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\begin{document}

\title{Statin-boosted cellular uptake and endosomal escape of penetratin due to
reduced membrane dipole potential}



\author[1]{Gyula Batta}%
\author[2]{Levente Karpati}%
\author[1]{Gabriela Fulaneto}%
\author[1]{Szabolcs Tarapcsak}%
\author[1]{Tamas Kovacs}%
\author[1]{Florina Zakany}%
\author[2]{Istvan Mandity}%
\author[1]{Peter Nagy}%
\affil[1]{University of Debrecen}%
\affil[2]{Semmelweis Egyetem}%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Since cell penetrating peptides are promising tools for delivery of
cargo into cells, factors limiting or facilitating their cellular uptake
are intensely studied. Using labeling with pH-insensitive and
pH-sensitive dyes we report that escape of penetratin from acidic
endo-lysosomal compartments is retarded compared to its total cellular
uptake. The membrane dipole potential, known to alter transmembrane
transport of charged molecules, is shown to be negatively correlated
with the concentration of penetratin in the cytoplasmic compartment.
Treatment of cells with therapeutically relevant concentrations of
atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol
synthesis, significantly increased endosomal escape of penetratin in two
different cell types. This effect of atorvastatin correlated with its
ability to decrease the membrane dipole potential. These results
highlight the importance of the dipole potential in regulating cellular
uptake of cell penetrating peptides and suggest a clinically relevant
way of boosting this process.%
\end{abstract}%



\sloppy


\textbf{Introduction}

During the past decades, there has been growing interest in cell
penetrating peptides (CPPs) that can traverse biological membranes. CPPs
are a diverse set of short peptide sequences that usually consist of 30
or fewer amino acids and can be classified as either cationic,
amphipathic or hydrophobic. CPPs are important because of their ability
to cross cell membranes in a nontoxic manner and because of their
capacity to support efficient delivery of cell-impermeable therapeutic
cargos with molecular weights several times greater than their own
(Guidotti, Brambilla \& Rossi, 2017). Several natural peptides with cell
penetration capability have been characterized including substance P
analogs, the Tat protein in HIV and the homeodomain of the Antennapedia
protein in Drosophila (Joliot, Pernelle, Deagostini-Bazin \& Prochiantz,
1991; Repke \& Bienert, 1987). The cell translocation sequence was
localized to the third helix of the homeodomain leading to the
development of a 16-amino acid oligopeptide rich in positively charged
amino acids. This peptide, penetratin, belongs to the cationic class of
CPPs, and is widely used in research aimed at defining the mechanisms of
cellular uptake of peptides (Derossi, Joliot, Chassaing \& Prochiantz,
1994).

While CPPs hold great promise in drug delivery, their clinical potential
is currently limited by low bioavailability, short half-life and lack of
specificity (Fominaya, Bravo \& Rebollo, 2015; Qian et al., 2016; Wang,
Wang, Zhang, Zhang, Guo \& Jin, 2014). This latter shortcoming can be
remedied by equipping the CPP with a homing domain or by fusing it to an
inhibitory domain made up of negatively charged residues that is removed
in the tumor microenvironment having increased proteolytic activity
(Jiang, Olson, Nguyen, Roy, Jennings \& Tsien, 2004; Wang, Wang, Zhang,
Zhang, Guo \& Jin, 2014). The mechanism of cellular entry of CPPs also
limits their efficiency putting it at the forefront of current
investigations. One of the two, well-established routes of cellular
entry for CPPs is direct plasma membrane translocation, which may
involve formation of inverted micelles, transient pores or increased
fluidity of the plasma membrane (Guidotti, Brambilla \& Rossi, 2017;
Ziegler, 2008). Another well-established route of cellular entry for
CPPs is endocytosis (Futaki, 2006). Unless the endocytic uptake itself
is followed by endosomal escape, the CPP does not gain access to the
cytosolic compartment and is digested in lysosomes. Many studies focused
on the release of CPPs from endosomes, leading to the insertion of
endosomolytic sequences into or covalent coupling of endosomolytic
compounds to CPPs (Erazo-Oliveras, Muthukrishnan, Baker, Wang \&
Pellois, 2012; Nakase, Kogure, Harashima \& Futaki, 2011).

