Translocation 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.

Translocation of cell-penetrating peptides using the weighted ensemble method within molecular dynamics simulations approach.

We report spontaneous translocation of a single cell-penetrating peptide (CPP) across a model membrane using the weighted ensemble (WE) method within the molecular dynamics (MD) simulations approach. Furthermore, we show that a water pore plays a crucial role in translocating the peptide and a tilt angle of the peptide also affects the translocation. Last, using umbrella sampling, we obtain the potential of mean force (PMF) along the translocation path and show that a single CPP's translocation is energetically unfavorable.

Cell penetrating peptides facilitate protein transport into the cell-sized asymmetric lipid vesicles at arbitrary timing.

Cell penetrating peptides (CPP) play an important role of drug delivery system. CPP have a strategy to overcame the membrane impermeability. There are two pathways to across the lipid bilayer via CPPs such as direct translocation and endocytosis. The direct translocation of CPPs was investigated using an artificial lipid membrane, such as planar lipid bilayers and nano-sized lipid vesicles. The direct translocation via CPPs using the planar lipid bilayers suggests that the negative transmembrane potential generating between planar lipid bilayers plays an important role for the direct translocation of β-gal via pep-1. In this study, the interactions between CPPs and cell-sized lipid vesicles were investigated using two peptides (Pep-1, penetratin). Next, the interaction between the CPP-DNase I complexes and membrane were observed. In addition, we investigated the DNase I translocation of CPP-DNase I complexes into the GUVs. Finally, we demonstrated the formation of a cross-linked actin network in GUVs using CPP-mediated translocation.CPPs-DNase I complexes was easily interacted with the asymmetric GUVs containing DOPG or DOPS in the inner leaflet. Therefore, CPPs mediated direct translocation of DNase I into the asymmetric GUVs with negatively charged lipids on the inner leaflet. These results indicate that the CPPs-mediated direct translocation could be triggered by the transmembrane potential generating by asymmetric GUV membrane. We found that the penetratin-mediated translocation of DNase I was more efficient than the Pep-1-mediated translocation. Using this asymmetric membrane component and CPP condition, we could translocate streptavidin into GUVs. Thus, highly versatile of CPP-mediated translocation was shown. This system will contribute to creations of well-defined artificial cell models with the capacity to control the start of inside reaction.

Synthetic Polypeptides with Cationic Arginine Moieties Showing High Antimicrobial Activity in Similar Mineral Environments to Blood Plasma.

Translocation of cell-penetrating peptides is promoted by incorporated arginine or other guanidinium groups. However, relatively little research has considered the role of these functional groups on antimicrobial peptide activity. A series of cationic linear-, star- and multi-branched-poly(L-arginine-co-L-phenylalanine) have been synthesized via the ring-opening copolymerizations of corresponding N-carboxyanhydride monomers followed by further modifications using the N-heterocyclic carbene organocatalyst. All the polymers are characterized by the random coiled microstructure. Antibacterial efficacy, tested by the gram-positive B. subtilis bacteria and the gram-negative E. coli bacteria, was sensitive to the structure and relative composition of the copolymer and increased in the order of linear- < star- < multi-branched structure. The multi-branched-p[(L-arginine)23-co-(L-phenylalanine)7]8 polymer showed the best antibacterial property with the lowest minimum inhibitory concentration values of 48 μg mL−1 for E. coli and 32 μg mL−1 for B. subtilis. The efficacy was prominent for B. subtilis due to the anionic nature of its membrane. All of the resultant arginine moiety-containing polypeptides showed excellent blood compatibility. The antibiotic effect of the copolymers with arginine moieties was retained even in the environment bearing Ca2+, Mg2+, and Na+ ions similar to blood plasma. The cationic arginine-bearing copolypeptides were also effective for the sterilization of naturally occurring sources of water such as lakes, seas, rain, and sewage, showing a promising range of applicability.

Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore.

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (-150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (-5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Cell-Penetrating Peptides: Applications in Tumor Diagnosis and Therapeutics.

