake studies are within this incubation time range. In addition, we determined the total uptake level after 2 h incubation (Figure 5A) because the luminescence for most CPPs used in this study was still increasing after 1 h. Judging by the kinetic profiles of the peptides, it is apparent that they can be divided into two categories. Tat and free luciferin displayed a fast cytosolic entry with a decaying exponential profile (Figure 4A) while EB1, TP10(Cys), M918, penetratin, and pVec showed a slower cytosolic entry with a sigmoidal profile (Figure 4B), which reached a maximal uptake rate after different time periods. MAP and TP10 fall into both categories depending on their concentrations; 10 µM TP10 and 10 µM and 5 µM MAP (Figure 4A) aligned with Tat, while other concentrations showed the same pattern as TP10(Cys) (i.e., “slow” uptake pattern). The uptake kinetic curves for the peptides in the fast uptake group look roughly the same. The internalization took place very rapidly; the uptake rate reached a maximum in a matter of seconds (Tat, 10 µM MAP) up to few minutes (TP10, 5 µM MAP) and decayed rapidly. On the

other hand, the peptides following a sigmoidal or slow uptake pattern can be further subdivided. The kinetic curves for EB1, TP10(Cys), and M918 are similar in shape with higher maximum uptake and faster internalization (steeper initial slope) than pVec and penetratin. Another way of categorizing the peptides can be according to the time of their initial internalization (i.e., cytosolic entry). EB1 and penetratin started to internalize immediately after their application to the cells. On the other hand, the first sign of internalization of pVec, M918, and TP10(Cys) was after 15 min. Furthermore, EB1 was the only peptide from the sigmoidal group that exhibited a deviation or a certain phase change within its kinetic curves; the maximal uptake rate for the 10 µM peptide occurred roughly 30 min earlier than at the lower concentrations (data not shown). Even though the peptides show heterogeneous total uptake (Figure 5A-D), the concentration dependence of uptake is quite similar but not linearly dependent on the concentration, although higher peptide concentrations naturally gave higher uptake. Interestingly, the total uptake of the peptides that display the sigmoidal uptake profile exceeded the total uptake of the peptides with the fast uptake profile if given enough time, e.g., 2 h (Figure 5A); however, care must be taken when interpreting these results since an unknown signal portion may have been left unregistered for the fast profile compounds due to short lag period between addition of the peptides and registration of the very first data point. In addition, the uptake rate of the slow internalization group remains high even after 2 h incubation (Figure 4B). This indicates that differences in total uptake of these two groups are strongly time dependent. EB1 is an analogue of penetratin, designed to have R-helical structure upon protonation at low pH, i.e., in endosomal compartments, and to promote endosomal escape. It is intriguing that, in 30 min, the uptake for EB1 indeed started to exceed that of penetratin (Figure 5C), and this difference increased over time. The kinetic profiles for these two peptides differ (Figure 4B)sEB1 reached its maximal internalization rate faster and to a higher extent. This indicates that the rationale behind its endosomolytic design is indeed effective and that EB1 escapes the endosomes better than penetratin, which is in line with earlier hypotheses and previously published results (27). TP10 showed a considerably higher uptake in the beginning of the incubation, but TP10(Cys) reached the uptake level of TP10 in 1 h and exceeded it by more than 2-fold after 2 h treatment. The only difference between the sequences of TP10 and TP10(Cys) is the localization of the cysteine residue, which is at the N-terminus in TP10 and on the side chain of Lys7 in TP10(Cys). TP10(Cys) is the milder peptide of the two in the

Figure 4. Internalization kinetics profiles of the CPPs. (A) Fast internalization profiles for 10 µM luciferin, Tat, and MAP in complete medium. The internalization profile for TP10 was omitted for clarity because it is analogous to the internalization profile for MAP. The inset describes the first 10 min of the uptake. (B) Slow internalization profiles for 10 µM EB1, TP10(Cys), M918, penetratin, and pVec in complete medium. The values display the means of three independent experiments performed in HeLa pLuc 705 cells in triplicates (mean ( SEM, n ) 3). The number of displayed data points is reduced for clarity.

