Uptake Of Cell-penetrating Peptides Research Articles

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

POSEIDON: Peptidic Objects SEquence-based Interaction with cellular DOmaiNs: a new database and predictor

Cell-penetrating peptides (CPPs) are short chains of amino acids that have shown remarkable potential to cross the cell membrane and deliver coupled therapeutic cargoes into cells. Designing and testing different CPPs to target specific cells or tissues is crucial to ensure high delivery efficiency and reduced toxicity. However, in vivo/in vitro testing of various CPPs can be both time-consuming and costly, which has led to interest in computational methodologies, such as Machine Learning (ML) approaches, as faster and cheaper methods for CPP design and uptake prediction. However, most ML models developed to date focus on classification rather than regression techniques, because of the lack of informative quantitative uptake values. To address these challenges, we developed POSEIDON, an open-access and up-to-date curated database that provides experimental quantitative uptake values for over 2,300 entries and physicochemical properties of 1,315 peptides. POSEIDON also offers physicochemical properties, such as cell line, cargo, and sequence, among others. By leveraging this database along with cell line genomic features, we processed a dataset of over 1,200 entries to develop an ML regression CPP uptake predictor. Our results demonstrated that POSEIDON accurately predicted peptide cell line uptake, achieving a Pearson correlation of 0.87, Spearman correlation of 0.88, and r2 score of 0.76, on an independent test set. With its comprehensive and novel dataset, along with its potent predictive capabilities, the POSEIDON database and its associated ML predictor signify a significant leap forward in CPP research and development. The POSEIDON database and ML Predictor are available for free and with a user-friendly interface at https://moreiralab.com/resources/poseidon/, making them valuable resources for advancing research on CPP-related topics. Scientific Contribution Statement: Our research addresses the critical need for more efficient and cost-effective methodologies in Cell-Penetrating Peptide (CPP) research. We introduced POSEIDON, a comprehensive and freely accessible database that delivers quantitative uptake values for over 2,300 entries, along with detailed physicochemical profiles for 1,315 peptides. Recognizing the limitations of current Machine Learning (ML) models for CPP design, our work leveraged the rich dataset provided by POSEIDON to develop a highly accurate ML regression model for predicting CPP uptake.Graphical

Examinations of cellular uptake of cell penetrating peptides in vitro and in vivo

Examinations of cellular uptake of cell penetrating peptides in vitro and in vivo

Cell-Penetrating Peptide-Bismuth Bicycles.

Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter human cells as demonstrated by live-cell confocal microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R8 ), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.

Application of Cell Penetrating Peptides as a Promising Drug Carrier to Combat Viral Infections.

Novel effective drugs or therapeutic vaccines have been already developed to eradicate viral infections. Some non-viral carriers have been used for effective drug delivery to a target cell or tissue. Among them, cell penetrating peptides (CPPs) attracted a special interest to enhance drug delivery into the cells with low toxicity. They were also applied to transfer peptide/protein-based and nucleic acids-based therapeutic vaccines against viral infections. CPPs-conjugated drugs or vaccines were investigated in several viral infections including poliovirus, Ebola, coronavirus, herpes simplex virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, Japanese encephalitis virus, and influenza A virus. Some studies showed that the uptake of CPPs or CPPs-conjugated drugs can be performed through both non-endocytic and endocytic pathways. Despite high potential of CPPs for cargo delivery, there are some serious drawbacks such as non-tissue-specificity, instability, and suboptimal pharmacokinetics features that limit their clinical applications. At present, some solutions are utilized to improve the CPPs properties such as conjugation of CPPs with targeting moieties, the use of fusogenic lipids, generation of the proton sponge effect, etc. Up to now, no CPP or composition containing CPPs has been approved by the Food and Drug Administration (FDA) due to the lack of sufficient in vivo studies on stability, immunological assays, toxicity, and endosomal escape of CPPs. In this review, we briefly describe the properties, uptake mechanisms, advantages and disadvantages, and improvement of intracellular delivery, and bioavailability of cell penetrating peptides. Moreover, we focus on their application as an effective drug carrier to combat viral infections.

Statin-boosted cellular uptake and endosomal escape of penetratin due to reduced membrane dipole potential.

Cell penetrating peptides are promising tools for delivery of cargo into cells, but factors limiting or facilitating their cellular uptake are largely unknown. We set out to study the effect of the biophysical properties of the cell membrane on the uptake of penetratin, a cell penetrating peptide. Using labelling with pH-insensitive and pH-sensitive dyes, the kinetics of cellular uptake and endo-lysosomal escape of penetratin were studied by flow cytometry. We report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared with its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.