Numerous other approaches have been adopted to increase the cellular
uptake of CPPs including backbone cyclization, unnatural amino acids,
pegylation and acylation (Erazo-Oliveras, Muthukrishnan, Baker, Wang \&
Pellois, 2012; Lonn et al., 2016; Najjar, Erazo-Oliveras, Brock, Wang \&
Pellois, 2017; Wallbrecher et al., 2014). The problem is further
complicated by the fact that cellular uptake in 3D tumor spheroids is
not strongly correlated with the uptake in monolayers (van den Brand,
Veelken, Massuger \& Brock, 2018). Other strategies for improving
cellular delivery are based on the realization that a CPP must cross a
membrane independent of its uptake mechanism. The direct translocation
mechanism involves crossing the plasma membrane, whereas the endocytic
mechanism relies on traversing membranes of the endolysosomal
compartment. Due to their charged nature, electrostatic interactions of
CPPs with anionic phospholipids and heparan sulphate proteoglycans have
been implicated in direct membrane translocation and endocytosis,
respectively (Poon \& Gariepy, 2007; Thoren, Persson, Esbjorner, Goksor,
Lincoln \& Norden, 2004). Transport of charged substances across the
plasma membrane is also influenced by the three different kinds of
membrane potentials, the transmembrane, the surface and the dipole
potential (O'Shea, 2003). The magnitude of the dipole potential,
generated by the preferential orientation of lipids and interfacial
water molecules, is approximately 200-300 mV, larger by a factor of at
least 4-5 than the widely known transmembrane potential (Wang, 2012).
Since the electric field associated with the dipole potential is
confined to the surface of the membrane, its strength is
10\textsuperscript{8}-10\textsuperscript{9} V/m, larger by 1-2 orders of
magnitude than the field associated with the transmembrane potential.
Therefore, the dipole potential exerts significant effects on the
conformation of transmembrane proteins (Clarke, 2015; Kov\selectlanguage{ngerman}ács et al.,
2016; Pearlstein, Dickson \& Hornak, 2017; Zákány, Kovács, Panyi \&
Varga, 2020), on the binding of molecules to the membrane (Asawakarn,
Cladera \& O'Shea, 2001) and their transmembrane transport (Flewelling
\& Hubbell, 1986). One of the most important factors determining the
dipole potential is the sterol content of membranes. Cholesterol has
been shown to increase the membrane dipole potential directly due to its
intrinsic dipole moment, and indirectly by increasing the order of
lipids and interfacial water molecules and by changing the dielectric
constant of the membrane (Haldar, Kanaparthi, Samanta \& Chattopadhyay,
2012; Sarkar, Chakraborty \& Chattopadhyay, 2017; Simon, McIntosh, Magid
\& Needham, 1992; Starke-Peterkovic, Turner, Vitha, Waller, Hibbs \&
Clarke, 2006; Zákány, Kovács, Panyi \& Varga, 2020). Due to this
correlation, the dipole potential has been shown to be larger in
raft-like membrane domains in cellular plasma membranes (Kovács, Batta,
Zákány, Szöll\selectlanguage{polish}ő\selectlanguage{english}si \& Nagy, 2017). Statins, inhibitors of
3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, decrease the
cholesterol content of cells in experimental and clinical settings, and
they were reported to decrease the dipole potential of the plasma
membrane (Bjorkhem-Bergman, Lindh \& Bergman, 2011; Sarkar, Chakraborty
\& Chattopadhyay, 2017). Statins are the most commonly used therapeutic
agents to treat hypercholesterolemia due to their beneficial effect on
cardiovascular morbidity and mortality (Aykan \& Seyithanoglu, 2019;
Crismaru et al., 2020; Endo \& Kuroda, 1976; Endo, Tsujita, Kuroda \&
Tanzawa, 1977). Although adverse effects, e.g. myopathy, liver
dysfunction and type 2 diabetes, have been associated with statins, they
are usually well tolerated and successfully used even in combination
with other drugs such as cholesterol absorption inhibitors or fibrates
(Crismaru et al., 2020; Fievet \& Staels, 2009; Luo, Wang, Zhu, Du, Wang
\& Ding, 2016; Schachter, 2005). While the primary mechanism of action
of all statins is identical, there are significant differences in their
efficacy and bioavailability (Schachter, 2005). Atorvastatin is superior
to other statins in requiring lower milligram equivalent doses to
achieve the same effect on LDL-cholesterol levels (Jones, Kafonek,
Laurora \& Hunninghake, 1998). As opposed to simvastatin and lovastatin,
which are pro-drugs of the active hydroxy-acid form, atorvastatin does
not require enzymatic activation, a property not to be overlooked in in
vitro applications (Corsini, Maggi \& Catapano, 1995).

Corollary to the aforementioned principles membrane potentials are
expected to influence the uptake of penetratin due to the charged nature
of the peptide. However, only a limited number of studies correlating
electrostatic potentials and CPP uptake have been reported.
Non-physiological abolishment of the transmembrane potential has been
shown to inhibit the uptake of positively charged cell-penetrating
peptides (Rothbard, Jessop \& Wender, 2005). Although a negative dipole
potential favors the incorporation of cell-penetrating peptides into
lipid monolayers in molecular dynamics simulations and in experiments
(Via, Del Popolo \& Wilke, 2018; Via, Klug, Wilke, Mayorga \& Del
Popolo, 2018), such effects have not been described in lipid bilayers or
living cells. Here, we not only show that the physiological, positive
dipole potential of cellular membranes inhibits the uptake and
endo-lysosomal escape of penetratin, but also report that an artificial
decrease of the dipole potential and treatment with atorvastatin at
concentrations corresponding to the clinical dose range stimulate entry
of penetratin into the cytosol.

\textbf{Results}

\emph{Fluorescence labeling reveals cellular uptake and delayed
endo-lysosomal escape of penetratin}