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Figure 5. Total cellular internalization of the CPPs. (A) Total cellular internalization of 1-10 µM luciferin-CPPs after 2 h incubation in complete medium. (B) 1 h incubation in complete medium. (C) 30 min incubation in complete medium. (D) 15 min incubation in complete medium. (E) 1 h incubation in HKRg buffer. All graphs are presented as fold increase compared to untreated cells. The values display the means of (A-D) three independent experiments performed in HeLa pLuc 705 cells in triplicates (mean ( SEM, n ) 3), (E) two independent experiments performed in HeLa pLuc 705 cells in triplicates (mean ( SEM, n ) 2).

sense that it provokes no membrane disturbance and has been reported to show higher uptake than TP10 (34). To verify that the uptake values we observed were not a consequence of extracellular cleavage of the conjugates or glutathione outflow from the cells, we monitored changes in extracellular free thiol content using Ellman’s reagent while incubating the cells with luciferin-CPP conjugates in PBSg buffer. Virtually no change in thiol concentration could be seen, except for TP10 and MAP between 0.5 and 1 h incubation, and also to some extent for Tat and TP10(Cys) peptide (Figure 3A). When incubating the cells with various concentrations of Ellman’s reagent without adding the peptide conjugates, it was apparent that its cleavage rate was significantly slower than the uptake rate of CPP conjugates. This was true even when using Ellman’s reagent at a concentration which was much higher than the determined Km value of the Ellman’s reagent cleavage reaction, i.e., when this reaction was ongoing at maximal rate (data not shown). Peptide Toxicity by Lactate Dehydrogenase (LDH) Leakage Assay. Cellular leakage of large Mw LDH has been shown to correlate with the leakage of deoxyglucosephosphate (a small molecule) (35). LDH leakage could therefore be used to predict leakage of a small, intracellular molecule like glutathione into the extracellular environment. This is an important factor in the releasable luciferin-CPP assay used herein, because extracellular glutathione could cause a reduction of extracellular luciferin-CPP conjugates. Released free luciferin would consequently enter cells, and the internalization efficacy of the corresponding CPP could be overestimated. Therefore, to exclude artifacts from a leaky membrane, the level of membrane disturbance or toxicity provoked by luciferin and the luciferinCPP conjugates in complete medium was measured by the LDH leakage assay (Figure 3B). None of the peptide concentrations used herein (1-10 µM) compromised the membrane integrity after 35 min incubation. Changes in the cell morphology after 2 h incubation were only noticeable for MAP (5 and 10 µM), TP10 (10 µM), and EB1

(10 µM), as judged by microscopy (data not shown). MAP and TP10 triggered a mild LDH leakage (13.0% and 8.2%, respectively) after 2 h incubation; however, EB1 can be considered nontoxic. Our results are thus in good agreement with previously published data (34, 35). Uptake Measurements in Media versus Buffer. Uptake measurements can be carried out in different incubation solutions, e.g., in complete or serum free cell growth media, or in various buffers such as HKR and PBS. Cellular uptake measurements using buffer as an incubation media do not closely represent physiological conditions. Buffer systems have nevertheless been applied in the past (23, 36, 37), which prompted us to compare our results carried out in complete media with those in HKRg buffer. As expected, the kinetic profiles for all of the peptides, except MAP and TP10, were different from the experiments carried out in media (data not shown). No peptide displayed a sigmoidal slow uptake profile; instead, a steady increase in the luminescence along with time was detected for pVec, M918, and EB1 (1-5 µM). Furthermore, Tat, TP10(Cys), penetratin, TP10, and 10 µM EB1 exhibited a dip followed by a steady increase, similar to penetratin when the uptake experiments were carried out in complete medium. The level of uptake was markedly higher for the buffer experiments where the most pronounced increases were 28- and 12-fold for 10 µM pVec and Tat, respectively (Figure 5B and E). The several-fold lower uptake for most of the CPPs in complete media can be explained by the presence of serum proteases and the affinity of CPPs toward negatively charged serum proteins. Data Fitting and Comparison of Uptake Kinetics Parameters. A modified logistic function according to eq 1 was fitted to the raw luminescence data of replicate samples measured during independent experiments. Only the peptides that belong to the slow-uptake group could be fitted using this model, i.e., Tat, and higher concentrations of TP10 and MAP conjugates were left out from this analysis. The goodness of fit was

