Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin

Cellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization.     

How to address CPP and AMP translocation? Methods to detect and quantify peptide internalization in vitro and in vivo (Review)

Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.     

Basic cell penetrating peptides induce plasma membrane positive curvature,lipid domain separation and protein redistribution

Basic cell penetrating peptides are tools for molecular cellular internalization of nonmembrane permeable molecules. Their uptake mechanisms involve energy-dependent and energy-independent pathways such as endocytosis, direct translocation or physical endocytosis. These mechanisms are ruled by both, the peptides physicochemical properties and structure and by the membrane lipids characteristics and organization. Herein we used plasma membrane spheres and membrane models to study the membrane perturbations induced by three arginine-rich cell penetrating peptides. Nona-arginine (R9) and the amphipathic peptide RWRRWWRRW (RW9) induced positive membrane curvature in the form of buds and membrane tubes. Membranous tubes underwent rolling resulting in formation of multilamellar membrane particles at the surface of the plasma membrane spheres. The amphipathic peptides RW9 and RRWRRWWRRWWRRWRR (RW16) provoked lipid and membrane associated protein domain separation as well as changes in membrane fluidity and cholesterol redistribution. These data suggest that membrane domains separation and the formation of multilamellar membranous particles would be involved in arginine-rich cell penetrating peptides internalization.     

Translocation and Endocytosis for Cell-penetrating Peptide Internalization

Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells.     

The application of pH-sensitive fluorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodified and conjugated dyes

Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.     

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.     

Arginine-rich cell-penetrating peptides

Arginine-rich cell-penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell-penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell-penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example.     

Translocation or membrane disintegration? Implication of peptide-membrane interactions in pep-1 activity.

The Cell membrane is impermeable for most peptides, proteins, and oligonucleotides. Moreover, some cationic peptides, the so-called cell-penetrating peptides (CPPs), are able to translocate across the membrane. This observation has attracted much attention because these peptides can be covalently coupled to different macromolecules, which are efficiently delivered inside the cell. The mechanism used by these peptides to pass across the membrane is a controversial matter of debate. It has been suggested that endocytosis is the main mechanism of internalization and this was confirmed by several studies for different peptides. Pep-1 is an exception worthy of attention for its ability to translocate cargo macromolecules without the need to be covalently attached to them. A preferential internalization by an endocytosis-independent mechanism was demonstrated both in vitro and in vivo. Pep-1 has a high affinity to lipidic membranes, it is able to insert and induce local destabilization in the lipidic bilayer, although without pore formation. No cytotoxic effects were found for pep-1 concentrations where translocation is fully operative. At much higher concentrations, membrane disintegration takes place by a detergent-like mechanism that resembles anti-microbial peptide activity. In this review, the ability of pep-1 to transverse the membrane by an endocytosis-independent mechanism, not mediated by pores as well as an ability to induce membrane disintegration at high peptide concentration, is demonstrated.     

Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.     

Studies on the internalization mechanism of cationic cell-penetrating peptides

A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43-58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43-58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation.     

Membrane interactions of two arginine-rich peptides with different cell internalization capacities

Cell penetrating peptides (CPPs) can cross cell membranes in a receptor independent manner and transport cargo molecules inside cells. These peptides can internalize through two independent routes: energy dependent endocytosis and energy independent translocation across the membrane, but the exact mechanisms are still unknown. The interaction of the CPP with different membrane components is certainly a preliminary key point that triggers internalization, such as the interaction with lipids to lead to the translocation process. In this study, we used two arginine-rich peptides, RW9 (RRWWRRWRR-NH(2)), which is a potent CPP, and RL9 (RRLLRRLRR-NH(2)) that, although binding tightly and accumulating on membranes, does not enter into cells. Using a set of experimental and theoretical techniques, we studied the binding, insertion and orientation of the peptides into different model membranes as well as the subsequent membrane reorganization. Herein we show that although the two peptides had rather similar behavior regarding lipid membrane interaction, subtle differences were found concerning the depth of peptide insertion, effect on the lipid chain ordering and kinetics of peptide insertion in the membrane, which altogether might explain their different cell internalization capacities. Molecular dynamics simulation studies show that some peptide molecules flipped their orientation over the course of the simulation such that the hydrophobic residues penetrated deeper in the lipid core region while Arg-residues maintained H-bonds with the lipid headgroups, serving as a molecular hinge in a conformation that appeared to correspond to the equilibrium one.     

