1 of 10Journal of Peptide Science, 2025; 31:e70014 https://doi.org/10.1002/psc.70014 Journal of Peptide Science RESEARCH ARTICLE No Evidence for Plasma Membrane Potential- Independent Cell Penetrating Peptide Direct Translocation Ali Hallaj | Francisco Tomas Ribeiro | Christian Widmann Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland Correspondence: Christian Widmann (christian.widmann@unil.ch) Received: 28 January 2025 | Revised: 19 March 2025 | Accepted: 24 March 2025 Funding: This work was supported by the Swiss National Science Foundation (310030_207464). Keywords: cell- penetrating peptides | direct translocation | Plasma membrane potential ABSTRACT Cell- penetrating peptides (CPPs) are small peptides that can carry bioactive cargoes into cells. CPPs access the cell's cytosol via direct translocation across the plasma membrane. We and others have shown that direct translocation of CPPs occurs through water pores that are formed upon hyperpolarization of the cell's membrane. Direct translocation through water pores can there- fore be blocked by depolarizing the plasma membrane. Other direct translocation mechanisms have been proposed that would not rely on membrane hyperpolarization. It has been reported, for example, that in HEK cells, CPP translocation occurs in a plasma membrane potential- independent manner, in contrast to HeLa cells, where CPP access to the cytosol required plasma membrane hyperpolarization. To address these apparent discrepant data, we have tested the requirement of plasma membrane hyperpolarization in a series of cell lines, including HEK and HeLa cells, for CPP direct translocation. Our data, obtained from a wide range of CPP concentrations, show that efficient direct translocation always requires plasma membrane hyperpolarization. We discuss the possible reasons why earlier studies have not evidenced the importance of the plasma membrane potential in the cytosolic uptake of CPPs in some cell lines. 1 | Introduction Cell- penetrating peptides (CPPs) are short cationic aminoacid sequences that have the capacity to enter cells. CPPs can trans- port various substances into cells, making them valuable tools for drug delivery and molecular research [1]. CPPs employ two mechanisms for cell entry: direct translocation across the plasma membrane and endocytosis. The entry is influenced by differ- ent factors including the concentration of the CPP, its charge, the pH of the medium, temperature, the presence of serum, and cell- membrane structure and curvature [2–5]. At lower concen- trations (≤ 1–10 μM), the main entry pathway is generally endo- cytosis [6–10], whereas direct translocation becomes prevalent at higher concentrations [6, 8, 9, 11–13]. Several studies have provided modeling, genetic, and cellular ev- idence that CPP direct translocation occurs through water pores, protein- free water columns spontaneously formed in highly polar- ized (megapolarized) membranes [11, 14–18]. Megapolarization is achieved through the combined activity of potassium channels and the accumulation of positively charged CPPs that can bring the membrane potential (Vm) down to values of −150 mV and lower [11]. CPPs appear to cross the plasma membrane at discrete re- gions called nucleation zones [19]. These zones are probably where the plasma membrane has been sufficiently polarized to allow the creation of water pores allowing CPP access to the cytosol [19]. Other models for CPP direct translocation are regularly mentioned in reviews (e.g., toroidal pore, barrel- stave pore, and inverted © 2025 European Peptide Society and John Wiley & Sons Ltd. https://doi.org/10.1002/psc.70014 https://orcid.org/0000-0002-1438-2842 https://orcid.org/0009-0002-5212-1739 https://orcid.org/0000-0002-6881-0363 mailto: mailto:christian.widmann@unil.ch http://crossmark.crossref.org/dialog/?doi=10.1002%2Fpsc.70014&domain=pdf&date_stamp=2025-04-04 2 of 10 Journal of Peptide Science, 2025 micelle). It is important to realize that these alternative models are derived from acellular approaches (e.g., neutron in- plane scat- tering) and that they are only supported by a handful of studies [20–23]. In contrast to the water pore Vm- dependent mechanism, no live cell or genetic evidence is