Uptake Kinetics of Luciferin-CPP Conjugates

Figure 6. Data fitting and comparison of uptake kinetic parameters. The raw luminescence data of replicate samples measured during independent experiments were fitted to a modified logistic function according to eq 1, and inflection point of the sigmoidal curve (tmax) was calculated for (A) MAP, TP10, and TP10(Cys); and for (B) pVec, M918, EB1, and penetratin. The values display the means of three independent experiments performed in HeLa pLuc 705 cells in triplicates (mean ( SEM, n ) 3).

quantified by the R2 parameter which was typically in the range 0.92-0.99. Thereafter, from the fitted curve we calculated the following kinetic parameters for three independent experiments: maximum slope, in other words the maximum uptake rate of the signal (amax), and the time at which the maximum slope occurs, i.e., the inflection point of the sigmoidal curve (tmax). Generally, the amax for all the CPPs increases linearly with increasing concentration. The change is most pronounced for EB1 peptide, exceeding at least two times the dependence between amax and concentration for all the other tested peptides; and interestingly, the amax for penetratin and pVec does not significantly depend on the peptide concentration at all (data not shown). When analyzing the time where the maximum slope of uptake (i.e., the maximal rate of uptake) occurs and how this depends on peptide concentration, we observed that the data points are clustered into two subareas. The maximal rate of uptake took place after 30-60 min incubation for most of the CPPs, but 10 µM EB1, 5 µM TP10, and 2.5 µM MAP displayed a maximal rate already after 1-10 min incubation despite that these peptides belong to the “slow” uptake group at these concentrations (Figure 6A,B).

DISCUSSION Comparison of the uptake potency of new and old CPPs is a flourishing and important research field. Several assays have been used for this purpose that rely on different readouts from distinct cellular compartments (38). This sometimes gives disparate results, which can be difficult to interpret and compare, especially if the whole study is not carried out within the same research group and with same experimental conditions. This is the first time a comparative uptake study on several CPPs is carried out with the releasable luciferin assay (23). We

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measured cytosolic cargo delivery kinetics of eight disulfide conjugated luciferin-CPP constructs (Table 1), where luciferin and the CPP are separated by a linker (Figure 1). The readout is produced by the processes described in Figure 2. We measured the rate of the CPP internalization by registering luminescence versus time and calculated cumulative uptake (total uptake) by integrating the kinetic curve for specific time-points. Free luciferin is capable of entering cells without any transport vector (39) with much faster and distinct kinetic profile than luciferin-transporter conjugates (23). Most CPPs, on the other hand, do not possess direct membrane translocation properties. Instead, various endocytic processes have been found to be involved in peptide-mediated cargo delivery. Nevertheless, several reports have concluded that direct translocation cannot be ruled out for unconjugated CPPs. For example, translocation of free Tat peptide has been reported to be independent of endocytosis, since the peptide frequently internalizes at low temperature and into genetically modified cells lacking certain endocytic pathways (40). MPG and Pep are two other peptides that also translocate cells via direct penetration mechanisms, even when conjugated to cargos (8, 15). Additionally, based on microscopy observations and flow cytometry experiments, there seems to be a certain concentration threshold above which the direct translocation could be favored (41, 42). This is consistent with our results where we observe similar kinetic profiles in the uptake of membrane permeable free luciferin and luciferin-Tat conjugate in complete media. However, numerous other studies have shown involvement of endosomal pathways for Tat (21, 43, 44) with an uptake profile that does not resemble our “fast” uptake curve (45). We also observe free luciferin-like “fast” uptake profile for 10 µM luciferin-TP10 and 5-10 µM luciferin-MAP. While both TP10 and MAP have been reported to be taken up by endocytosis (21, 37), the behavior of TP10 in our case does not reflect that. It raises a question whether it can partially be explained by the minor cytotoxic effects observed in the LDH assay after 2 h incubation and also with specific TP10 membrane interactions (46). However, minor leakage-induced glutathione outflow should not have a significant impact on the extracellular cleavage of the conjugates, since the volume of incubation media compared to volume of a cell is very large. Additionally, our direct measurement of glutathione outflow after treatment with TP10 indicated no effect after 30 min when the incubation was carried out in PBSg buffer. Hence, we hypothesize that the amount of internalized peptide can be overestimated only if the conjugates leak into the cell. It has been observed in the past that TP10, depending on the cargo molecule, induces different levels of LDH leakage from HeLa cells (34). Additionally, under certain circumstances this peptide forms transient pores in large unilamellar vesicles (LUVs) and promotes calcein leakage from these vesicles (46). However, when small membrane disturbances (lower concentrations in our case) are experienced, the main translocation mechanism is still endocytosis (46). In the case of MAP peptide, the free luciferin-like uptake behavior is somewhat more complicated to interpret, and care must be taken when analyzing the results. We observe slight toxicity of luciferin-MAP treated cells, although previous results have shown high toxic effects of MAP peptide, which have been attributed to its high amphipaticity (35). However, the toxicity was negligible in the first 0.5 h incubation, while we still observed an increase in extracellular thiol content in this time frame which was not present after 15 min, as measured in PBSg buffer. It is therefore unlikely that membrane disturbance and/or extracellular glutathione explain the exponential profiles that both peptides, TP10 and MAP, displayed at these concentrations, because that fast uptake process started instantly after the peptide treatment. On the other hand, it has previously

