Identification and characterization of a novel cell-penetrating peptide

Cell-penetrating peptides (CPPs) are short amino acid sequences that promote their own translocation across cell plasma membrane. When linked with cargo such as polypeptides, nucleic acid, or liposomes, CPPs can facilitate the transport of these entities across the cell membrane. Therefore, CPPs are receiving increased interest in drug delivery and gene therapy. The majority of CPPs identified so far are polycationic peptides which interact with heparin sulfate chains of plasma membrane for internalization. Here, we report the identification and characterization of a conformationally constrained 13 amino acid peptide (CVQWSLLRGYQPC, designated as S41) which is clearly distinct from classical polycationic peptides. Immunofluorescence assay was employed to test the cellular uptake of S41 in mouse neuroblastoma cell line Neuro2A (N2A) and rat cerebellar granule neurons (CGNs). Internalization of S41 was further examined in N2A cells by means of mutational analysis, flow cytometry and confocal microscopy. Our results demonstrate that S41 can enter cells through lipid rafts dependent endocytosis.     

Cell-penetrating peptides and antimicrobial peptides: how different are they?

Some cationic peptides, referred to as CPPs (cell-penetrating peptides), have the ability to translocate across biological membranes in a non-disruptive way and to overcome the impermeable nature of the cell membrane. They have been successfully used for drug delivery into mammalian cells; however, there is no consensus about the mechanism of cellular uptake. Both endocytic and non-endocytic pathways are supported by experimental evidence. The observation that some AMPs (antimicrobial peptides) can enter host cells without damaging their cytoplasmic membrane, as well as kill pathogenic agents, has also attracted attention. The capacity to translocate across the cell membrane has been reported for some of these AMPs. Like CPPs, AMPs are short and cationic sequences with a high affinity for membranes. Similarities between CPPs and AMPs prompted us to question if these two classes of peptides really belong to unrelated families. In this Review, a critical comparison of the mechanisms that underlie cellular uptake is undertaken. A reflection and a new perspective about CPPs and AMPs are presented.     

Translocation of cell‐penetrating peptides into Candida fungal pathogens

Cell‐penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)₃K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration‐dependent manner, and an additional peptide (TP‐10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)₃K, and TP‐10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.     

Elucidating cell-penetrating peptide mechanisms of action for membrane interaction,cellular uptake,and translocation utilizing the hydrophobic counter-anion pyrenebutyrate

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with varying hydrophobicity and arginine content is investigated. The cellular CPP uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell-based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2′-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.     

Secondary structure of cell-penetrating peptides controls membrane interaction and insertion

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.     

Fatty acyl moieties: improving Pro-rich peptide uptake inside HeLa cells.

In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro-rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro-rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.     

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this review, we will summarize high-resolution structural and dynamic findings towards the understanding of the structure–activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

The cell penetrating peptides pVEC and W2-pVEC induce transformation of gel phase domains in phospholipid bilayers without affecting their integrity

The cell penetrating peptide (CPP) pVEC has been shown to translocate efficiently the plasma membrane of different mammalian cell lines by a receptor-independent mechanism without exhibiting cellular toxicity. This ability renders CPPs of broad interest in cell biology, biotechnology, and drug delivery. To gain insight into the interaction of CPPs with biomembranes, we studied the interaction of pVEC and W2-pVEC, an Ile --> Trp modification of the former, with phase-separated supported phospholipid bilayers (SPB) by atomic force microscopy (AFM). W2-pVEC induced a transformation of dipalmitoyl phosphatidylcholine (DPPC) domains from a gel phase state via an intermediate state with branched structures into essentially flat bilayers. With pVEC the transformation followed a similar pathway but was slower. Employing fluorescence polarization, we revealed the capability of the investigated peptides to increase the fluidity of DPPC domains as the underlying mechanism of transformation. Due to their tighter packing, sphingomyelin (SM) domains were not transformed. By combination, AFM observations, dynamic light scattering studies, and liposome leakage experiments indicated that bilayer integrity was not compromised by the peptides. Transformation of gel phase domains in SPB by CPPs represents a novel aspect in the discussion on uptake mechanisms of CPPs.     

pVEC hydrophobic N‐terminus is critical for antibacterial activity

Cell‐penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure‐activity relationship and sequence alignment studies have suggested that the hydrophobic N‐terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N‐terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.     