available to support these models. Although CPP entry into cells through Vm- dependent water pores is now a widely accepted mechanism for CPP direct translocation, there are data in the literature that point to other possible CPP entry modes across the plasma membrane. For example, it has been reported that cytosolic acquisition of the R9 CPP (a CPP made of nine arginine residues) in the HEK cell line is not affected by membrane depolarization, whereas it is in HeLa cells [19]. It was therefore suggested that CPP di- rect translocation in HEK cells proceeds in a Vm- independent manner. In the present work, CPP direct translocation was evaluated in four different cell lines, including the HEK cells, using a broad range of CPP concentrations. At high CPP concentrations, CPP cytosolic acquisition was systematically inhibited by cell de- polarization, even in HEK cells. At lower CPP concentrations, a very weak CPP- derived cytosolic signal could sometimes be detected, but this signal was not affected by cell depolarization. This weak signal amounted to only a few percent of the cytoso- lic CPP signal acquired through the Vm- dependent mechanism, and it was not different from the background fluorescence sig- nal found outside of cells after extensive washing. This work supports the notion that there is only one efficient mechanism of CPP translocation across the plasma membrane and that this mechanism crucially depends on adequate plasma membrane polarization. 2 | Materials and Methods 2.1 | Peptides Peptides were synthesized by SB- PEPTIDE, Lyon France in the L- conformation. CPPs were coupled to 5- TAMRA at their N terminus through an aminohexanoic acid linker (AHX). The peptides used were TAMRA- R9 (NH2- TAMRA- AHX- RRRRRRRRR- COOH), TAMRA- Penetratin (NH2- TAMRA- AHX- RQIKIWFQNRRMKWKK- COOH), and Cy5- R9 (NH2- Cy5- RRRRRRRRR- COOH). 2.2 | Cell Culture and Culture Media HeLa cells were cultured in 5% CO2, 37°C incubators in RPMI 1640 (Invitrogen, #61870) supplemented with 10% heat- inactivated fetal bovine serum (FBS) (Invitrogen, A5256701). HEK293T (HEK cells), LAN1, and U2OS were cultured in DMEM supplemented with FBS. Cells were grown up to 80% confluency and then trypsinized as follow. The culture me- dium was aspirated, and the cells were washed with 10 mL phosphate buffer solution (PBS) (Bichsel; #1000324) at room temperature. Then, 1 mL of trypsin–EDTA (Gibco; #25300– 054) was added to the plate and incubated for 5 min at 37°C. Then, 4 mL of medium was added, the cells were resuspended by up and down pipetting, and the cells were collected in Falcon tubes (Corning; #430829) and centrifuged for 3 min at 192g. The supernatant was then aspirated, and the pelleted cells were resuspended in 8 mL complete medium. An aliquot was then transferred to a Neubauer cell to determine the cells' concentration. For all experiments, 2 × 105 cells were seeded on glass bottom plates (MatTek; #P35G- 1.5- 14- C). Cells used in this study were routinely tested for mycoplasma and found not contaminated. 2.3 | CPP Treatment and Plasma Membrane Depolarization For nondepolarized samples: 2 × 105 cells on MatTek plates were treated with different concentrations of the CPPs ranging from 2 to 20 μM for 10 min. After treatment, the cells were washed three times with 1 mL of PBS. Finally, fresh complete medium was added to the plates (RPMI for HeLa and DMEM for U2OS, HEK, and LAN- 1). To induce plasma membrane depolarization, cells were treated with 4 μg/μl of gramicidin for 10 min before the addition of the CPPs. 2.4 | Confocal Imaging Images were taken with a Leica Stellaris 8 confocal microscope using a Leica HC PL APO CS2 63x/1.40 oil immersion objec- tive (Leica Microsystems, Buffalo Grove, IL, United States). Image settings were as follow: scan speed 400 Hz, image size 2048 × 2048 pixels (uncropped), 16- bit acquisition (allowing 65,536 gray level acquisition), zoom 1×, pixel size 0.142 μm. Laser settings were 2% intensity and 2.5% gain, except for HEK cells where the laser intensity was set to 5%. In Figure 1B, two views of the same images are shown but with different contrast adjustments. In the left images, the contrast was adjusted to a wider range (0–20,751 gray levels) to clearly show the cytosolic signal without oversaturation. In the right images, the contrast was adjusted to a narrower range (0–6000 gray levels) to high- light endosomes. It is important to note that these contrast ad- justments were applied only for visualization purposes. When quantifying the CPP signal, the original unadjusted image data were used to ensure accurate measurements. 