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been shown (41, 42, 47) that the mechanism of cellular entry sometimes changes from endocytic to direct translocation with increasing CPP concentration, which could also be the key factor here. In contrast, it has also been observed that MAP is highly endocytosed into cells, specifically into the nuclei (48), although MAP remains a quite inefficient peptide in some nuclear delivery assays (21). This controversy might be explained, to some extent, by calcium inflow triggered membrane repair response (MRR) which may mask membranolytic effects of this peptide (49). The MRR could lead to less observed LDH leakage while allowing high and fast direct uptake of MAP peptide. None of the other peptides (EB1, penetratin, M918, pVec, TP10(Cys), 1-5 µM TP10, and 1-2.5 µM MAP) that we tested follow free luciferin-like fast uptake. However, the uptake profile of penetratin displayed a characteristic dip in the first minutes of incubation, which indicates that a small portion of this CPP enters the cytosol quickly. This is consistent with previously published results that state that direct translocation for this peptide cannot account for its efficient uptake (50). These results emphasize the importance of endocytosis in this process (50). The peptides which fall into the “slow” internalization category display similar uptake characteristics. In most cases, the recorded luminescence flux slowly increased in time, but after a certain time point, it reached a plateau and started to decrease. This indicates that the internalization rate reaches a characteristic point where the increase in uptake starts to decrease (i.e., the uptake rate starts to approach a certain equilibrium) and that the luciferin-luciferase reaction occurs more rapidly than the uptake of luciferin-CPP constructs. On the other hand, when interpreting these results it is important to remember that side products of the luciferin-luciferase reaction, namely, oxyluciferin and dehydroluciferyl adenylate, can influence the readout, since these compounds may inhibit the reaction to some extent (51). The key factor of the releasable luciferin assay is a cytosolic release of luciferin from the carrier molecule. It seems to be a common belief that a disulfide bond is suitable when cytosolic delivery is studied, because it is supposed to be cleaved only in the cytoplasm due to high concentration of glutathione (52). However, there are some discrepancies in the literature. The disulfide bridge approach has been extensively used in many studies in the field (21, 53-56) despite some concerns that disulfide bonds may be reduced partially on the cell membrane of some cells and in vesicular compartments by protein disulfide isomerase or gamma-interferon-inducible lysosomal thiol reductase (57). It has been shown that the main reduction, nevertheless, takes place in the cytoplasm (58). Then again, a recent publication claims that the reduction occurs extracellularly and is dependent on the peptide sequence (59), which means that the reduction potency may have to be established for each peptide. Despite all these controversies, we did not observe any increase in extracellular sulfhydryl content (i.e., disulfide bond cleavage or glutathione outflow) in the extracellular environment in a relevant time frame, except in the case of TP10(Cys) after 1 h (Figure 3A). MAP and Tat undeniably showed some increase in extracellular sulfhydryl content after 30 min (Figure 3A), which was not observed after 15 min incubation (data not shown). However, due to technical reasons this experiment was carried out in PBSg buffer and not in complete media as the uptake experiments, and it is known that the peptides could be considerably more toxic in buffers than in cell growth media. Also, the Ellman’s reagent cleavage rate by cells in complete media was much slower than the uptake rate of the CPP conjugates (data not shown). Our uptake results (Figure 5A) are in accordance with previous results from the splice correction assay with TP10(Cys), M918, and penetratin, showing high cytosolic entry while pVec,