Penetratin was labeled with two different fluorescent dyes in order to
study the mechanism and kinetics of its cellular uptake. The
fluorescence of naphthofluorescein (NF) is quenched at acidic pH
enabling studying the release of penetratin from the acidic
endo-lysosomal compartment (Qian, Dougherty \& Pei, 2015). In order to
complement the information on endo-lysosomal escape we chose labeling
with AFDye532, a dye exhibiting pH-independent fluorescence to report on
the total cellular content of penetratin. The cellular fluorescence of
AFDye532-penetratin is proportional to the total cellular uptake of
penetratin, the intensity of NF-penetratin characterizes its
concentration in non-acidic compartments (mainly the cytosol), whereas
the ratio of NF-labeled and AFDye532-labeled penetratin intensities
reveals the fractional escape of the cell-penetrating peptide from
acidic compartments. The molecular weight and purity of labeled
penetratin was checked by mass spectrometry and high performance liquid
chromatography (Suppl. Fig. 1). We developed an approach based on flow
cytometric measurement of cell-associated fluorescence signals to
separately measure the kinetics of cellular uptake and endo-lysosomal
release of these fluorescently labeled penetratins. Cells were incubated
in the continuous presence of an equimolar mixture of NF-penetratin and
AFDye532-penetratin (5 \selectlanguage{greek}µ\selectlanguage{english}M of each) at 37\selectlanguage{ngerman}°C, and the fluorescence
intensity of cells was measured in a time-correlated manner. After
removing cells with compromised membrane permeability based on DAPI
staining from the dataset and compensating for spectral crosstalk, the
time-dependent change of fluorescence intensity was plotted using moving
average smoothing. The signal of AFDye532-penetratin, corresponding to
the total cellular content of penetratin, reached saturation at 200-400
seconds in two different cell lines, while a significantly delayed
saturation of penetratin concentration in the neutral, cytoplasmic
compartment at 800-1000 seconds was observed based on the pH-sensitive
fluorescence of NF-penetratin (Fig. 1). The ratio of NF to AFDye532
intensities, characterizing the fraction of penetratin in neutral
compartments, initially declined, corresponding to a preferential
presence of penetratin in acidic endosomes, followed by a gradual
increase reaching saturation at approximately 800-1000 seconds (Fig. 1).
Since flow cytometry lacks subcellular resolution, we correlated the
fluorescence intensities of the two reporters with their cellular
location. Confocal microscopic investigation of the distribution of the
fluorescent penetratin derivatives revealed that AFDye532-penetratin
exhibits bright fluorescence even in endosomes where the fluorescence of
NF is quenched and that the cell-associated fluorescence of NF
originates from outside the endo-lysosomal compartment (Fig. 2). This
observation confirms that interpretation of the fluorescence intensity
of AFDye532-penetratin and NF-penetratin, measured by flow cytometry, as
total cellular penetratin uptake and the amount outside endosomes,
respectively, is indeed correct. Since AFDye532 and NF form a Förster
resonance energy transfer pair, anticorrelation between their
fluorescence intensities could also have been caused by energy transfer,
i.e. a high local concentration of the acceptor (NF) quenching the
fluorescence of the donor (AFDye532). In order to exclude this
possibility, the equimolar mixture of fluorescent penetratins was
supplemented with 10 \selectlanguage{greek}µ\selectlanguage{english}M unlabeled penetratin. If energy transfer is to
blame for the anti-correlation between the fluorescence intensities of
the two dyes, dilution of their local concentration with unlabeled
penetratin is expected to eliminate or reduce this anti-correlation.
However, the time dependence of the fluorescence intensities and their
ratio were not changed significantly by the presence of unlabeled
penetratin allowing us to conclude that the fluorescence intensities of
AFDye532 and NF correctly report total cellular uptake of penetratin and
its concentration in neutral compartments, respectively (Suppl. Fig. 2).
In summary, we established a flow cytometric approach revealing the
different kinetics of cellular uptake and endo-lysosomal release of
penetratin setting the stage for further analysis of the effect of
dipole potential-modifying agents on penetratin uptake.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. Cellular uptake and endo-lysosomal escape of penetratin. Two
different cell types were incubated at 37\selectlanguage{ngerman}°C in the continuous presence
of fluorescently labeled penetratins (5 \selectlanguage{greek}µ\selectlanguage{english}M AFDye532-penetratin and 5 \selectlanguage{greek}µ\selectlanguage{english}M
NF-penetratin). Time-correlated flow cytometric recording of the
fluorescence intensity of cells was performed. The average intensity of
AFDye532, characterizing total cellular uptake, and the average
intensity of NF-penetratin, characteristic of the amount of penetratin
present in pH-neutral compartments, were calculated as a function of
time after gating out debris and non-viable cells. The ratio of the two
fluorescence intensities is plotted in the panel on the right. The
continuous lines show the average fluorescence intensity calculated for
20-sec time periods. The symbols with error bars, representing the
standard error of the mean calculated from 13-15 samples from five
biological replicates, are only shown for every
7\textsuperscript{th}data point for clarity.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. Confocal microscopic images of live cells dual-labeled with
AFDye532- and NF-penetratin. Cells were incubated with
AFDye532-penetratin (A) and NF-penetratin (B) at 37\selectlanguage{ngerman}°C for 20 min and
images were captured by confocal microscopy. Panel C displays a
differential interference contrast image of the cells. The arrows in the
fluorescence images point at areas showing the anti-correlation between
the pH-insensitive fluorescence of AFDye532 and the fluorescence of NF
quenched at acidic pH.

\emph{Reduction of the membrane dipole potential enhances uptake and
endo-lysosomal release of penetratin}

Since the strong intramembrane dipole potential is expected to affect
the uptake and/or endo-lysosomal release of positively-charged
penetratin, we treated cells with 6-ketocholestanol and phloretin,
agents known to increase and decrease, respectively, the positive,
intramembrane dipole potential (Gross, Bedlack \& Loew, 1994; Kovács et
al., 2016). Flow cytometric analysis of the fluorescence intensity ratio
of the dipole potential sensitive dye, di-8-ANEPPS, confirmed that
6-ketocholestanol significantly increased the dipole potential. On the
other hand, phloretin decreased the dipole potential although its effect
did not reach statistical significance (Fig. 3A). These observations are
in accordance with our previous results (Kovács et al., 2016).

