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.