Automated high-content imaging for cellular uptake, from the Schmuck cation to the latest cyclic oligochalcogenides.

Recent progress with chemistry tools to deliver into living cells has seen a shift of attention from counterion-mediated uptake of cell-penetrating peptides (CPPs) and their mimics, particularly the Schmuck cation, toward thiol-mediated uptake with cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs), here exemplified by asparagusic acid. A persistent challenge in this evolution is the simultaneous and quantitative detection of cytosolic delivery and cytotoxicity in a high-throughput format. Here, we show that the combination of the HaloTag-based chloroalkane penetration assay (CAPA) with automated high-content (HC) microscopy can satisfy this need. The automated imaging of thousands of cells per condition in multiwell plates allows us to obtain quantitative data on not only the fluorescence intensity but also on the localization in a very short time. Quantitative and statistically relevant results can be obtained from dose–response curves of the targeted delivery to selected cells and the cytotoxicity in the same experiment, even with poorly optimized cellular systems.

Newly synthesized peptide, Ara-27, exhibits significant improvement in cell-penetrating ability compared to conventional peptides.

Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 μM Ara-27 in both HDF and hDPSC. In contrast, 2 μM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 μM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.

How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides

Cell-penetrating peptides (CPP) are broadly recognized as efficient non-viral vectors for the internalization of compounds such as peptides, oligonucleotides or proteins. Characterizing these carriers requires reliable methods to quantify their intracellular uptake. Flow cytometry on living cells is a method of choice but is not always applicable (e.g. big or polarized cells), so we decided to compare it to fluorescence spectroscopy on cell lysates. Surprisingly, for the internalization of a series of TAMRA-labeled conjugates formed of either cationic or amphipathic CPPs covalently coupled to a decamer peptide, we observed important differences in internalization levels between both methods.We partly explained these discrepancies by analyzing the effect of buffer conditions (pH, detergents) and peptide sequence/structure on TAMRA dye accessibility. Based on this analysis, we calculated a correction coefficient allowing a better coherence between both methods. However, an overestimated signal was still observable for both amphipathic peptides using the spectroscopic detection, which could be due to their localization at the cell membrane. Based on several in vitro experiments modeling events at the plasma membrane, we hypothesized that fluorescence of peptides entrapped in the membrane bilayer could be quenched by the tryptophan residues of close transmembrane proteins. During cell lysis, cell membranes are disintegrated liberating the entrapped peptides and restoring the fluorescence, explaining the divergences observed between flow cytometry and spectroscopy on lysates.Overall, our results highlighted major biases in the fluorescently-based quantification of internalized fluorescently-labeled CPP conjugates, which should be considered for accurate uptake quantification.

19F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by 19F NMR.

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

Population-based analysis of cell-penetrating peptide uptake using a microfluidic droplet trapping array.

Cell-penetrating peptides (CPPs) have garnered significant attention as a method to introduce reporters and therapeutics into intact cells. While numerous studies have been performed identifying new CPP sequences, relatively little is known about their uptake efficiency at the single-cell level. Here, a droplet microfluidic trapping array was used to characterize CPP uptake across a population of single intact cells. The microfluidic device allowed for facile and rapid isolation and analysis of single-cell fluorescence in a 787-member overhead trapping array with > 99% droplet trapping efficiency. The permeability efficiencies of four different CPPs were studied and compared in HeLa cells. Population analysis was performed using linkage hierarchical cluster analysis by R programming to bin cells into subpopulations expressing very low to very high peptide uptake efficiencies. CPP uptake was observed to be heterogeneous across the population of cells with peptide concentration and sequence both playing important roles in the diversity of CPP uptake, the overall peptide uptake efficiency, and the intracellular homogeneity of peptide distribution. This microfluidic-based analytical approach finds application in personalized medicine and provides new insight in the heterogeneity of CPP uptake which has the potential to affect both biosensor and drug internalization in intact cells. Graphical abstract .

Intracellular Delivery of Nanoparticles Mediated by Lactoferricin Cell-Penetrating Peptides in an Endocytic Pathway.

