The generally agreed upon first step of the translocation mechanism involving GRTs is the association of the oligoguanidinium transporter with the membrane. Each positively-charged guanidinium head group features a specialized, rigid planar array of hydrogen bond donors that allows for highly effective bidentate hydrogen bond formation with negatively-charged carboxylates, phosphates, and sulfates (). These negatively-charged functional groups are subunits of many cell membrane constituents such as phospholipids, fatty acids, proteins, and heparan sulfate proteoglycans (HSPGs). As charged (polar) groups they often extend beyond the cell surface or membrane inner leaf into the surrounding polar milieu. Relative to the single hydrogen bond that would be possible with the ammonium group in lysine, the bidentate hydrogen bond network formed from guanidinium groups is stronger and thus provides a structural rationalization for the differing abilities of oligolysines and oligoarginines to associate with and cross cellular membranes [50
]. The decreased translocation ability of lysine and ornithine relative to arginine also supports the observation that, while charge is necessary, it is not sufficient, as both the guanidinium and the ammonium group have a single positive charge. Obviously, the degree of protonation of the systems under assay conditions is another factor that could be of consequence as oligomers of the more basic guanidinium group would be more protonated (have more cationic sites) relative to lysine analogs.
Representative associations of a polycationic guanidinium transporter with anionic cell membrane constituents.
Combining the positive charge with a bidentate hydrogen bond donor results in a functional moiety ideally poised to interact with the negatively charged cell surface constituents. The driving force for this pairing is the associated decrease in free energy in going from solution to the surface of a membrane. While a guanidinium group has no recognizable hydration shell [51
] and a very weak association with a counterion such as a phosphate group in an aqueous environment (~ 0.2 kcal/mol) [52
], as the guanidinium group approaches the surface of a lipid bilayer and local polarity decreases, its association with a phosphate counterion increases dramatically (~2.6 – 6.0 kcal/mol) [53
]. This association is further strengthened as the complex moves into the less polar membrane. The magnitude of this association is expected to increase with guanidinium content. Indeed, arginine oligomers containing fewer than six amino acids are relatively ineffective in entering cells, while oligomers of six or more arginine residues enter readily. Maximal uptake is seen for an oligomer of 15 arginines [1
]. The inefficient entry of longer oligomers (>20 arginines) is also explained by this association as the increased interaction resulting from additional guanidinium groups suppresses release of the oligomer from the membrane.
To further explore the importance of this bidentate hydrogen bond, oligomers of arginine containing monomethyl- and dimethylguanidinium head groups were synthesized and attached to fluorescein [54
]. A single methyl substitution on each guanidinium group of an oligomer resulted in a compound with conserved charge but with a reduced ability to form a bidentate hydrogen bond. This compound, when assayed for cellular uptake, is 80% less effective than the unalkylated arginine oligomer. Furthermore, dimethylation of each guanidinium group provides a compound unable to form a bidentate hydrogen bond. Significantly, the cellular translocation of this compound is reduced by greater than 95% (). In short, the decreased ability of mono- and dimethylated oligomers of arginines to form hydrogen bonds correlates with their decreased ability to enter cells, suggesting that hydrogen bond formation plays an important role in the entry process.
The effect of mono and dimethylation of the guanidinium group on cellular uptake.