Eirı´ksdo´ttir et al.

Tat, and MAP fall into the low efficacy group (21). pVec has been shown to display excellent uptake according to fluorometric methods but fails to induce oligonucleotide-mediated splice correction, indicating that pVec remains entrapped in endosomal vesicles. Here, we find that pVec matches Tat, MAP, and TP10 in cytosolic entry after 2 h treatment. However, it should be noted that the uptake of peptides belonging to the fast exponential internalization group may be somewhat underestimated due to approximately 30 s lag between adding the peptide to cells and starting the measurement, during which large signals may be left unregistered. The advantage of using the luciferin-releasable linker-CPP method over the splice correction assay is that the results of the former assay can be obtained faster, and more importantly, information about kinetic behavior can also be obtained. A caveat with the assay is possible artifacts emanating from potential premature reduction of the disulfide bridge in the luciferin-CPP conjugate. However, we observe the same trend in uptake as with the splice correction assay, and thus, extracellular reduction does not appear to be a problem here. In fact, while extracellular reduction of luciferin-CPP conjugate would generate a high readout, corresponding CPP-PNA reduction would give a low readout, because free PNA does not enter cells. Reduction of the conjugates should therefore give opposite results, which is not the case. Furthermore, when establishing the releasable luciferin-transporter assay, Jones et al. verified that the main portion of the measured CPP internalization was due to uptake of the luciferin-CPP conjugate and subsequent release of the luciferin rather than direct translocation of luciferin after extracellular reduction of some portion of the luciferin-CPP conjugate (23). Due to serum proteases or negatively charged serum proteins, CPPs are likely degraded or retained in extracellular environment to some extent in the presence of serum (60-62). CPPs are also susceptible to extracellular degradation in buffer, probably caused by membrane-bound proteases (37). However, owing to the relevance of serum in in vivo studies, we measured the uptake of CPPs in complete media containing 10% serum and compared it to the uptake in HKRg buffer. The differences in results were apparent; the peptides showed higher internalization in buffer than in media with up to 28-fold difference. Also, the CPPs that showed the best characteristics in terms of uptake rate and total uptake when incubating the conjugates in complete media were not necessarily the best cargo carriers when the incubations were carried out in HKRg. It has been widely noted that peptides that are more positively charged and/or have higher amphipathic moments often display more cell-penetrating capabilities than others. However, there is no obvious correlation between the peptide uptake (30 min, 60 min, or 120 min) or LDH leakage with their hydropathy score or amphipathic moments according to our measurements (data not shown). Nevertheless, the two peptides with the highest above-mentioned parameters, MAP and TP10, were the only ones showing significant LDH leakage. As mentioned before, only one time-point measurement may not reflect the overall properties of a certain carrier molecule. In order to draw conclusions from the kinetic behavior of our conjugates, a modified logistic function according to eq 1 was fitted to the raw luminescence data for the slow uptake profiles. Next, we calculated the average values and SEM of the maximum slope of the signal (amax) and the time at which the maximum slope occurred (tmax), i.e., the inflection point of sigmoidal uptake curves, which is often compared with halflife of uptake in the literature. The amax differs among CPPs, but the value is generally higher at higher peptide concentrations. However, we did not observe any drastic changes in amax, and tested CPPs could not be