2.5 | Measuring Cell Viability In a six- well plate (Corning Costar, #3516) , 2 × 105 HeLa cells were seeded. The following day, the medium was changed; the cells were then treated with increasing concentrations of TAMRA- R9 for 10 min. The cells were then washed three times with 1 mL of PBS and then trypsinized. The cells were collected in Eppendorf tubes using fresh culture medium. The cells were centrifuged at 192g for 3 min, the supernatant was then aspirated, and the cells were washed with 1 mL of PBS and centrifuged again at 192g. The supernatant was then removed, and the cells were resus- pended in 50 μL PBS. In a separate tube, 25 μL of Trypan Blue Stain (0.4%) (Gibco; #15250- 061) was added, followed by 25 μL of the cell suspension, and after mixing, the cells were transferred to a Neubauer chamber where the number of live (refringent), and dead (blue) cells were counted. The % cell viability was cal- culated as 100 − [blue cells/(blue + refringent cells)*100]. 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 3 of 10 FIGURE 1 | Legend on next page. 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 4 of 10 Journal of Peptide Science, 2025 2.6 | Resting Membrane Potential Measurement At 37°C, 200,000 HEK, HeLa, U2OS, or LAN1 cells were trans- ferred to Eppendorf tubes and incubated 30 min, in 5% CO2 in- cubator, with 100 nM of DiBAC4 [3] (Thermo Fisher, #B438), a slow- response potential- sensitive probe. A standard curve specific to each cell line was established. Six tubes contain- ing 200,000 cells were incubated with 4 μg/mL gramicidin for 10 min. This was followed by the addition of DiBAC4 [3] in in- creasing concentrations across the six tubes (0, 50, 100, 200, 400, and 800 nM). Samples' fluorescence intensity was then mea- sured by flow cytometry. The curve allows for the attribution of a fluorescence intensity value to a given cytosolic concentration of DiBAC4 [3]. The final membrane potential readings were cal- culated using the Nernst equation as described before [24, 25]. 2.7 | Flow Cytometry In 24- well plates (Corning Costar, #3526), 60,000 cells were plated. The following day, depolarization was induced by incu- bating cells with 4 μg/mL gramicidin for 10 min. Cells were then incubated with the indicated CPP concentrations for 10 min at 37°C. Cells were then washed and trypsinized. After cen- trifugation, the pellet was resuspended in sorting buffer (PBS, 0.5 mM EDTA, 5% FBS). The buffer, before the addition of FBS, was filtered using PES membrane 0.22 μm filter (Cobetter Lab; #SFM33PE0022S). Flow cytometry was then performed using a CytoFLEX S (Beckman Coulter) apparatus, and 10,000 cells were recorded per time point. 2.8 | Image Analysis and Quantification All image analysis was performed using Fiji (Version 1.53q, NIH, United States). To visualize the cytosol, LUTs (look- up tables) of images were set to 0–6000 grey levels . A region of interest (ROI) in the cytosol was selected avoiding any endosomal/vesicular structure and then the unadjusted raw value in the ROI was quantified. The median fluorescence intensity was plotted in the graphs. To visualize endosomes in cells, the LUTs of images were adjusted to 0–2000 grey levels, and then the number of endosomes per cells was manually determined. 2.9 | Statistical Analysis All graphs and statistical analysis were done using GraphPad Prism version 10.1.0 (316). 3 | Results It has been reported in HEK cells that the CPP endosomal up- take and the CPP cytosolic signal increase at similar rates as the CPP concentration rises, in contrast to what is seen in HeLa cells where the rate of cytosolic signal acquisition outruns the rate of CPP endosomal uptake as CPP concentrations increase [19]. It was also reported that CPP direct translocation in HeLa cells could be blocked by plasma membrane depolarization, whereas CPP translocation in HEK cells was not affected by depolariza- tion. These two sets of results ([i] variations between the rates of CPP endocytosis and CPP translocation and [ii] differential ef- fects of depolarization on CPP translocation) were interpreted as providing evidence for two distinct modes of CPP translocation. 