Another parameter to consider in the association of a GRT with the membrane is the spatial orientation of the guanidinium head groups. Due to electrostatic repulsion, there is a low probability of having two adjacent anionic cell membrane constituents. Consequentially, an improved association would be found in transporters with a similar spacing in their cationic functionalities. Additionally, an increase in the flexibility of the scaffold presenting the guanidiniums would increase the percentage of guanidiniums capable of interacting with species of complementary charge on the surface of the cell. The hypothesis that increases in guanidinium group spacing and flexibility would improve cell membrane association and cellular uptake is supported by experiments using oligomers of arginine interdigitated with a series of non α-amino acids of increasing length (glycine, β-alanine, 4-aminobutyric acid, 6-aminohexanoic acid). For these systems, an increase in the spacing between arginine residues results in a concomitant increase in cellular uptake, with a maximal level observed with 6-aminohexanoic acid [23
]. This increase in cellular uptake is also observed for oligocarbamate transporters with increased spacing of the guanidinium-containing sidechains along the linear backbone [3
], as well as in peptoid transporters, where a lengthened spacing between the guanidinium head group and the backbone results in an increase in uptake [1
]. Dendrimer transporters also exhibit this property: branches of increasing length and thus flexibility show greater uptake [4
The favorably aligned bidentate hydrogen bond between a guanidinium-containing transporter and a carboxylate, sulfate, or phosphate on the surface of a cell and the electrostatic attraction of such charged species is expected to produce an association between the transporter and the lipid bilayer. This association strengthens with increasing numbers of and spacing (up to a point) between guanidiniums. With a stronger association between a membrane and a transporter, there is a greater chance of the transporter being internalized, whether through an active or passive transport mechanism. At the same time, this association must be reversible to allow release after uptake. In short, guanidinium groups are well-suited for adherence to cell surfaces bearing negatively-charged groups. The association would allow for increased residence time and therefore favor internalization. The association, while relatively strong, must be reversible to allow for release after entry as discussed below.
3.2. Mechanisms of Uptake
CPPs, and more specifically GRTs, can enter cells through multiple mechanisms. Confocal microscopy is often used to visualize their uptake. It has been proposed that the observed punctate staining is due to endocytosis and that the diffuse cytosolic staining arises from nonendosomal uptake. Other studies suggest that the diffuse cytosolic staining cannot be due solely to escape from the endosomes as it occurs more quickly than the punctate endosomal fluorescence [55
]. Maiolo et al.
reported that at 4 °C, while there is a significant decrease in the amount of punctate staining of U2OS cells with fluoresceinated R7
W, there is an increase of diffuse cytosolic staining. They suggest that the lack of punctate staining indicates an inhibition of endocytosis and that the diffuse cytosolic staining, not inhibited by the cold, is due to a second, non-endocytotic mechanism at work [55
]. They have also observed a different degree of endosomal uptake in different cell types with some cell types displaying more diffuse staining than others.
Though there are many possible mechanisms for GRT uptake, there is little doubt that endocytosis is involved with several systems, as many have observed a decrease in cellular uptake at 4 °C, a condition known to inhibit endocytosis. This has been observed with cargoes such as fluorescent tags [27
], peptide cargoes [58
], fusion proteins [59
], DNA-Tat complexes [61
], gold particles, [62
] liposomes [63
] and FITC-avidin complexed CPPs [64
], though in the last case the authors state that Tat conjugates were less impacted by the low temperature than the other CPPs studied. It has also been shown that chemical means to induce energy depletion result in decreased uptake of CPPs [27
], adding further evidence that a significant portion of uptake could involve an energy-dependent mechanism. This energy dependence appears to be cargo sensitive. Exemplifying this, in a comparison between a DNA-Tat complex and a much smaller fluorescently labeled Tat conjugate, the Tat complex exhibited an almost complete loss of uptake into cells pretreated with sodium azide [61
], while the smaller conjugate showed no such sensitivity. This will be more fully discussed later in the review.
Within the large category of endocytosis there are several possible mechanisms of uptake (). The most studied form of endocytosis is clathrin-mediated endocytosis. Several authors have proposed that this process is the primary mechanism of uptake of arginine-rich transporters. It has been reported that in HeLa cells, labeled CPPs colocalize with transferrin [27
] a glycoprotein marker for endocytosis, while others have reported that fluorescently labeled r8
] and fusion proteins [59
] do not colocalize with transferrin. Inhibition of endocytosis was also attempted with varying results. A Tat-avidin conjugate shows only a modest decrease in uptake upon treatment with hyperosmolar medium, a condition that was shown to decrease clathrin-dependent endocytosis. This led the authors to suggest that other mechanisms might be at play [64
]. Other authors report that treatment with the endocytosis inhibitor chlorpromazine results in a 50% decrease of uptake of fluorescently-tagged Tat, while a potassium-free buffer results in a 40% decrease in HeLa cells [67
Proposed endocytotic mechanisms of uptake, from left to right: clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis.