Uptake Kinetics of Luciferin-CPP Conjugates

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Figure 7. Hypothetical entry routes of luciferin-CPP conjugates according to their internalization kinetics. The conjugates belonging to the “fast” internalization group (Tat, TP10*, MAP*) cross the cell membrane rapidly (iA), while representatives of the “slow” kinetics group (pVec, penetratin, EB1, M918, TP10(Cys), TP10*, MAP*) go through endocytosis (iB) and reach the cytosol after endosomal escape (iC) followed by the release of free luciferin (ii) and production of light (iii). The data reveal that CPPs marked with * (see text) differ in their endosomal escape rate and ability to move between the “fast” and the “slow” internalization groups according to their concentration.

grouped into subpopulations based on this parameter. The only exception was perhaps EB1, which displayed the most pronounced dependence between amax and concentration, exceeding at least 2-fold the other CPPs we tested. This may indicate the presence of a certain peptide concentration threshold level above which endosomal escape of the conjugate is initiated more rapidly. The time at which amax is found (tmax) differs among CPPs as well. According to our model, tmax for higher concentrations of EB1, TP10, and MAP conjugates is very short, less than 10 min, whereas in the case of the other tested CPPs, the tmax is in the range 30-60 min and it remains at similar levels for all concentrations. Interestingly, for lower concentrations of EB1, TP10, and MAP conjugate the tmax increased to comparable levels with the other CPP conjugates (Figure 6A,B). This indicates that the delay in uptake increases significantly, which in turn might show that at lower concentrations the mode of uptake for these peptides could be different or there could be a phase change in the endosomal escape rate. The tmax value can be compared with presented half-life of uptake from the literature. For example, 1 µM penetratin in a work which addressed cytosolic delivery of CPPs into Bowes cells using a quenched fluorescence assay had a half-life of uptake of ∼60 min (45). In the same work, the half-lives of Tat, transportan, and MAP were approximately 30, 10 and 7 min, respectively (45). This is in line with earlier works where the half-life of radioactively labeled transportan was found to be around 10 min (4, 63), and this is analogous to our results except in the case of the Tat-luciferin conjugate. In addition, sigmoidal uptake curves were not observed in the abovementioned studies, which may reflect involvement of more processes in our case (such as linker cyclization and enzymatic conversion of free luciferin), different cell lines and incubation conditions. Interestingly, 1 µM NBD-labeled penetratin internalized in a similar time frame into K562 cells, but the signal could have arisen largely from a portion of conjugates trapped in endosomes due to experimental setup (64). In opposition to this, penetratin has been observed to have rapid uptake in the first 15 min, and the authors discuss involvement of a direct penetration mechanism in addition to endocytosis (65); but a penetratin analogue entered CHO cells with a half-life of

approximately 30 min, as assessed from an uptake kinetics graph measured by mass spectrometry, while the entry into GAGdeficient CHO cells occurred slightly more rapidly but yet to a remarkably lower extent (66). It must be noted that the authors find the internalization of naked penetratin to be a multimechanism process with a clear temperature-independent component which was more pronounced at lower concentrations in the case of GAG-deficient cells, which does not completely agree with our results and hypotheses. Kinetics of unconjugated 5 µM penetratin, Tat, polyarginine, and transportan have been compared in A549, HeLa, and CHO cells in another study from 2005, but unfortunately, the authors do not provide any numerical data regarding kinetics, and the comparisons can only be made on a qualitative basis (67). However, it is apparent that in different cell lines the kinetics, and also the total uptake, is different. For instance, in their case the half-life of uptake appears to be 1-2 h for penetratin, 0.5-1 h for Tat, and ∼0.5 h for polyarginine and transportan (67). Additionally, the authors emphasize involvement of various endocytic pathways in the internalization process; however, the internalization kinetics seems to depend also on the presence/ absence of a peptide cargo (67), which should be considered when comparing our data. FACS analysis with 10 µM Tat showed similar internalization kinetics as endocytosis markers in another study, both at physiological temperature and at +4 °C. The uptake had not reached a plateau after 60 min, and the half-life of uptake could not be estimated (68). However, the uptake of Tat was saturated after 90 min incubation in another experiment where it took place quickly, allowing internalization of half of the final peptides in less than 10 min (69). This adds more controversy to uptake kinetics studies of this peptide in light of our results where we observe evidence of direct penetration of this peptide. The kinetic curves of free and plasmid complexed Tat peptide have been also compared. In a work conducted on CHO cells, the authors observed different kinetic profiles for free and plasmid-complexed Tat peptide (70). While the uptake of free Tat peptide resembled a first-order process with a half-life about 15 min, as assessed from the figures, the uptake of plasmid complexes increased linearly with time. In addition, more interestingly, unlike in the case of Tat-plasmid complexes, the