3.1 | CPP Entry Into Cells in Response to Increasing CPP Concentrations To evaluate the first argument further, that is variations be- tween the rates of CPP endocytosis and CPP translocation, we established a dose–response curve of cytosolic acquisition of the R9 CPP into four different cell lines incubated (Figure 1A). Because CPPs do not escape endosomes or very inefficiently [9, 26–28], the values in this figure mostly, or only, correspond to CPP cytosolic acquisition by direct translocation. At the lowest concentration tested (2 μM), if a cytosolic signal was detected, it matched that of the background extracellular signal. At CPP concentrations ≥ 5 μM, the cytosolic CPP signals sharply in- creased, by a factor of 10 or more at the highest concentration tested (20 μM). A dose–response curve of the number of CPP- containing endosomes acquired by cells as the CPP concen- tration was increased was also established (Figure  1A). The number of CPP- containing endosomes per cell already reached its maximum at the lowest CPP concentration tested. Our interpretation of the data shown in Figure 1 is as follows. As indicated in Section 1, CPP enters cells by endocytosis and by di- rect translocation. The amount of CPP that cells can take up does not generally increase linearly as the CPP concentration in the ex- tracellular medium increases. This stems from the fact that at low CPP concentrations, CPP enters cells mainly through endocytosis. At higher concentrations, endocytosis does not increase further, whereas CPP direct translocation starts to occur, and the amount of CPP entering cells via this route quickly exceeds the amount of CPP that was endocytosed (Figure 1B). Hence, CPP uptake occurs in two stages as the CPP concentration increases: a first stage cor- responding mainly to endocytosis and a second stage where direct translocation predominates. The amount of CPP taken up by cells FIGURE 1 | No parallel increase in CPP cytosolic acquisition and endosomal uptake. (A) LAN1, HeLa, HEK, and U2OS cells were treated with increasing concentrations of TAMRA- R9 (0–20 μM) for 10 min before three PBS washes. The fluorescence cytosolic signal was then measured. Blue circles correspond to the cytosolic CPP signal; red circles correspond to the extracellular signal (background signal). The number of endosomes per cell was also determined (green circles). Data were acquired from three independent experiments for a total of 85–446 measurements for cytosolic quantitation and from 40 cells for endosome counting. (B) Representative confocal images of the four cell lines used. The images on the left- hand side allow the visualization of the cytosolic signal without saturation. The images on the right- hand side represent the same cells but in conditions favoring the visualization of endosomes at low CPP concentrations. These conditions lead to saturation of the cytosolic signal at higher CPP concen- trations (see Section 2 for details). Scale bar 10 μm. 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 5 of 10 in the first stage can be order of magnitude lower than in the sec- ond stage. These two stages were observed in the four cell lines tested, in compliance with previous work that have shown that CPP concentration affects the uptake mechanism of CPPs (endo- cytosis vs direct translocation) [8, 9, 29, 30]. The CPP used in Figure 1 was R9. To determine if the two stages described above in HEK cells could also be seen with another CPP, we repeated the experiment with Penetratin. As shown in Figure 2, there was again a sharp increase in the cytosolic signal at Penetratin concentrations above 5 μM. The pattern of cyto- solic acquisition in HEK cells is therefore similar when R9 and Penetratin are used, and this pattern is not different from what is seen in other cell lines. 