Having established that the treatments modify the dipole potential
according to expectations, we set out to characterize the effect of an
altered dipole potential on the uptake of penetratin. A diminished
dipole potential significantly enhanced both the total cellular uptake
of penetratin and its concentration in non-acidic compartments in both
SKBR-3 and MDA-MB-231 cells (Fig. 4). Since both of these processes were
enhanced at the lower dipole potential achieved by phloretin treatment,
the ratio of the NF and AFDye532 intensities, characterizing the
fractional release from acidic compartments, remained unchanged. On the
other hand, 6-ketocholestanol, which significantly increased the dipole
potential (Fig. 3A), had a statistically non-significant effect on both
the total cellular uptake and endo-lysosomal release of penetratin
(Fig.~4). In conclusion, we have established that the physiological
level of the intramembrane, positive dipole potential significantly
inhibits the uptake of penetratin.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3. Characterization of the effects of phloretin,
6-ketocholestanol and atorvastatin on the biophysical properties of the
membrane. Cells were pretreated with phloretin, 6-ketocholestanol or two
different concentrations of atorvastatin (ATO) as described in Materials
and methods. The dipole potential was measured with di-8-ANEPPS, whose
excitation intensity ratio correlates positively with the dipole
potential (A). The fluorescence anisotropy of TMA-DPH is inversely
related to membrane fluidity (B), whereas the generalized polarization
(GP) of Laurdan is proportional to the compactness of the membrane (C).
The error bars display the standard error of the mean of five biological
replicates. Asterisks indicate statistical significance
(p\textless{}0.05) reported by ANOVA followed by Tukey's HSD test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4. Decreased dipole potential enhances cellular uptake and
endo-lysosomal escape of penetratin. Cells (SKBR-3, MDA-MB-231,
displayed on the left) were treated with a mixture of
AFDye532-penetratin and NF-penetratin at 37\selectlanguage{ngerman}°C and their fluorescence
intensity was measured by flow cytometry. The membrane dipole potential
of cells was decreased and increased by pre-treatment with phloretin and
6-ketocholestanol, respectively. Time-dependent intensities and their
ratio were determined after gating out debris and dead cells. The error
bars represent the standard error of the mean calculated from 12-14
samples from five biological replicates. Asterisks indicate significant
difference of the phloretin-treated sample compared to the control at 20
min (p\textless{}0.05).

\emph{Statins increase the endo-lysosomal release of penetratin due to
decreased dipole potential}

Increasing the uptake of cell penetrating peptides has great potential
medical benefit. Since the treatment used for decreasing the dipole
potential in the previous section cannot be applied in humans, we sought
an alternative approach to enhance the uptake of penetratin by
modulating the dipole potential. The dipole potential correlates with
membrane cholesterol content (Kovács, Batta, Zákány, Szöll\selectlanguage{polish}ő\selectlanguage{english}si \& Nagy,
2017; Sarkar, Chakraborty \& Chattopadhyay, 2017), and statins are known
to decrease the dipole potential (Sarkar, Chakraborty \& Chattopadhyay,
2017). We opted for atorvastatin since it is one of the most effective
statins and it is the active substance not requiring enzymatic
activation (Corsini, Maggi \& Catapano, 1995; Jones, Kafonek, Laurora \&
Hunninghake, 1998; Schaefer et al., 2004). Atorvastatin, used at a
concentration of 1-10 nM corresponding to the serum concentration in
human patients (Bjorkhem-Bergman, Lindh \& Bergman, 2011), significantly
decreased the total cholesterol content of MDA-MB-231 cells by 40-50\%.
Albeit to a lesser extent, atorvastatin also decreased the cholesterol
content of SKBR-3 cells (Suppl. Fig. 3). In perfect agreement with these
results, atorvastatin decreased the dipole potential in both cell lines,
but SKBR-3 displayed lower sensitivity (Fig. 3A). Treatment of
MDA-MB-231 cells with the same concentration range of atorvastatin
significantly enhanced the endo-lysosomal release of penetratin with
only a marginal effect on total cellular uptake (Fig. 5). At the same
time, the effect of atorvastatin on SKBR-3 cells was smaller and it did
not reach statistical significance. Since the extent of decrease in the
cholesterol content of this cell line turned out to be smaller compared
to MDA-MB-231, we also tested the effect of higher atorvastatin
concentrations. 100~nM and 10 \selectlanguage{greek}µ\selectlanguage{english}M of atorvastatin decreased the total
cellular uptake of penetratin in SKBR-3 cells. Although cells with
increased membrane permeability were discarded from the analysis, we
attribute this finding to compromised cell viability. However, the
amount of penetratin leaving the endo-lysosomal compartment was
significantly higher in cells treated with these high atorvastatin
concentrations even though the total cellular uptake was lower (Fig. 5).
This finding is evidenced by the almost two-times higher NF/AFDye532
intensity ratio characterizing the fraction of penetratin escaping from
endosomes. In conclusion, we have convincingly shown that release of
penetratin from the endo-lysosomal compartment is the step that is the
most significantly increased by atorvastatin treatment.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5. Atorvastatin enhances endo-lysosomal escape of penetratin. Two
different cell types were pretreated with the indicated concentrations
of atorvastatin for 72 h and the uptake of AFDye-532-penetratin and
NF-penetratin were followed by flow cytometry. Time-dependent
intensities and their ratio were determined after gating out debris and
dead cells. The error bars represent the standard error of the mean
calculated from 13-15 samples from five biological replicates. Asterisks
indicate significant difference of the sample treated with the highest
concentration of atorvastatin in the figure compared to the control at
20 min (p\textless{}0.05).

\emph{Characterization of the effects of atorvastatin and the dipole
potential modifying compounds on membrane biophysical properties}

The previous experiments revealed that treatments reducing the membrane
dipole potential enhance the total cellular uptake and the release of
penetratin from endosomes, while increasing the dipole potential results
in an opposite, weak effect. The validity of this conclusion was checked
by calculating the rank-correlation between the dipole potential and
penetratin uptake (Suppl. Fig. 4). A significant, negative correlation
was revealed between the dipole potential and the total uptake (AFDye532
fluorescence), the concentration of penetratin in non-acidic
compartments (NF fluorescence) and the fractional release of penetratin
from the endo-lysosomal compartment (ratio of NF and AFDye532
intensities). Since the experimental data corresponding to the effect of
phloretin on total cellular uptake of penetratin seemed to deviate from
the trend established by the other conditions, the correlation was also
tested by removing these outlier data points. The significant and
biologically relevant negative correlation between the endo-lysosomal
release of penetratin and the dipole potential was retained. However,
removing the data points corresponding to phloretin treatment eliminated
most of the dipole potential-dependent changes in total cellular uptake
(Suppl. Fig. 4). This observation indicates that although both
atorvastatin and phloretin decrease the dipole potential, their cellular
effects and their influence on penetratin uptake are different. Since
our measurement approach based on the pH-dependent fluorescence of NF
assumes that the pH of the endo-lysosomal compartment is equally acidic
in all compared samples, a possible explanation for the different
results obtained with atorvastatin and phloretin is that they alter the
pH of lysosomes. Therefore, we performed fluorescence ratiometric
measurements of lysosomal pH, which revealed no significant effect of
any of the treatments on lysosomal pH (Suppl. Fig. 5). These results
imply that the ratio of the fluorescence of AFDye532-penetratin and
NF-penetratin correctly reports the release of penetratin from acidic
compartments. The issue of the different effect of phloretin and
atorvastatin on penetratin release will be further considered in the
Discussion.