Other forms of endocytosis have also been implicated with a great deal of current interest being directed toward macropinocytosis. Macropinocytosis is a type of endocytosis that is mediated by lipid rafts and is clathrin-, caveolae-, and receptor-independent. The specific process involves the enclosing of actin-containing membrane protrusions to form vesicles called macropinosomes [68
]. These vesicles can often be greater than 1 μm in size and it has been suggested that they are leaky [69
]. Specific monitoring of this endocytotic pathway can be achieved by pretreatment with amiloride, an inhibitor of the Na+
exchange that is required in macropinocytosis [70
]. Additionally, an F-actin elongation inhibitor, cytochalasin D, can also be used [71
]. A number of research groups have proposed macropinocytosis as the mechanism of uptake for CPPs. Dowdy and coworkers have recently reported that both a Tat-Cre fusion protein [58
] and FITC-labeled Tat [72
] enter cells through lipid raft-mediated macropinocytosis, which is supported by the dose-dependent inhibition of uptake observed when cells are pretreated with amiloride or cholesterol is removed with β-cyclodextrin. However, cargo and cell type must be considered when examining mechanisms of uptake. For example, Ferrari and coworkers observed uptake into 3T3 cells (in contrast to the HeLa and CHO cells in Dowdy’s work) of a different Tat construct, a GST-Tat-GFP fusion protein, through a caveolae-mediated pathway [60
], observing colocalization with the caveolar endocytosis marker, caveolin-1. In an excellent mechanistic study of fluorescently labeled octaarginine, Futaki et al.
observed a considerable role of macropinocytosis in peptide uptake into HeLa cells, finding that both cytochalasin-D and the macropinocytosis inhibitor ethylisopropylamiloride significantly suppress uptake of the peptide into HeLa cells [57
]. Interestingly, in a more recent study, Futaki and Harashima discovered that when octaarginine-modified liposomes containing a rhodamine dye are incubated with NIH3T3 cells, the mechanism of uptake depends upon the density of the peptide on the surface of the liposome [73
]. The authors observed a shift from clathrin-mediated endocytosis to a macropinocytosis uptake mechanism as the density of the octaarginine increased. In contrast to the aforementioned groups, Shen et al.
reported that the translocation of I125
in HeLa cells is not inhibited by amiloride pre-treatment or incubation at 16 °C, which argues for an uptake mechanism distinct from macropinocytosis [74
]. They support their claim with a subcellular fractionation method they developed to separate the vesicular from the cytosolic compartments [75
]. Additionally, the authors found that coincubation with EGF, a known stimulator of macropinocytosis, does not significantly increase the amount of oligoarginine found in the cytosol.
In addition to macropinocytosis, other lipid raft-mediated forms of endocytosis have been implicated, in particular caveolin-dependent endocytosis. Tat-GFP in HeLa [59
] cells and in CHO-K1 and HL3T1 cells [60
] have been shown to colocalize with caveolin-1. Both Tat-rhodamine [66
] and Tat-GFP complexes [60
] have also been shown to colocalize with cholera toxin. Cholera toxin is known to proceed through a caveolin-dependent pathway [76
]. β-cyclodextrin, which is known to disrupt lipid rafts, has been reported to have differing effects on uptake. FITC-avidin complexed with biotin-CPP exhibits only a 20% drop in uptake [64
] upon treatment with β-cyclodextrin, while the uptake of a Tat-GFP fusion peptide is shown to be significantly inhibited by methyl β-cyclodextrin [59
] as was a Tat-rhodamine compound whose uptake is substantially reduced in HeLa cells [66
]. Also, nystatin, a compound known to inhibit caveolae formation, only decreased the amount of uptake in some cell lines (CHO1) and not others (BGM) and only for some cargoes such as a large DNA-Tat complex and not for fluorescently labeled Tat alone, suggesting that not only is caveolae-mediated uptake cell dependent, but also cargo dependent [61
]. Other authors have found that nystatin and filipin III have little impact on the uptake of fluorescently labeled Tat into HeLa cells or CHO cells [67
]. Some have proposed that whether a specific conjugate is taken up by clathrin or caveolae endocytosis is highly dependent on cargo and cell type. Indeed, a clathrin-mediated endocytosis mechanism predominates with fluorescent beads less than 200 nm in diameter, but as the size of the beads increases the mode of uptake becomes increasingly caveolae-mediated. For beads of 500 nm in diameter, the caveolae pathway dominates uptake [77
Another type of mechanism that has been proposed for cellular entry is non-endocytotic, direct diffusion through the membrane. The uptake of a charged (polar) species into a highly lipophilic (non-polar) environment such as a membrane would seem to oppose dogma. However, natural examples of such a process do exist as demonstrated in the groundbreaking research by MacKinnon on potassium ion channels, in which four highly conserved arginine residues in the channel’s voltage sensor are shown to move into the lipid environment of the membrane [84
]. Numerous models for understanding how direct diffusion of GRTs could occur have been suggested. Derossi et al.