3.2 | CPP Entry Into Cells at High CPP Concentrations Is Plasma Membrane Potential (Vm)- Dependent To address the second argument, that is some cell lines take up CPPs by direct translocation independently of the plasma mem- brane potential [ 19, 31], we determined in the four cell lines tested in Figure 1, the effect of plasma membrane depolarization on R9 cytosolic acquisition at various CPP concentrations. As seen in Figure 1, at the lowest concentration tested (2 μM), if a cytosolic signal was detected, it matched that of the background extracellular signal (Figure 3A). At 5 μM of the CPP, a few cells started to acquire the CPP in the cytosol, and this cytosolic sig- nal could be abrogated by cell depolarization. At 15 μM, where direct translocation predominates, plasma membrane depolar- ization induced by gramicidin blocked CPP direct translocation in all the tested cell lines, including HEK cells (Figure 3A,B). Cytosolic acquisition of Penetratin at 15 μM in HEK cells was also inhibited by cell depolarization (Figure 2). As an alternative method to assess CPP cellular uptake, we used flow cytometry. Note, however, that this technique does not discriminate the cytosolic CPP signal from the endosomal CPP signal. The flow cytometry signal obtained after incubat- ing the four cell lines with 10 μM of the R9 CPP was efficiently reduced by gramicidin- induced depolarization (Figure  4). At 5 μM, gramicidin did not affect the cellular CPP signal. This was expected because at this concentration, CPP acquisition proceeds mostly through endocytosis (see Figures 1B and 3A,B). The point to note here is that plasma membrane depolarization greatly affected direct translocation in the HEK cell line. There is therefore no indication that the mechanism of CPP direct translocation is different in HEK cells compared to HeLa cells. 3.3 | Analysis of Various Parameters That Can Potentially Affect CPP Direct Translocation A report by the laboratory of Hanne Mørck Nielsen has suggested that TAMRA dye coupled to CPPs induces some cytotoxity and/or membrane perturbations [32]. In this report, viability was assessed using small molecules of less than 1 kDa. Such small molecules, especially if they are positively charged as is the case for the anth- racycline derivative DRAQ7 used in the mentioned report, have the potential to cross the plasma membrane through the water pores created as the plasma membrane gets megapolarized by the presence of CPPs [11]. The movement of these viability dyes across the plasma membrane can therefore be favored by CPPs, and this can possibly generate a confounding signal that artefactually ex- aggerates CPP cytotoxicity. To monitor the cytotoxic potential of the TAMRA- R9 compound used in the present study, we used try- pan blue, a negatively charged viability dye. Figure 5A shows that TAMRA- R9 does not display cytotoxicity at the doses used in the present work (≤ 20 μM) to monitor its uptake by cells. It appears therefore that cytotoxicity is not a confounding factor impeding the interpretation of the present data on CPP translocation. To fur- ther explore a possible uniqueness of TAMRA in favoring a plasma membrane potential- dependent CPP translocation, we coupled R9 to Cy5, another fluorophore. Figure 5B shows that the transloca- tion of Cy5- R9 into the cytosol of HeLa cells can also be blocked by depolarization induced by gramicidin. Hence, the plasma mem- brane potential dependence for the direct translocation of R9 into cells does not rely on the fluorophore the CPP is coupled to. We have shown that the activity of potassium channels that sets the resting membrane potential can be critical for the ability of CPPs to translocate into cells [11]. Figure  5C reports that the FIGURE 2 | Penetratin direct translocation operates as for R9. HEK cells were treated with increasing concentrations of TAMRA- Penetratin (0–15 μM) with or without gramicidin. The fluorescence cytosolic signal was then measured. Blue and purple circles correspond to the cytosolic signal of cells that were not treated or treated with gramicidin, respectively. Red circles correspond to the signal measured in extracellular regions (background signal). Data were acquired from three independent experiments for a total of 58–107 measurements. 