Since treatments altering the dipole potential could potentially also
modify other biophysical properties of the membrane, the correlations
between penetratin uptake and two other fluorescent indicators were also
tested. Membrane fluidity, inversely proportional to microviscosity and
the fluorescence anisotropy of TMA-DPH (Kuhry, Fonteneau, Duportail,
Maechling \& Laustriat, 1983), was not changed by any of the treatments
in the SKBR-3 cell line, and phloretin and 6-ketocholestanol were also
without effect in MDA-MB-231 cells (Fig. 3B). In contrast, an unexpected
decrease in the membrane fluidity of this cell line was induced by
atorvastatin, an effect most likely attributable to the compensatory
increase in the synthesis of certain lipid species in order to
compensate for inhibited cholesterol synthesis (Dawaliby et al., 2016).
Membrane fluidity and total cellular uptake of penetratin did not
correlate with each other, but there was a strong correlation between
membrane fluidity and the fractional escape of penetratin from acidic
compartments (Suppl. Fig. 4). However, the power of membrane fluidity to
predict endo-lysosomal escape of penetratin is limited by two
observations: (i) the comparable effects of phloretin and atorvastatin
on penetratin uptake are not correlated with their distinct effects on
membrane fluidity in MDA-MB-231 cells; (ii) none of the treatments
induced any significant change in the membrane fluidity of SKBR-3 cells,
while phloretin increased endosomal release of penetratin.

The effect of the treatments on the generalized polarization of Laurdan,
known to be proportional to membrane compactness and inversely related
to membrane hydration (Parasassi, De Stasio, Ravagnan, Rusch \& Gratton,
1991), was also tested. None of the conditions led to any significant
change in membrane compactness in the two cell lines (Fig. 3C). The
generalized polarization of Laurdan did not show appreciable correlation
with total penetratin uptake or its concentration in non-acidic
compartments (Suppl. Fig. 4). Given the fold-changes induced and the
statistical significance of the correlations we can conclude that the
dipole potential has the largest power for predicting penetratin uptake,
and that release of the cell-penetrating peptide from endosomes and its
concentration in the cytoplasm are both strongly correlated with the
dipole potential.

\textbf{Discussion}

In this study, correlative measurement of the biophysical properties of
the plasma membrane and the uptake of penetratin allowed us to reach the
following major conclusions: (i) The physiological, positive membrane
dipole potential inhibits the total cellular uptake of penetratin and
its release from acidic endo-lysosomes. These conclusions are based on
temporal measurement of the cellular intensity of AFDye532-labeled and
NF-labeled penetratin, respectively. The effect of the dipole potential
on both steps of penetratin uptake is most likely mediated by an
alteration in the membrane insertion of the cell-penetrating peptide,
since incorporation and penetration of peptides and small, hydrophobic
molecules into the membrane are known to depend on the dipole potential
(Asawakarn, Cladera \& O'Shea, 2001; Cladera \& O'Shea, 1998). (ii)
Treatment with phloretin and atorvastatin reduced the membrane dipole
potential and led to a significantly enhanced cytoplasmic concentration
of penetratin. Since the applied, nanomolar concentration of
atorvastatin is identical to that used in the clinical setting, the
finding of statin-boosted uptake of penetratin is of potential medical
significance. (iii) By analyzing the correlation between several
membrane biophysical properties and penetratin uptake the dipole
potential turned out to be the only characteristic of sufficient value
for predicting penetratin uptake.

While both phloretin and atorvastatin decreased the dipole potential and
increased penetratin uptake, their differential effects raise question
about which step in the uptake process is the most sensitive to the
dipole potential. In particular, although atorvastatin decreased the
dipole potential more substantially than phloretin, only the latter
induced an increase in the total cellular uptake of penetratin, while
atorvastatin only increased the escape from acidic endo-lysosomes. These
apparent contradictions can be resolved by the following three points:
(i) The fact that phloretin does not increase the fraction of penetratin
released from the endo-lysosomal compartment is explained by the fact
that this compound is unlikely to reach sufficient concentrations in
intracellular membranes during the brief, 10-minute incubation and
therefore the dipole potential of these compartments remains largely
unaffected. On the other hand, the three-day treatment with atorvastatin
is sufficiently long so that substantial decrease in the cholesterol
content and consequent reduction in the dipole potential of
endo-lysosomal membranes can take place. (ii) The minuscule,
phloretin-induced decrease of the dipole potential and the significantly
elevated uptake of penetratin after phloretin treatment also require
clarification. We assume that the physiological level of the dipole
potential already limits penetratin uptake as much as potentially
achievable by the dipole potential. This conclusion is supported by the
fact that the significantly enhanced dipole potential after
6-ketocholestanol treatment had hardly any effect on the characteristics
of penetratin uptake. On the other hand, even the relatively minor
decrease in the dipole potential, achieved by phloretin, may be
sufficient to facilitate penetratin uptake. However, the magnitude of
this change in the dipole potential is not sufficiently large to reach
statistical significance given the measurement errors. (iii) Although
the reduction in the dipole potential achieved by atorvastatin is
significantly larger than the phloretin-induced change, atorvastatin had
no effect on the total cellular concentration of penetratin. The sudden
rise in AFDye532-penetratin intensity and the protracted increase in the
intensity of NF-penetratin indicate that initial uptake is mediated by
endocytosis, a characteristic left unaltered by any of the treatments.
Therefore, the lack of statin-induced increase in the total uptake of
penetratin implies that endocytosis must be hindered in
atorvastatin-treated samples. Indeed, it has been shown that statins
inhibit endocytosis by interfering with the prenylation-dependent
function of certain G proteins (Sidaway et al., 2004; Yilmaz et al.,
2006). Alternatively, the compensatory increase in membrane viscosity
after atorvastatin treatment may also impede endocytosis.