proposed an inverted micelle model where positively-charged peptides interact with negatively-charged membrane constituents to form an inverted micelle structure which can open on the inside or outside of the cell [86
]. A carpet model, in which aggregates of peptides coat the cell surface and disrupt its structure, has also been proposed [87
]. While pore formation in the membrane is also possible, this mechanism can be ruled out by testing for membrane leakage of lactate dehydrogenase or propidium iodide staining [88
]. Our group has proposed a mechanism of uptake called adaptive translocation, which involves the recruitment of negatively-charged cell surface constituents by the positively-charged GRT peptide as the latter contacts a cell surface, transiently forming an ion pair complex with attenuated polarity that is able to adaptively diffuse into the membrane and subsequently into the cell () [54
]. This mechanism is supported by a simple water-octanol partitioning experiment in which it is shown quantifiably that highly water-soluble arginine oligomers partition into the non-polar membrane surrogate phase (octanol) upon addition of sodium laurate, a surrogate of a membrane constituent. Solubilization is readily quantified by phase separation and weighing of the dissolved residue. Remarkably, the once water soluble (>95%) GRT becomes membrane soluble (>95%) by forming a complex with membrane constituents. This does not require micelles or vesicles, as only 1.2 laurate counterions are needed on average per guanidinium group.
Adaptive translocation mechanism of uptake and octanol-water partitioning.
The driving force for the passage of this ion pair complex across the cell membrane is proposed to be the membrane potential. Since this potential favors the movement of positively-charged species into a cell, if the ion pair complex formed from the polyguanidinium transporter retains any time-averaged cationic character its passage across the membrane would be favorable. Since the ability of a cell to maintain a membrane potential is dependent on ATP, this mechanism is consistent with studies that show an energy dependence on cellular uptake. To probe this mechanism, it was shown that the cellular uptake of Fl-r8 was reduced by more than 90% by eliminating the membrane potential with an extracellular buffer containing a concentration of potassium ion equivalent to that found intracellularly. Additionally, varying the membrane potential by the use of potassium ion buffers with different concentrations results in differing amounts of cellular uptake. Notably, cellular uptake possessed a linear relationship with the potassium Nernst potential calculated based on the extracellular potassium ion concentration. Finally, treatment of cells with gramicidin A, known to reduce the membrane potential through its pore-forming activity, reduces cellular uptake of guanidinium-based transporters by greater than 90%. Conversely, hyperpolarizing the cells with valinomycin, a peptide that selectively shuttles potassium into the cell, significantly increases cellular uptake.