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.     

Kinetic uptake profiles of cell penetrating peptides in lymphocytes and monocytes

Background

Nucleolar targeting peptides (NrTPs), resulting from structural minimization of the rattlesnake toxin crotamine, are a novel family of cell-penetrating peptides (CPPs) shown to internalize and deliver cargos into different cell types.

Methods

In this study, we address NrTP kinetics of translocation into primary cells. We used flow cytometry to measure the intracellular uptake of rhodamine B-labeled NrTPs in peripheral blood mononucleated cells (PBMCs).

Results

The kinetic profiles for each peptide are concentration-independent but significantly different among NrTPs, pointing out for the amino acid sequence importance. Arginine-containing peptides (NrTP7 and Tat_48–60, used for comparison) were found to be more toxic than lysine-containing ones, as expected. On the other hand, one same peptide behaves differently in each of the lymphocyte and monocyte cell populations, suggesting differences in entry mechanism that in turn reflect diversity in cell functionality. Uptake results obtained at 4 °C or using chemical endocytosis inhibitors support the importance of non-endocytic mechanisms in the cellular internalization of NrTP1 and NrTP5, while confirming endocytosis as the main mechanism of NrTPs entry.

Conclusion

Overall, both direct translocation and endocytosis mechanisms play a role in NrTP entry. Yet, there is predominance of endocytosis-mediated mechanisms. NrTPs (especially NrTP6) are an excellent intracellular delivery tool, with efficient internalization and no toxicity.

General significance

This work validates NrTPs as potential therapeutic tools for, e.g., cancer or inhibition of viral replication and establishes a new comparative and quantitative method to test CPP efficiency.     

Amphipathic peptides and drug delivery

The discovery of cell-penetrating peptides as gene delivery systems and the interest in the mechanism by which these vectors cross the cell membrane have generated a large number of studies. Among the parameters involved in the translocation process, controversy has arisen about the role of the amphipathicity of the carriers in the interaction and reorganization of the cell membrane. In this review we have summarized the vectors with primary or secondary amphipathicity related to secondary structure. Some of the insights into the relationship between the aggregation state of the peptide at the concentrations used for internalization studies and its interaction with the cell membrane result from our contribution to the field with a new family of amphipathic proline-rich peptides.     

Measuring peptide translocation into large unilamellar vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4(13)-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4(13)-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4(13)-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4(13)-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4(13)-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Effect of the electrostatic potential on the internalization mechanism of cell penetrating peptides derived from TIRAP

In order to develop future therapeutic applications for cell penetrating peptides (CPPs), it is essential to characterize their internalization mechanisms, as they might affect the stability and the accessibility of the carried drug. Several internalization mechanisms have been described in literature, such as endocytosis and transduction. In this work we study the internalization mechanism in HeLa cells of two TIRAP derived peptides: pepTIRAP and pepTIRAP^ALA, where some of the cationic amino acids were replaced with alanines. Detailed analysis of internalization and the peptides electrostatic potential was carried out, to shed light on the internalization mechanism involved. Molecular modeling studies showed that the main difference identified between pepTIRAP and pepTIRAP^ALA is the distribution of their electrostatic potential field. The structure of pepTIRAP displays a predominantly positive potential when compared to pepTIRAP^ALA, which has a more balanced potential distribution. In addition, docking experiments show that interactions between pepTIRAP and negatively charged molecules on the cellular surface such as heparan sulfate are stronger than the ones exhibited by pepTIRAP^ALA. A mathematical model was proposed to quantify the amount of peptide internalized or non-specifically bound to the membrane. The model indicates a stronger interaction of pepTIRAP with the plasma membrane, compared to pepTIRAP^ALA. We propose these discrepancies are related to the differences in the electrostatic potential characteristics of each peptide. In the case of pepTIRAP, these interactions lead to the formation of nucleation zones, which are the first stage of the transduction internalization mechanism. These results should be considered for effective design of a cell penetrating peptide.     

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.     

A critical reassessment of penetratin translocation across lipid membranes

Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10(-13) m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.     

A comprehensive model for the cellular uptake of cationic cell-penetrating peptides

The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so-called cell-penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis-independent cellular entry. In this study, we provide evidence that the Antennapedia-homeodomain-derived antennapedia (Antp) peptide, nona-arginine and the HIV-1 Tat-protein-derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis-independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant-negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.