Despite the aforementioned uncertainties, two of the treatments,
phloretin and atorvastatin, significantly enhanced the concentration of
penetratin in the cytosol, the compartment most relevant from a
therapeutical point of view. Analysis of the correlation between
penetratin uptake and different biophysical properties of the membrane
revealed that the dipole potential is the strongest predictor of
penetratin uptake. Although the dipole potential, membrane compactness
and viscosity characterize different membrane properties, all of them
are related to membrane order. Therefore, it is not surprising that
besides the dipole potential, other membrane characteristics also
correlate with penetratin uptake. Membrane viscosity is correlated with
endo-lysosomal release of penetratin. However, the predictive value of
this correlation is undermined by the following two points: (i) While
phloretin exerts no effect on membrane fluidity, it significantly
enhances penetratin uptake in both SKBR-3 and MDA-MB-231 cells. (ii)
Although penetratin uptake was modulated by the treatments in SKBR-3,
none of them altered membrane viscosity in this cell line. Membrane
compactness or hydration, characterized by the generalized polarization
of Laurdan, was correlated weakly and in a statistically non-significant
way with penetratin uptake. The predictive value of this correlation is
further deteriorated by the fact that the generalized polarization of
Laurdan was non-significantly modified by any of the treatments in
MDA-MB-231 cells, while large and significant effects in penetratin
uptake were observed.

In the present manuscript, not only did we identify the positive,
intramembrane dipole potential inhibiting the uptake and endo-lysosomal
release of penetratin, but we could also enhance the cytoplasmic
concentration of the cell-penetrating peptide in a medically relevant
way by statin treatment. In order for a treatment modality to be of
potential medical importance, it must be well-tolerated and
therapeutically effective. In contrast to many in vitro studies, in
which statins are often overdosed, the nanomolar concentration range of
atorvastatin used in the presented experiments is identical to the serum
concentration at therapeutic doses (Bjorkhem-Bergman, Lindh \& Bergman,
2011). By applying atorvastatin not requiring metabolic activation, we
also circumvented another pitfall of in vitro cellular studies, the
application of statins in a prodrug form, which are unlikely to be
converted to the active metabolite in cell cultures. Although certain
side-effects are associated with statin treatment, extensive experience
indicates that they are safe, well-tolerated even in long-term
applications in combinations with other drugs (Fievet \& Staels, 2009;
Luo, Wang, Zhu, Du, Wang \& Ding, 2016). These circumstances further
support the potential medical relevance of our findings.

Although reduction of the dipole potential in both MDA-MD-231 and SKBR-3
cells resulted in enhanced penetratin accumulation in the cytosol after
phloretin treatment, the latter cell line exhibited lower sensitivity to
atorvastatin. Sensitivity to statins correlates inversely with HMG-CoA
reductase activity (G\selectlanguage{ngerman}öbel, Breining, Rauner, Hofbauer \& Rachner, 2019;
Kimbung, Lettiero, Feldt, Bosch \& Borgquist, 2016). While the
expression of this enzyme is higher by \selectlanguage{english}~20-30\% in SKBR-3 cells
according to a publication (Kimbung, Lettiero, Feldt, Bosch \&
Borgquist, 2016) and the Expression Atlas of the European Bioinformatics
Institute (https://www.ebi.ac.uk/gxa/home), the confidence intervals of
the expression of HMG-CoA reductase in the two cell lines completely
overlap according to the Genevestigator platform comparing
transcriptomic data from several public repositories, with the level of
expression corresponding to the MDA-MB-231 cell line spanning three
orders of magnitude. In addition, atorvastatin, albeit at a micromolar
concentration, resulted in 4-fold induction of HMG-CoA reductase
expression in SKBR-3 cells, while no change in enzyme expression was
observed in MDA-MD-231 cells (Kimbung, Lettiero, Feldt, Bosch \&
Borgquist, 2016). Cellular uptake of statins is believed to be mediated
by transporters to a large extent. Organic anion transporter
polypeptides (OATP) have been implicated in transmembrane import of
statins (Dobson \& Kell, 2008; Kalliokoski \& Niemi, 2009; Wu, Whitfield
\& Stewart, 2000). However, none of the OATP transporters is expressed
significantly differently according to the previous databases.
Therefore, the most likely cause for the different atorvastatin
sensitivity of the two cell lines in terms of penetratin uptake and
reduction in cellular cholesterol content seems to be the difference in
the baseline and statin-induced expression of HMG-CoA reductase
expression, but solid conclusions cannot be drawn due to inconsistencies
in the literature.

In conclusion, we have shown that a decreased, positive membrane dipole
potential significantly increases both the total cellular uptake and
endocytic escape of penetratin depending on what kind of treatment is
used for modifying the dipole potential. As a result, both medically
relevant (atorvastatin) and irrelevant (phloretin) treatments decreasing
the dipole potential enhance the concentration of penetratin in the
cytoplasm, the compartment most relevant for its therapeutic action.
This discovery could permit the delivery of drugs and drug candidates
exhibiting low or no transmembrane permeability into cells in animal
experiments, human trials or in the clinical setting after further
studies clarify the cell type dependence and the in vivo potential of
this treatment.