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uptake of free Tat peptide did not depend on sodium azide, an inhibitor of ATP-dependent cellular transport (70) which supports our data of this peptide belonging to the “fast” internalization group. When Tat peptide was used to deliver a cargo peptide into cytosol, it was shown to enter cells following a sigmoidal uptake curve with the maximal rate of uptake occurring approximately at 30 min starting from the incubation (52). To demonstrate the role of a cargo peptide to the uptake kinetics even more, the existing well-established peptides were modified with short penetration accelerating sequence (Pas), and the uptake curve, approximately linear in time, was changed to a first-order like curve with an approximate half-life of 15 min for polyarginine in full media (71). This work, as all the other mentioned kinetics studies, emphasizes quite strongly how important the experimental conditions are when comparing different measurement results. Just as the cell lines (e.g., suspension cells vs adherent cells) used in the discussed experiments could change the results, different cargoes play an important role here too. Therefore, when comparing the uptake kinetics of CPPs this should be especially strongly kept in mind. In summary, our results demonstrate that in complete media there are two types of uptake kinetic profiles for the CPPs tested herein: either an early uptake that reached maximum within minutes or a later type of uptake with maximum rate that varied from 30 to 60 min. Tat, luciferin, 10 µM TP10, and 5-10 µM MAP belong to the fast uptake group. The rest of the peptides (EB1, penetratin, M918, pVec, TP10(Cys), 1-5 µM TP10, and 1-2.5 µM MAP) fall into the slower uptake category. We hypothesize that, since free luciferin can cross plasma membranes directly without being endocytosed, the CPPs whose uptake profiles are similar to the profile of free luciferin (i.e., the fast uptake group) are also capable of crossing cell membranes directly. CPPs with the slow uptake profile, on the other hand, are presumably exploiting endocytic routes when entering cells (Figure 7). The clustering and behavior of luciferin-CPP conjugates fall in line with a CPP structural versatility and polymorphism study except for the Tat peptide (72). Precisely which endocytic routes are exploited will be tested in a follow-up study, and the work is ongoing regarding this matter. The peptide conjugates whose kinetic parameters amax and tmax differ to large extent at various concentrations might display dual uptake modes as opposed to other CPPs. This is not surprising for the higher concentration conjugates of TP10 and MAP, which shift from the fast internalization group into the slow internalization group when their concentration is decreased, but for the EB1 conjugate, this finding is very interesting. It seems that indeed there might be a group of CPPs whose mode of uptake could depend significantly on their concentration and that a phase change of uptake might explain deviating results found in the literature. In conclusion, we find that, when comparing CPPs in terms of their uptake and internalization mechanisms, kinetic data provide a valuable additional tool to conventional end-point measurements with various biological assays. Our data also indicate that this luciferin-CPP system as a (semi)biological assay is a promising tool for studying uptake mechanisms of CPPs more thoroughly, since it is capable of shedding more light onto CPP subgroups which could remain hidden if kinetic data of conventional biological readout assays are discarded.

ACKNOWLEDGMENT The work presented in this article was supported by Swedish Research Council (VR-NT); by SSF (SwedensJapan); by Center for Biomembrane Research, Stockholm; by Knut and Alice Wallenberg’s Foundation; by the EU trough the European Regional Development Fund through the Center of Excellence in Chemical Biology, Estonia; by the targeted financing

Eirı´ksdo´ttir et al.

SF0180027s08 from the Estonian Government; by the DoRa Program of The European Social Fund; and by Archimedes Foundation. Conflicts of interest: none.

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