These mechanistic explanations for direct penetration arose from the observation by many groups that uptake of the CPPs was not significantly reduced either by incubation at 4 °C or in the presence of metabolic inhibitors, both of which suggest a non-endocytotic mechanism [1
]. This low temperature, non-endocytotic pathway has been demonstrated with multiple peptides, including truncated Tat peptide sequences [89
], HIV-1 Tat [90
], octaarginine [1
], and Tat-decorated liposomes [93
]. It has been reported, however, that cell fixation leads to artifacts in both uptake and intracellular distribution of peptides [27
], leading some researchers to propose that the observed results need to be reevaluated and endocytosis is the actual uptake pathway. It should be noted, however, that a number of groups have reported non-endocytotic uptake with live (unfixed) cells [1
]. Thoren et al.
studied the uptake of fluorescent analogs of Tat(48–60) and heptaarginine in live PC-12 cells, reporting that R7
W is efficiently internalized at both 37 °C and 4°C [91
]. Additionally, the authors note that depletion of ATP with rotenone and 2-deoxy-D-glucose inhibited entry of FM 4–64 (a commercial marker of endocytosis), but not the uptake of R7
W. Rothbard et al.
reported that Fl-R7
uptake into Jurkat cells is inhibited by pre-incubation with sodium azide, but rapid and efficient uptake at 3 °C along with the absence of fluorescence in endocytotic vesicles provided support for a mechanism distinct from endocytosis [50
]. Representative of how the various pathways are not mutually exclusive, a study by Jones et al. with fluorescently tagged R8 in KG1a cells reported staining in both endocytotic vesicles and the cytoplasm at intermediate temperatures (12–30 °C), supporting the idea that multiple pathways of uptake for a given GRT can occur simultaneously [95
As has already been alluded to above, uptake of a given GRT is dependent on a variety of factors, all of which could influence and direct the uptake towards a certain pathway. This interplay of conditions should be considered when attributing a mechanism to a given CPP. The size of the cargo attached to the transporter is one factor that affects the mechanism. It has been shown that complexes or fusions of Tat with large 20 nm quantum dots or proteins (>50 amino acids) enter cells primarily through an endocytotic, vesicle-associated pathway whereas a small peptide (<50 amino acids) cargo can transduce cells through an additional, non-endocytotic pathway [15
]. Another study has shown that fluorescently labeled, unconjugated R7
W are taken up efficiently by A431 and U2OS cells, with both diffuse and vesicular staining observed; however, conjugating negatively-charged amino acid sequences to the GRTs dramatically reduces uptake and the little staining observed is solely vesicular [55
]. A temperature dependence on the mechanism of uptake has also been demonstrated. While some studies have shown inhibition of uptake at 4 °C, others have proposed a temperature-independent mechanism for GRT translocation. A combination of the two has also been reported in a study of the uptake of Alexa488-R8
(both L and D stereoisomers) into KG1a cells. At temperatures between 4 and 12 °C, fluorescence is observed in both the cytoplasm and the nucleus. At 12–30 °C, however, additional labeling is observed in endocytotic vesicles. Finally, when the cells are warmed to 37 °C fluorescence is observed solely in vesicles [95
]. GRTs have also been shown to enter one cell type selectively over others. In a study involving both ex vivo
and in vivo
applications, a modified Tat peptide conjugated to Alexa594 is taken up by renal ganglion cells and a subset of inner nuclear layer cells of the retina, but no fluorescence is observed in any outer retina cells [96
]. As further evidence that not all cell types are equivalent, one research group observed different amounts of uptake for biotinylated R9
in glycosaminoglycan-deficient CHO cells and in wild-type cells [11
]. Furthermore, in contrast to the R9
case, internalization of a guanidinylated aminoglycoside is inhibited in the former cell type as compared to the normal uptake in cells containing glycosaminoglycans. Yet another factor to consider when examining the uptake of charged GRTs involves the associated counterions. In the absence of the aromatic counteranion pyrenebutyrate, Alexa488-R8
is internalized into HeLa cells primarily by endocytosis; however, preincubation with pyrenebutyrate provides rapid, diffuse cytosolic labeling, arguing for direct membrane translocation of the peptide [97
]. These examples call attention to only a few of the many factors that can affect the contribution of a given internalization pathway to the overall uptake of a GRT.