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 6 of 10 Journal of Peptide Science, 2025 FIGURE 3 | Cell- depolarization inhibits direct translocation in all the tested cell lines. (A) LAN1, HeLa, HEK, and U2OS cells were treated with increasing concentrations of TAMRA- R9. The fluorescence cytosolic signal was measured in cells preincubated or not with gramicidin as shown in Figure 2. Data were acquired from three independent experiments for a total of 53–270 measurements. (B) Representative confocal images of the four cell lines used (scale bar 10 μm). 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 7 of 10 resting membrane potential of the four cell lines used in the pres- ent work is comprised between −30 and −15 mV, with a relatively high variability between experiments. The Vm of HeLa and HEK cells were similar at around −28 mV. Intriguingly, the Vm of U2OS cells was higher than the other cells lines even though these cells were found to be more efficient than the others in R9 cytosolic acquisition (see Figure 1 for example). This indicates that the Vm is not the only parameter that modulates CPP direct transloca- tion. Other factors, such as the lipid composition of the plasma membrane [17, 33, 34], its stiffness and curvature [4, 35], and the capacity of membrane components to bind to CPPs [17, 36–38], can modulate the efficacy of CPP translocation. FIGURE 4 | Alternative measurement of fluorescence signal through flow cytometry. Quantification of fluorescence signal obtained by flow cy- tometry performed on cells treated with the indicated CPP concentrations following or not gramicidin treatment; data obtained from three to seven independent experiments. Examples of flow cytometry profiles are provided for each condition below the graphs. 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 8 of 10 Journal of Peptide Science, 2025 4 | Discussion It has been suggested by Wallbrecher and collaborators that HEK and HeLa cells take up CPPs in their cytosol using two different mechanisms (see Section  1), one regulated by the plasma membrane potential (HeLa cells) and one not (HEK cells) [19]. However, the present study provides evidence that cytosolic acquisition proceeds similarly in these two cells lines (and two additional ones) and that this cytosolic acqui- sition requires that the cells are polarized. Hence, our data do not support the existence of a CPP direct translocation mech- anism that would not be regulated by the plasma membrane potential of cells. What could be the reasons for the apparent discrepant findings between the earlier studies and the present one? One explana- tion could be that Wallbrecher and collaborators did not use high enough CPP concentrations to favor direct translocation over other forms of CPP cellular acquisition, such as endocytosis, or surface binding. In the present study, in HEK cells, strong CPP direct translocation in most of the cells was only seen at concen- trations above 10 μM (at 20 μM in particular). In the Wallbrecher study, the highest CPP concentration used was 10 μM [19]. As endocytosis already operates efficiently at a CPP concentration of 2 μM (see Figure 1A), one could argue that at 10 μM, CPP en- docytosis contributes significantly to the overall amount of CPP acquired by HEK cells when CPP uptake is quantitated by flow cytometry, which was the case in Figure 5 of the Wallbrecher study [19]. CPP endocytosis is not blocked by cell depolarization [10], and consequently, the CPP cell- associated signal in HEK cells incubated with 10 μM R9 might not be much affected by gramicidin. In HeLa cells, CPP direct translocation is already maximal at 15 μM (Figure  1), indicating that HeLa cells have a better capacity to acquire CPPs by direct translocation com- pared to HEK cells. This could explain why in the Wallbrecher study, the R9 cytosolic acquisition at the 10 μM concentration was inhibited by gramicidin in HeLa cells and not in HEK cells. Note, however, that in our hands, the cytosolic acquisition of R9 at the 10 μM concentration can be inhibited by depolarizing HEK cells (Figure  4). Variations in the culture conditions be- tween the Wallbrecher study and ours (at the level of the serum for example) may explain the difference in CPP uptake at a given concentration. One