In virus studies in the late 80ies, it was found that HIV-1 trans-activator of transcription (Tat), a protein of 101 amino acids essential for HIV-1 replication, is rapidly taken up from the culture medium by cultured cells (Frankel and Pabo 1988; Green and Loewenstein 1988). It turned out that only a small proportion of the protein was responsible for the internalization of the whole protein. The relevant peptide comprises residues 49-57, eight of which are basic amino acids (Fawell et al. 1994; Pepinsky et al. 1994; Vives et al. 1997a; Vives et al. 1994). In the first examinations of the potential of the basic HIV-1 Tat domain (Tat peptide) to mediate the transport of other proteins into cells, large proteins like β-galactosidase,
horseradish peroxidase, RNase A and domain III of Pseudomonas exotoxin A were successfully incorporated when coupled to full-length HIV-1 Tat and Tat49-57 peptide
(Anderson et al. 1993). A fusion protein of Tat peptide with β-galactosidase was injected into mice to monitor the bioavailability of a Tat-coupled protein. The functional protein could be detected in various tissues like heart, liver, and spleen but also lung and skeletal muscle (Fawell et al. 1994; Schwarze et al. 1999). Later studies showed that Tat peptide exhibits toxic effects at concentrations above 5 µM in the extracellular medium (Hallbrink et al. 2001; Vives et al. 1997b). Considering the good delivery properties, Tat peptide was soon discussed as a delivery vector for clinical applications.
Similar properties were found for short basic domains in the Drosophila Antennapedia homeobox protein (Derossi et al. 1994) and in the Herpes Simplex protein VP22 (Elliott and Ohare 1997). Thus, a rapid development was triggered in which more peptides with the capability to enter cells were discovered and broad variety of cargo molecules was delivered into cultured cells.
Initial assays suggested that these peptides could directly penetrate the plasma membrane by a novel mechanism that became known as protein transduction. Therefore, the new class of peptides was referred to as protein transduction domains (PTDs). After the development of synthetic delivery peptides the term cell-penetrating peptides (CPP) was coined to refer to naturally occurring PTDs and their functional homologs from organic synthesis. (Jeang et al. 1999; Lindgren et al. 2000b; Nagahara et al. 1998). As they help to deliver otherwise impermeable molecules into cells, they were also called Trojan peptides.
By the time of the discovery of the CPPs, fluorescence activated cell sorting (FACS) and fluorescence microscopy were the solely applied methods to estimate the amount of internalized fluorescently labeled CPPs, so that the absolute amount of compound and its distribution inside the cell was not quantitatively assessed. It was found that the fluorescent compounds were taken up by almost all of the treated cells within minutes, which was evaluated and reported as a “high uptake efficiency”. Shuffling of the amino-acid sequence and the use of D-amino acids did not inhibit the internalization, so that a receptor-dependent mechanism was ruled out (Derossi et al. 1996). Since low temperatures and inhibitors of endocytosis, vesicular trafficking, and energy metabolism did not interfere with the observed rapid uptake, a non-endocytotic mechanism for protein transduction was proposed (Derossi et al. 1996; Polyakov et al. 2000). Reports from fluorescence microscopy indicated that fluorescently labeled CPPs reached the cytosol and accumulated in the nucleus (Vives et al. 1997a). Therefore, it was proposed that protein transduction acted by direct passage of the plasma-membrane, and the mechanistic explanations for this phenomenon ranged from penetration by passive diffusion through the membrane to pore formation and the generation of inverted micelles (Green et al. 2003; Schwarze et al. 1999).
1.2.8.1 Artifacts in early uptake studies
In 2003, two independent reports pointed out, that most of the previously obtained results in mechanistic studies had to be attributed to artifacts (Richard et al. 2003; Thoren et al. 2003). As the basic residues of the CPPs are positively charged under physiological conditions, CPPs interact strongly with negatively charged phospholipids and proteoglycans on the
surface of the plasma membrane. Thus, they cannot be removed by washing and are counted as internalized compounds in FACS experiments. Treated cells need to be incubated with trypsin prior to measurements to remove these artifactual CPPs from the exterior of the cell (Richard et al. 2003). Alternatively, the internalized CPPs can be distinguished from adhering ones by fluorescence quenching experiments, in which the fluorescently labeled peptide is quenched by externally added, non-permeant agents if it resides outside the cell (Drin et al. 2001; Hallbrink et al. 2001). In a novel method, extracellular peptides are chemically modified by a non-permeant reagent to be distinguishable from internalized peptides after cell lysis and HPLC analysis (Hallbrink et al. 2004).
a) b)
Figure 1-17 Artifacts in mechanistic studies on CPP uptake. a) Cells with CPPs attached to their surface are counted as cells with internalized CPPs. b) Fixation procedures allow an intracellular reorganization of CPPs.