Elucidating the mechanism of uptake is of more than just scholarly importance. The mechanism of uptake impacts intracellular trafficking, which in turn determines how quickly a cargo is either degraded or delivered to various organelles. These various fates will have a large impact on the therapeutic efficiency of the cargo. Fischer et al
. have demonstrated colocalization of a fluoresceinated Tat with BODIPY, a Golgi tracer, and failed to see any colocalization with Lysotracker [98
], a lysosome tracer. Fretz et al
. have observed colocalization with Lysotracker in the case of a Tat-modified liposome [63
]. Fuchs et al.
have also observed colocalization of fluoresceinated R9
with FM 1–43, a marker for endocytotic vesicles, in CHO cells [100
]. Geisler and Chmielewski have also reported colocalization of an arginine-rich CPP with Lysotracker in MCF-7 cells, though they do report extensive cytosolic staining that does not colocalize with the lysosomes, suggesting either endosomal escape or a different mode of uptake [99
]. Al-Taei et al
. found that disruption of the Golgi in K562 with brefeldin A resulted in no difference in staining with fluorescently labeled Tat [90
]. However, the authors performed a dextran pulse-chase experiment and observed colocalization of Tat and R8
with dextran, suggesting that both peptides are trafficked to lysosomes. Still others have observed no colocalization of a Tat-GFP fusion protein with Lysotracker or with early endosome antigen-1 in HeLa cells but it does colocalize with caveolae-associated markers [59
]. These studies suggest that not only mechanism, but also trafficking, is cargo- and cell type-dependent.
In the cases where endosomal uptake predominates, in order for the cargo to be therapeutically active, it is necessary for the GRT conjugate to escape the endosomes. Attempts have been made to study endosomal release by inhibiting it. Potocky et al
. demonstrated that if HeLa cells are incubated with NH4
Cl little to no fluorescence is observed in the cytoplasm compared to untreated cells, suggesting that acidification of the endosomes is necessary for escape [65
]. They also found that at low concentrations (1.5 μM) Fl-Tat has mainly vesicular staining while at higher concentration (7 μM) cytosolic staining is observed, which they propose is due to a concentration dependence of endosomal escape. Fuchs et al.
have also suggested that there is a concentration dependence on endosomal escape, observing that fluorescently labeled R9
-induced leakage from vesicles occurs only at high concentration [100
]. Some have built endosomal escape into their conjugates. Dowdy et al
. reported that treatment with a Tat fusogenic peptide to facilitate escape from the endosomes increases the amount of recombination with a Tat-Cre fusion protein in NIH3T3 cells [58
]. Still others have found if they treat cells with a soluble photosensitizer that creates reactive oxygen species a significant increase in endosomal release of various GRTs is observed. They found in the case of Tat a greater than 75-fold increase in escape when the cells are treated with the photosensitizer and light [101
]. Once endosomal escape has been achieved in many cases it is still necessary to release the cargo from the transporter. This has been achieved, through a variety of mechanisms including disulfide reduction that selectively releases the cargo inside of the cell [102
As described in the example above, for many conjugates it is necessary to release the drug/probe cargo from the transporter after uptake to achieve the activity of the free cargo. Recently, we have also reported an imaging method that provides, for the first time, quantification of transporter-conjugate uptake and cargo release in real-time in both cells and animal models [103
]. This method uses luciferase-transfected cells and transgenic (luciferase) reporter mice and whole-body imaging, allowing non-invasive quantification of transporter-conjugate uptake and probe (luciferin) release in real-time. This process also effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications. In our study we selected a disulfide linker because its cleavage would occur only after cell entry upon encountering a high glutathione concentration (15 mM inside vs. 15 μM outside [104
]) (). The resultant thiol would then undergo cyclization, releasing free luciferin which would be converted by luciferase to oxyluciferin and a photon of light [105
]. Because luciferin derivatives alkylated on the phenolic oxygen do not generate light [106
], only free luciferin is measured. Importantly, the signal-to-noise ratios are excellent relative to fluorescence because there is essentially no background tissue luminescence. The approach is readily applied to cells and to live animals [107
]. This new procedure allows one to quantitatively compare the performance of different transporters for a constant linker design or different linkers and their release kinetics for a given transporter system. Because signal arises only when free drug surrogate is turned over by its intracellular target (luciferase), it is a true measure of effective cargo delivered.
Scheme for the measurement of the uptake of GRTs and the intracellular release of their cargo.