Since their identification over twenty-five years ago, the plethora of cell-penetrating peptides (CPP) and their applications has skyrocketed. These 5 to 30 amino acid in length peptides have the unique property of breaching the cell membrane barrier while carrying cargoes larger than themselves into cells in an intact, functional form. CPPs can be conjugated to fluorophores, activatable probes, radioisotopes or contrast agents for imaging tissues, such as tumors. There is no singular mechanism for translocation of CPPs into a cell, and therefore, many CPPs are taken up by a multitude of cell types, creating the challenge of tumor-specific translocation and hindering clinical effectiveness. Varying strategies have been developed to combat this issue and enhance their diagnostic potential by derivatizing CPPs for better targeting by constructing specific cell-activated forms. These methods are currently being used to image integrin-expressing tumors, breast cancer cells, human histiocytic lymphoma and protease-secreting fibrosarcoma cells, to name a few. Additionally, identifying safe, effective therapeutics for malignant tumors has long been an active area of research. CPPs can circumvent many of the complications found in treating cancer with conventional therapeutics by targeted delivery of drugs into tumors, thereby decreasing off-target side effects, a feat not achievable by currently employed conventional chemotherapeutics. Myriad types of chemotherapeutics such as tyrosine kinase inhibitors, antitumor antibodies and nanoparticles can be functionally attached to these peptides, leading to the possibility of delivering established and novel cancer therapeutics directly to tumor tissue. While much research is needed to overcome potential issues with these peptides, they offer a significant advancement over current mechanisms to treat cancer. In this review, we present a brief overview of the research, leading to identification of CPPs with a comprehensive state-of-the-art review on the role of these novel peptides in both cancer diagnostics as well as therapeutics.

Elementary processes for the entry of cell-penetrating peptides into lipid bilayer vesicles and bacterial cells

Cell-penetrating peptides (CPPs) can translocate across the plasma membrane of living eukaryotic cells and enter the cytosol without significantly affecting cell viability. Consequently, CPPs have been used for the intracellular delivery of biological cargo such as proteins and oligonucleotides. However, the mechanisms underlying the translocation of CPPs across the plasma membrane remain unclear. In this mini-review, we summarize the experimental results regarding the entry of CPPs into lipid bilayer vesicles obtained using three methods: the large unilamellar vesicle (LUV) suspension method, the giant unilamellar vesicle (GUV) suspension method, and the single GUV method. The advantages and disadvantages of these methods are also discussed. Experimental results to date clearly indicate that CPPs can translocate across lipid bilayers and enter the vesicle lumen. Three models for the mechanisms and pathways by which CPPs translocate across lipid bilayers are described: (A) through pores induced by CPPs, (B) through transient prepores, and (C) via formation of inverted micelles. Both the pathway of translocation and the efficiency of entry of CPPs depend on the lipid composition of the bilayer and the type of CPP. We also describe the interaction of CPPs with bacterial cells. Some CPPs have strong antimicrobial activities. There are two modes of action of CPPs on bacterial cells: CPPs can induce damage to the plasma membrane and thus increase permeability, or CPPs enter the cytosol of bacterial cells without damaging the plasma membrane. The information currently available on the elementary processes by which CPPs enter lipid bilayer vesicles and bacterial cells is valuable for elucidating the mechanisms of entry of CPPs into the cytosol of various eukaryotic cells.

Continuous detection of entry of cell-penetrating peptide transportan 10 into single vesicles

Entry of cell-penetrating peptides (CPPs) into living cells by translocating across plasma membranes is an important physiological phenomenon. To elucidate the mechanism of the translocation of CPPs across lipid bilayers, it is essential to reveal its elementary processes. For this purpose, here, we have developed a new method for the continuous, quantitative detection of the entry of CPPs into giant unilamellar vesicles (GUVs), where we investigate the interaction of fluorescent probe-labeled CPPs with single GUVs containing large unilamellar vesicles (LUVs) and fluorescent probes in their lumens using confocal microscopy. Using this method, we investigated the interaction of carboxyfluorescein (CF)-labeled transportan 10 (CF-TP10) with single GUVs comprised of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) containing LUVs of the same membrane and Alexa Fluor 647 hydrazide (AF647) in their lumens. At low concentrations of CF-TP10, first the fluorescence intensity (FI) of the GUV membrane increased with time, and then after some lag time the FI of the GUV lumen due to CF-TP10 increased continuously with time without leakage of AF647. At higher concentrations of CF-TP10, after the FI of the GUV lumen due to CF-TP10 increased significantly, leakage of AF647 started. These results indicate that CF-TP10 entered the GUV lumen by translocating across the GUV membrane and then bound to the LUVs there without pore formation and that CF-TP10 concentration in the lumen increased with time. The rate of entry of CF-TP10 into GUV lumen increased with CF-TP10 concentration. We discussed the kinetics of entry of CF-TP10 into single GUVs.