possibility of having a fluorescent signal in the cytosol at low concentrations or following depolarization is endosomal es- cape. However, we think that this is unlikely because CPPs are trapped in endosomes after entering cells by endocytosis [9, 39]. Although some cytosolic signal may be detected in some studies and attributed to endosomal escape, this signal can arise from direct translocation across the plasma membrane. Of note, such cytosolic signal only accounts for a fraction (< 3%) of what can be obtained in conditions allowing for efficient direct transloca- tion [9, 27, 28] and therefore remains marginal. FIGURE 5 | Analysis of various parameters that can potentially affect CPP direct translocation. (A) HeLa cells were treated with increasing con- centrations of TAMRA- R9. Cell viability analysis was assessed by Trypan blue staining. Data were acquired from three independent experiments. (B) Increasing concentrations of Cy5- R9 were added to HeLa cells pretreated or not with gramicidin and analyzed as in Figure 2. Data were acquired from three independent experiments for a total of 82- 204 measurements. (C) Resting membrane potential measurements of LAN- 1, HeLa, HEK, and U2OS cells. Data were acquired from three independent experiments (indicated by different color shades), each including three replicates (10,000 cells were analyzed per replicate). 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 9 of 10 An alternative hypothesis, which we would like to favor, is that the residual signal still occurs via partial membrane po- larization generated by cell surface- located CPPs even in the presence of gramicidin [11]. As mentioned above, the residual CPP translocation in gramicidin- treated cells only accounts for a tiny fraction of the total CPP direct translocation signal. Thus, in conditions of efficient CPP translocation, most CPP cytosolic acquisition relies on adequate plasma membrane hyperpolarization. 5 | Conclusion The present work demonstrates that plasma membrane depolar- ization inhibits CPP direct translocation in all tested cell lines. We therefore obtained no evidence that cell penetrating peptide direct translocation operates in a plasma membrane potential- independent manner. Author Contributions Conception and design of study: AH and CW. Acquisition of data: AH and FR. Analysis and/or interpretation of data: AH and CW. Drafting the manuscript: AH and CW. Revising the manuscript and approval of the submitted version: AH and CW. Acknowledgments This work was supported by the Swiss National Science Foundation (Grant 310030_207464 to CW). We are grateful to Dr. Evgeniya Trofimenko for critically reading the manuscript. We thank Luigi Bozzo and the Cellular Imaging Facility of the University of Lausanne for their support in image acquisition. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References 1. C. Bechara and S. 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Ohtsuki, “Cellular siRNA Delivery Medi- ated by a Cell- Permeant RNA- Binding Protein and Photoinduced RNA Interference,” Bioconjugate Chemistry 19 (2008): 1017–1024. 10991387, 2025, 5, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1002/psc.70014 by B cu L ausanne, W iley O nline L ibrary on [11/07/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://doi.org/10.3390/ph5111177 No Evidence for Plasma Membrane Potential-Independent Cell Penetrating Peptide Direct Translocation ABSTRACT 1   |   Introduction 2   |   Materials and Methods 2.1   |   Peptides 2.2   |   Cell Culture and Culture Media 2.3   |   CPP Treatment and Plasma Membrane Depolarization 2.4   |   Confocal Imaging 2.5   |   Measuring Cell Viability 2.6   |   Resting Membrane Potential Measurement 2.7   |   Flow Cytometry 2.8   |   Image Analysis and Quantification 2.9   |   Statistical Analysis 3   |   Results 3.1   |   CPP Entry Into Cells in Response to Increasing CPP Concentrations 3.2   |   CPP Entry Into Cells at High CPP Concentrations Is Plasma Membrane Potential (Vm)-Dependent 3.3   |   Analysis of Various Parameters That Can Potentially Affect CPP Direct Translocation 4   |   Discussion 5   |   Conclusion Author Contributions Acknowledgments Conflicts of Interest Data Availability Statement References