Furthermore, the uptake of CPPs into the cytosol and their accumulation in the nucleus was put into doubt. Upon fixation, cellular membranes are ruptured and CPPs attached to the cell surface or residing in the cytosol are redistributed. Reaching the nucleus they interact with the negatively charged nucleic acids thus mimicking a nuclear accumulation (Leifert et al. 2002; Lundberg et al. 2003; Lundberg et al. 2002). To examine the real intracellular distribution, imaging has to be carried out in living cells (Drin 2003; Richard et al. 2003; Umezawa et al. 2002).
Studies using corrected procedures (i.e. imaging of living cells) to avoid artifacts revealed that fluorescently labeled CPPs were taken up on a much slower time scale. Inside the cells they were found localized in vesicular structures that suggested an endocytosis-like mechanism. However, a leaking of these compounds to the cytosol was also detectable after long incubation times. Fluorescently labeled CPPs were linked to a quencher via a disulfide bond, so that the fluorescence was quenched. Upon addition to cultured cells, fluorescence
was detected which was attributed to an entry of the compound into the cytosol, where the disulfide bond was cleaved and the fluorescent probe released from the quencher (Hallbrink et al. 2001). Furthermore, novel studies pointed out a nuclear localization of CPPs (Chiu et al. 2004a).
Moreover, up to 90% of fluorescent probe was found to be associated with degradation products of the peptides, which occur after cellular uptake. This was seen as a proof of lysosomal degradation by one group (Saalik et al. 2004), while others could show that the addition of chloroquine to inhibit lysosomal function did not interfere with the degradation (Hallbrink et al. 2004). These differing results have to be attributed to differing methods of preparation ranging from the study of single cells (Soughayer et al. 2004) to the preparation of lysates (Hallbrink et al. 2004), but also to the nature of the peptides themselves (Saalik et al. 2004). More publications dealing with the intracellular distribution of CPPs have been announced for the beginning of 2005.
Over the last months, novel attempts to quantify the intracellular concentration of CPPs and to determine their intracellular distribution have been reported (Chiu et al. 2004a; Hallbrink et al. 2004; Soughayer et al. 2004). Yet, the existing results are highly controversial: The reported overall intracellular concentrations of the CPPs range from an equilibrium with the extracellular medium to the 35fold of the external CPP concentration, which can be attributed to the peptide to cell ratio, the cell density, the cell-type, the used peptide and the attached cargo (Hallbrink et al.; Hallbrink et al. 2004; Lindsay). However and especially notable, none of the studies discriminates between the CPP content of cytosol or nucleus and the vesicular structures. As long as the decisive parameters have not been identified and standard protocols have been developed, the data from CPP uptake studies will be difficult to compare and evaluate. At present, the results obtained from mechanistic investigations do not fit into one consistent picture.
However, the literature confers to the uptake of CPPs as highly efficient. This means, that the peptide vector is taken up by all cells as opposed to some transfection methods in which only a small fraction of the treated cells incorporates the applied effector molecule. Internalized compounds are found in endosomes, lysosomes as well as in the cytosol and nucleus. The uptake efficiency as determined by FACS and fluorescence quenching experiments reflects the overall amount of labeled compound found in endosomes, lysosomes and the cytosol. The first experiments to quantify the amount of internalized compound and to determine its distribution are expected in the upcoming months. It may turn out that the fraction of cargo- coupled-CPPs reaching in the cytosol or leaving the endosomal compartment is small. However, in numerous experiments it was sufficient to exhibit the desired biological function as shall be discussed in the following chapter. None of the current studies explains how the “endosomal escape” or endosomal exit might occur. Like the polycationic dendrimers, most of the CPPs are protonated within the acidic compartments and might aggregate with membranous structures leading to an accumulation in these compartments. Is a high concentration of these polycations responsible for the rupture of the lysosome/endosome or are there transporters or secretion processes responsible for the cytosolic release? These questions have to still be answered. For now, most of the studies refer to “endosomal escape” to describe the phenomenon.