Cell-penetrating peptides (CPPs) containing a preponderance of basic amino acids are able to deliver biologically active macromolecules and nanomaterials into live cells. Quantum dots (QDs) are nanoparticles with unique fluorescence properties that have found wide application in biomedical imaging. In this study, we demonstrate transduction of an L6 CPP (RRWQWR) derived from bovine lactoferricin (LFcin) into human lung cancer cells. L6 noncovalently interacts with QDs to form stable complexes. L6/QD complexes enter cells most efficiently when prepared at a nitrogen/phosphate ratio of 60. Mechanistic studies indicate that L6/QD complexes enter cells by endocytosis. Treatment with 1,2-benzisothiazol-3(2H)-one (BIT), an industrial preservative that enhances uptake of certain CPPs, does not affect L6 CPP-mediated protein transduction efficiency. L6 and L6/QD complexes are not cytotoxic. These results indicate that L6 LFcin might be an efficient and safe nanoshuttle for nanoparticles or nanomedicines in biomedical applications.

Spectroscopic and Microscopic Approach to Monitor the Changes in Bilayer Rigidity during Cell Penetrating Peptide Induced Self-Reproduction of Phospholipid Vesicles

Understanding peptide-membrane interaction in view of structural change of lipid bilayer is essential to develop new therapeutic strategies. Though different mechanisms are proposed for cell penetrating peptides (CPPs) penetration in lipid bilayer, the exact mechanism of cellular uptake of CPPs remains a significant unanswered piece of puzzle. Here, we report a novel observation that model phospholipid vesicles self-reproduce to generate unilamellar daughter vesicles in presence of a CPP, nonaarginine (R9). Fluorescence lifetime image analysis (FLIM) exquisitely captures the changes of L-α-phosphatidylcholine (LAPC) bilayer in different times and also quantifies the changes through valuable lifetime distributions obtained. Mean square displacement (MSD) calculations from FLIM images of single vesicle trajectory establish CPP induced self-reproduction of LAPC vesicles. Heterogeneous nature of lipid bilayer after 1 hour of R9 addition is observed from fluorescence correlation spectroscopy (FCS) which supposed to consider as the starting point of “mother vesicles” reproduction. Solvation dynamic study delineates the change in dynamics of the encapsulated water inside vesicles which also states different rigidity of lipid bilayer in different states of vesicle self-reproduction. Fluorescence anisotropy image analysis provides information regarding change in lipid bilayer rigidity by quantifying the rotational motion of the fluorophore in presence of perturbation of CPPs. From these experimental observations, we are able to establish phospholipid vesicles self-reproduction in presence of CPPs which may be considered as a possible route of CPPs action to deliver cargos to cell interior through hydrophobic barriers. Overall, our results provide an experimental underpinning of the role of lipid bilayer structural changes in self-reproduction which can exhibit great potential to interpret the protein-lipid interaction in emergence of life.

Selective Uptake Into Drug Resistant Mammalian Cancer by Cell Penetrating Peptide-Mediated Delivery.

Research over the past decade has identified several of the key limiting features ofmultidrug resistance (MDR) incancer therapy applications, such as evolving glycoprotein receptors at the surface of the cell that limit therapeutic uptake, metabolic changes that lead to protection from multidrug resistant mediators which enhance degradation or efflux of therapeutics, and difficulty ensuring retention of intact and functional drugs once endocytosed. Nanoparticles have been demonstrated to be effective delivery vehicles for a plethora of therapeutic agents, and in the case of nucleic acid based agents, they provide protective advantages. Functionalizing cell penetrating peptides, also known as protein transduction domains, onto the surface of fluorescent quantum dots creates a labeled delivery package to investigate the nuances and difficulties of drug transport in MDR cancer cells for potential future clinical applications of diverse nanoparticle-based therapeutic delivery strategies. In this study, eight distinct cell penetrating peptides were used (CAAKA, HSV1-VP22, HIV-TAT, HIV-gp41, Ku-70, hCT(9-32), integrin-β3, and K-FGF) to examine the different cellular uptake profiles in cancer versus drug resistant melanoma (A375 & A375-R), mesothelioma (MSTO & MSTO-R), and glioma (rat 9L and 9L-R, and human U87 & LN18) cell lines. The results of this study demonstrate that cell penetrating peptide uptake varies with drug resistancestatusand cell type, likely due to changes in cell surface markers. This study provides insight into developing functional nanoplatform delivery systems in drug resistant cancer models.

Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates

The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors.

