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
108-109 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; Kovács 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, Szöllősi & 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.