Simulation study of interaction mechanism between peptide and asymmetric membrane

Cell-penetrating peptides (CPPs) are widely used as drug carriers, owing to their superior ability to cross cell membrane both alone and with cargos, such as genes and other particles. Understanding the translocation mechanism of CPP is significant for many therapeutic purposes, including targeting drug and gene delivery. In this study, we performed a coarse-grained molecular dynamics simulation to investigate the interaction mechanism between polyarginine peptides and asymmetric membranes. Results showed that peptides can penetrate through the lipid bilayer by inducing a hydrophilic hole formation in the asymmetric membrane. Furthermore, the lengthy peptide chain length (R4–R16 peptides) and high membrane asymmetry positively affect peptide penetration. Our study provides insights into the molecular-level interactions between peptides and asymmetric membranes, as well as suggestions for targeted gene and drug delivery.     

Re-evaluating the role of strongly charged sequences in amphipathic cell-penetrating peptides: a fluorescence study using Pep-1

Cell-penetrating peptides (CPPs) are able to translocate across biological membranes and deliver bioactive proteins. Cellular uptake and intracellular distribution of CPPs is commonly evaluated with fluorescent labels, which can alter peptide properties. The effect of carboxyfluorescein label in the Lys-rich domain of the amphipathic CPP pep-1, was evaluated and compared with non-labelled pep-1 in vitro and in vivo. A reduced membrane affinity and an endosomal-dependent translocation mechanism, at variance with non-labelled pep-1, were detected. Therefore, the charged domain is not a mere enabler of peptide adsorption but has a crucial role in the translocation pathway of non-labelled pep-1.     

Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly

In recent years, cell-penetrating peptides have proven to be an efficient intracellular delivery system. The mechanism for CPP internalisation, which first involves interaction with the extracellular matrix, is followed in most cases by endocytosis and finally, depending on the type of endocytosis, an intracellular fate is reached. Delivery of cargo attached to a CPP requires endosomal release, for which different methods have recently been proposed. Positively charged amino acids, hydrophobicity and/or amphipathicity are common to CPPs. Moreover, some CPPs can self-assemble. Herein is discussed the role of self assembly in the cellular uptake of CPPs. Sweet Arrow Peptide (SAP) CPP has been shown to aggregate by CD and TEM (freeze-fixation/freeze-drying), although the internalised species have yet to be identified as either the monomer or an aggregate.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of ζ-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

Mechanistic aspects of CPP-mediated intracellular drug delivery: relevance of CPP self-assembly

In recent years, cell-penetrating peptides have proven to be an efficient intracellular delivery system. The mechanism for CPP internalisation, which first involves interaction with the extracellular matrix, is followed in most cases by endocytosis and finally, depending on the type of endocytosis, an intracellular fate is reached. Delivery of cargo attached to a CPP requires endosomal release, for which different methods have recently been proposed. Positively charged amino acids, hydrophobicity and/or amphipathicity are common to CPPs. Moreover, some CPPs can self-assemble. Herein is discussed the role of self assembly in the cellular uptake of CPPs. Sweet Arrow Peptide (SAP) CPP has been shown to aggregate by CD and TEM (freeze-fixation/freeze-drying), although the internalised species have yet to be identified as either the monomer or an aggregate.     

Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides

The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R₇, and R₇W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.     

Bioportides: Bioactive cell-penetrating peptides that modulate cellular dynamics

The study and exploitation of cell-penetrating peptides (CPPs) now extends into a third exciting decade. Pharmacokinetic modulators, including the more common sequences Tat, penetratin and transportan-10, markedly enhance the intracellular delivery of small drugs, peptides, oligonucleotides and proteins. We introduced the term bioportide to distinguish cell penetrant peptides with intrinsic bioactivities from more typically inert CPP vectors. Our first examples included rhegnylogically organised bioportides, monomeric peptides presenting pharmacophores for both cellular internalization and bioactivity discontinuously distributed within the primary sequence. However, it is conceptually expedient to employ the same terminology to encompass s ychnologic bioportides that comprise an inert CPP vector conjugated to an otherwise impermeable bioactive peptide. In such cases the CPP provides an obvious address function whilst the bioactive cargo, often a protein mimetic sequence, is the message. Additional targeting sequences, usually added as chimeric extensions, can also be accommodated within the design of CPPs and bioportides to enable cell- and tissue-selective targeting. Thus, the identification and exploitation of bioportides provides further scope to employ CPPs as research tools, diagnostics and therapeutics spanning a range of pathologies.     

Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides

The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R7, and R7W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.