\textbf{Materials and methods}

\emph{Synthesis of penetratin}

Penetratin (RQIKIWFQNRRMKWKK-amide, molecular weight 2245.75 g/mol) was
synthesized manually on TentaGel R RAM (Rapp Polymere, Tubingen,
Germany), a low crosslinked polystyrene PEG copolymer resin (the
substitution was 0.2 mmol/g) by the solid-phase method of Merrifield
with standard Fmoc chemistry. Amino acids for peptide synthesis were
purchased from Iris Biotech (Marktredwitz, Germany). Amino acid side
chains were protected as follows: Trt for Gln and Asn,\emph{t}
-butoxycarbonyl (Boc) for Trp and Lys, and Pbf for Arg. A typical
coupling reaction contained 3 equivalents of Fmoc-amino acid, 3
equivalents of
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) and 6 equivalents of diisopropylethylamine
(DIPEA), and was allowed to proceed with mixing in dimethylformamide
(DMF) for at least 2.5 hours. Completion of the coupling was assessed by
Kaiser test. The Fmoc group was removed by treatment with 2\%
piperidine, 2\% 1,8-Diazabicyclo{[}5.4.0{]}undec-7-ene (DBU) in DMF,
followed by six washes with
DMF-MeOH-CH\textsubscript{2}Cl\textsubscript{2}. Completion of
deprotection was assessed by Kaiser test.

\emph{Fluorescence labeling of penetratin}

After coupling the last amino acid, arginine, its Fmoc protecting group
was replaced by Fmoc-6-aminohexanoic acid in order to introduce a
6-carbon linker followed by removing the Fmoc group. Half of penetratin,
still on the resin, was treated with 1.5 equivalents of AFDye532
N-hydroxysuccinimide ester (molecular weight 723.77 g/mol, Fluoroprobes,
Scottsdale, AZ) and 1.5 equivalents of DIPEA in DMF overnight. The other
half of penetratin was reacted with 1.5 equivalents of
5(6)-carboxynaphthofluorescein \emph{N} -succinimidyl ester (molecular
weight 573.51 g/mol, Darmstadt, Germany) in the presence of DIPEA in
DMF. Completion of the coupling was assessed by Kaiser test. The labeled
peptides were deprotected and released from the resin by treatment with
95:2.5:2.5 (v/v) trifluoroacetic acid (TFA)/triisopropylsilane/water and
dithiothreitol for 3 hours. The solutions were filtrated, and the
peptides were precipitated with a 15-fold volume of cold diethyl ether.
After filtration the crude products were purified by preparative
reversed-phase HPLC (JASCO, Victoria, Canada) on a C18 column and
lyophilized. The purity of the products (\textgreater{}95\%) was
assessed by reversed-phase HPLC (JASCO) equipped with an analytical C18
column. The authenticity of each peptide was confirmed by Bruker
electrospray ionization mass spectrometry. The predicted and measured
molecular mass of the (M+H)\textsuperscript{+} form of
AFDye532-penetratin was 2983.59 and 2983.548, respectively, whereas the
predicted and measured molecular mass of the
(M+H)\textsuperscript{+}variant of NF-penetratin was 2833.33 and 2832.5,
respectively.

\emph{Cell culture}

The SKBR-3 cell line was obtained from the American Type Culture
Collection (Manassas, VA), while MDA-MB-231 cells were a kind gift of
Peter Bay (University of Debrecen). The cell lines were selected based
on their different atorvastatin sensitivity (Gobel, Breining, Rauner,
Hofbauer \& Rachner, 2019; Kimbung, Lettiero, Feldt, Bosch \& Borgquist,
2016; Zhang et al., 2019). Both cell lines were propagated in DMEM
containing 10\% fetal bovine serum supplemented with antibiotics. Cells
were harvested at a confluency of 80-90\% before measurements.

\emph{Treatments for modifying the dipole potential or inhibiting
HMG-CoA reductase}

6-ketocholestanol and phloretin (Sigma-Aldrich, St. Louis, MO) were used
for measuring penetratin uptake at increased and decreased dipole
potentials, respectively. Both compounds were applied at a concentration
of 100 \selectlanguage{greek}µ\selectlanguage{english}M at room temperature in the presence of 0.05\% (v/v) Pluronic
F-127 for 10 min (Clarke \& Kane, 1997; Kov\selectlanguage{ngerman}ács, Batta, Zákány, Szöll\selectlanguage{polish}ő\selectlanguage{english}si
\& Nagy, 2017). In order to inhibit HMG-CoA reductase atorvastatin
(Sigma-Aldrich) was applied at final concentrations of 1 nM-10 \selectlanguage{greek}µ\selectlanguage{english}M for 72
hours. The low nanomolar concentrations represent ranges found in human
serum during atorvastatin treatment of patients with
hypercholesterolemia (Bjorkhem-Bergman, Lindh \& Bergman, 2011).

\emph{Membrane dipole potential measurement with di-8-ANEPPS}

After treatment with atorvastatin, 6-ketocholestanol or phloretin cells
were incubated with di-8-ANEPPS (Thermo Fisher, D3167) at a final
concentration of 2 \selectlanguage{greek}µ\selectlanguage{english}M on ice for 20 minutes (Clarke \& Kane, 1997;
Gross, Bedlack \& Loew, 1994; Kov\selectlanguage{ngerman}ács, Batta, Zákány, Szöll\selectlanguage{polish}ő\selectlanguage{english}si \& Nagy,
2017). Cells were kept on ice until measurement. Right before
spectrofluorimetry, the sample was warmed to 37\selectlanguage{ngerman}°C, and spectra were
acquired with a Fluorolog-3 spectrofluorimeter (Horiba Jobin Yvon,
Edison, NJ). The temperature of the cuvette holder was adjusted to 37°C
by a circulating water bath. Excitation spectra were registered between
410 nm and 520, while the emission wavelength was set to 660 nm in order
to minimize effects of membrane fluidity on the fluorescence of the
indicator (Clarke \& Kane, 1997). The ratio of fluorescence intensities
integrated between excitation wavelengths 410-416 nm and 503-510 nm
correlates positively with the dipole potential (Clarke \& Kane, 1997;
Kovács, Batta, Zákány, Szöll\selectlanguage{polish}ő\selectlanguage{english}si \& Nagy, 2017).