Effects of N-terminal and C-terminal modification on cytotoxicity and cellular uptake of amphiphilic cell penetrating peptides

Purpose To assess the effect of “N-Acetylation and C-Amidation” on the cellular uptake, cytotoxicity and performance of amphiphilic cell penetrating peptides (CPP) loaded with methotrexate (MTX). Methods Several CPPs were synthesized by solid phase peptide synthesis method. Some of these sequences were modified with pyroglutamic acid at N-terminus and benzylamine or memantine at C-terminus. The resultant nanomaterials were prepared due to the physical linkage between CPPs and MTX. The internalization and cytotoxicity of both CPP-MTX bioconjugates and unmodified CPPs against MCF-7 human breast adenocarcinoma cells was evaluated. Results N-l and C-terminal modification did not alter the toxicity of CPPs. Physical linkage of CPPs with MTX resulted in a lower drug loading efficiency in comparison with chemically conjugated CPP-MTX bio-conjugates. Both nano-complexes increase the toxic effect of MTX on MCF-7 cells. Furthermore, N- and C-terminal modification may cause a tangible reduction in cellular uptake of CPPs. Conclusion In conclusion, it was shown that cytotoxicity of modified peptides which were physically linked with MTX, considerably higher than both physically loaded unmodified peptides and chemically conjugated peptides with MTX. Also, cell internalization was reduced after peptide end-protection. These findings confirmed the effectiveness of N- and C-terminal modifications on cell viability and CPPs internalization.

Cell Penetrating Peptides: A Promising Tool for the Cellular Upta ke of Macromolecular Drugs

The lipid bilayer of the plasma membrane is a selective impermeable barrier for the internalization of most macromolecules. Cell penetrating peptides (CPPs) could cross the plasma membrane barrier to deliver various molecules into cells and are considered as a promising tool to deliver macromolecular drugs. However, the exact cellular uptake mechanisms of CPPs are still ambiguous. It was reported that the exact cellular uptake pathway was determined by numerous factors such as the amino acid sequences (hydrophobicity and net charge), extracellular CPP concentration, cargoes' properties, cell type and the temperature. No matter what kind of mechanisms, the electrostatic interaction between the positive charged amino acids and the membrane with negatively charged glycosaminoglycans (GAGs), especially heparan sulphate proteoglycans (HSPGs), was supposed to be the first crucial step for CPPs uptake. The first recognition triggers cytoskeletal remodeling via activating Rho/Rac GTPases and kinase C, followed by the cell surface microdomains changing, ligand binding and cellular uptake. This review briefly discusses the classification, structure-activity relationships, cellular uptake mechanisms and biomedical applications of CPPs.

Cell-penetrating peptides and their utility in genome function modifications (Review)

For almost 30 years, studies have confirmed the effectiveness of cell-penetrating peptides (CPPs) in the facilitation of the intracellular delivery of various cargo molecules, including RNA, DNA, plasmids, proteins or nanoparticles, under in vitro and in vivo conditions. The cellular uptake of CPPs occurs via energy-dependent, as well as -independent mechanisms. In this relatively new direction of research, studies have attempted to introduce genome modification systems into cells by CPPs. Cellular uptake of CPPs carrying either covalently bound or electrostatically conjugated cargo, has several advantages over viral delivery systems, as it does not lead to any significant cytotoxicity or immunogenicity, and simultaneously it is more efficient than other non-viral systems. So far, CPPs have been successfully used to introduce Cre recombinase, zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats systems into cells. The present article systematically reviewed the information obtained from studies on CPPs and assessed their utility with regard to their effectiveness and safety of use.

Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane fluidity

Cell-penetrating peptides (CPPs) are prominent delivery vehicles to confer cellular entry of (bio-) macromolecules. Internalization efficiency and uptake mechanism depend, next to the type of CPP and cargo, also on cell type. Direct penetration of the plasma membrane is the preferred route of entry as this circumvents endolysosomal sequestration. However, the molecular parameters underlying this import mechanism are still poorly defined. Here, we make use of the frequently used HeLa and HEK cell lines to address the role of lipid composition and membrane potential. In HeLa cells, at low concentrations, the CPP nona-arginine (R9) enters cells by endocytosis. Direct membrane penetration occurs only at high peptide concentrations through a mechanism involving activation of sphingomyelinase which converts sphingomyelin into ceramide. In HEK cells, by comparison, R9 enters the cytoplasm through direct membrane permeation already at low concentrations. This direct permeation is strongly reduced at room temperature and upon cholesterol depletion, indicating a complex dependence on membrane fluidity and microdomain organisation. Lipidomic analyses show that in comparison to HeLa cells HEK cells have an endogenously low sphingomyelin content. Interestingly, direct permeation in HEK cells and also in HeLa cells treated with exogenous sphingomyelinase is independent of membrane potential. Membrane potential is only required for induction of sphingomyelinase-dependent uptake which is then associated with a strong hyperpolarization of membrane potential as shown by whole-cell patch clamp recordings. Next to providing new insights into the interplay of membrane composition and direct permeation, these results also refute the long-standing paradigm that transmembrane potential is a driving force for CPP uptake.

Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.