\emph{Measurement of membrane fluidity, hydration and cellular
cholesterol content}

After treatment with atorvastatin, phloretin or 6-ketocholestanol,
150,000 cells were stained with 10 \selectlanguage{greek}µ\selectlanguage{english}M
4'-(trimethylammonio)-diphenylhexatriene (TMA-DPH) or 2 \selectlanguage{greek}µ\selectlanguage{english}M Laurdan
(6-dodecanoyl-N,N-dimethyl-2-naphthylamine; both obtained from
Sigma-Aldrich) for 30 minutes at room temperature. Anisotropy of TMA-DPH
was measured with a Fluorolog-3 spectrofluorimeter with the temperature
of the cuvette holder adjusted to 37\selectlanguage{ngerman}°C. TMA-DPH was excited at 352\selectlanguage{english} nm
and its emission was measured at 430\selectlanguage{english} nm. Fluorescence anisotropy
(\emph{r} ) was measured in the L-format according to the following
formula (Kuhry, Fonteneau, Duportail, Maechling \& Laustriat, 1983):

where \emph{I} \textsubscript{vv} and \emph{I} \textsubscript{vh} are
the vertical and horizontal components, respectively, of the
fluorescence excited by vertically polarized light, and G is an
instrument-specific correction factor characterizing the different
sensitivity of the detection system for vertically and horizontally
polarized light. Laurdan was excited at 350\selectlanguage{english} nm and its emission was
detected in the blue range of its emission spectrum between 430-440\selectlanguage{english} nm
(\emph{I} \textsubscript{blue}) and at the red edge between 495-505 nm
(\emph{I} \textsubscript{red}) to measure generalized polarization
(\emph{GP} ) according to the following formula (Parasassi, De Stasio,
Ravagnan, Rusch \& Gratton, 1991):

Total cholesterol content of cellular samples was determined using
Cholesterol Quantification Kit (MAK043, Sigma-Aldrich).

\emph{Flow cytometric measurement of penetratin uptake and
endo-lysosomal release}

Cells were incubated in the continuous presence of 5 \selectlanguage{greek}µ\selectlanguage{english}M
AFDye532-penetratin, 5 \selectlanguage{greek}µ\selectlanguage{english}M NF-penetratin and 0.25 \selectlanguage{greek}µ\selectlanguage{english}g/ml DAPI at 37\selectlanguage{ngerman}°C in
the thermostated sample holder of a Becton Dickinson FACSAria III flow
cytometer (Becton Dickinson, Mountain View, CA). DAPI was excited at 405
nm, and its emission was measured between 430-470 nm. Both AFDye532 and
NF were excited at 561 nm, and their fluorescence was recorded at
515-545 nm and 663-677 nm, respectively. Apart from background
measurements every experiment was carried out for 20 min and the
measurement started right after adding the DAPI/penetratin mixture,
preheated to 37°C, to the cells. After spectral compensation and gating
out dead cells based on DAPI positivity the time-correlated fluorescence
intensities were exported using FCS Express (De Novo Software, Pasadena,
CA). A custom-written Matlab program was used for calculating a moving
average with a window size of 20 seconds. The pH-insensitive
fluorescence of AFDye532 is proportional to the total cellular uptake of
penetratin. Due to quenching of NF at acidic pH, the ratio of
NF-penetratin to AFDye532 fluorescence intensities reports the
endo-lysosomal escape of the cell-penetrating peptide (Qian, Dougherty
\& Pei, 2015). The fluorescence intensities of both indicators were
normalized to the mean intensity measured in the first time window.
Normalized mean intensities were divided by each other to obtain the
NF-penetratin/AFDye532-penetratin ratio. The standard error of the mean
(\emph{SEM} ) of the ratio parameter was calculated using error
propagation analysis assuming independence of the two fluorescence
intensities:

designate the mean intensity of the respective parameter.

\emph{Investigation of penetratin uptake by confocal microscopy}

Penetratin uptake and endo-lysosomal release was visualized using a
Zeiss LSM880 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Fluorescently labeled penetratins were used at the same concentration as
in flow cytometry. The temperature of the microscope stage was adjusted
to 37°C. AFDye532 and NF were excited at 514 nm and 633 nm,
respectively. Detection of AFDye532 and NF fluorescence was carried out
between 525-629 nm and 637-759 nm, respectively.

\textbf{Acknowledgements}

This work was supported by research grants from the National Research,
Development and Innovation Office, Hungary (K120302,
GINOP-2.3.2-15-2016-00020, GINOP-2.3.2-15-2016-00044). The Lendület
grant from the Hungarian Academy of Sciences is gratefully acknowledged.
This work was completed in the ELTE Thematic Excellence Program
supported by the Hungarian Ministry for Innovation and Technology.
Project no. 2018-1.2.1-NKP-2018-00005 has been implemented with the
support provided from the National Research, Development and Innovation
Fund of Hungary, financed under the 2018-1.2.1-NKP funding scheme.

\textbf{Author contributions}

GB carried out most of the cellular experimental work. LK synthesized
penetratin and its fluorescently-labeled derivatives. ST and GFH
contributed significantly to the flow cytometric experiments. TK and FZ
measured cellular cholesterol content and some of the experiments about
the biophysical properties of the membrane. IMM and PN conceived and
funded the project. GB and LK wrote the initial version of the
manuscript.

\textbf{Conflict of interest}

The authors declare no conflict of interest.

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