Negative Dipole Potentials and Carboxylic Polar Head Groups Foster the Insertion of Cell-Penetrating Peptides into Lipid Monolayers.

Cell-penetrating peptides (CPPs) are polycationic sequences of amino acids recognized as some of the most effective vehicles for delivering membrane-impermeable cargos into cells. CPPs can traverse cell membranes by direct translocation, and assessing the role of lipids on the membrane permeation process is important to convene a complete model of the CPP translocation. In this work, we focus on the biophysical basis of peptide-fatty acid interactions, analyzing how the acid-base and electrostatic properties of the lipids determine the CPP adsorption and incorporation into a Langmuir monolayer, focusing thus on the first two stages of the direct translocation mechanism. We sense the binding and insertion of the peptide into the lipid structure by measuring the changes in the surface pressure, the surface potential, and the reflectivity of the interface. We show that, beyond the presence of anionic moieties, negative dipole potentials and carboxylic polar head groups significantly promote the insertion of the peptide into the monolayer. On the basis of our results, we propose the appearance of stable CPP-lipid complexes whose kinetics of formation depends on the length of the lipids' hydrocarbon chains.

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)3 K, 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)3 K, 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.

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells

AbstractDespite extensive use of arginine‐rich cell‐penetrating peptides (CPPs)—including octaarginine (R8)—as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity‐sensitive dye (di‐4‐ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature‐inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells.

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Effects of Mechanical Properties of Lipid Bilayers on the Entry of Cell-Penetrating Peptides into Single Vesicles.

The translocation of cell-penetrating peptides (CPPs) through plasma membranes of living cells is an important physiological phenomenon in biomembranes. To reveal the mechanism underlying the translocation of a CPP, transportan 10 (TP10), through lipid bilayers, we examined the effects of the mechanical properties of lipid bilayers on the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into a giant unilamellar vesicle (GUV) using the single GUV method. First, we examined the effect of lateral tension in membranes on the entry of CF-TP10 into single GUVs comprising a mixture of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8). CF-TP10 entered the GUV lumen before the membrane permeation of Alexa Fluor 647 hydrazide (AF647) from the GUV and thus before pore formation in the membrane. The fraction of entry of CF-TP10 before pore formation and the rate of membrane rupture increased with tension. The CF-TP10-induced fractional change in the membrane area increased continuously with time until membrane rupture, but it increased more slowly than did the CF-TP10 concentration in the GUV membrane. A high mole fraction of cholesterol inhibited the entry of CF-TP10 into single GUVs by suppressing the translocation of CF-TP10 from the external to the internal monolayer, although higher concentrations of CF-TP10 induced the formation of pores through which CF-TP10 rapidly translocated. Suppression of the translocation of CF-TP10 by cholesterol can be reasonably explained by the large line tension of a prepore. We discussed the role of mechanical properties in membranes on the entry of CF-TP10 into single GUVs and proposed a hypothesis of the mechanism that CF-TP10 translocates across a bilayer through transient hydrophilic prepores in the membrane.

Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes

Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways — endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides.

Cholesterol Influence on the Interaction of Cell Penetrating Peptides (CPPs) with Model Membranes

A number of high efficiency peptidic transporters are known, which are called CPPs, and which use three mechanisms of cell incorporation. The models proposed to explain the direct translocation of CPPs across biological membranes include the “inverted micelle model”, models involving the formation of membrane pores and the “carpet model”1. Studies have demonstrated that cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes2. Other studies show that higher cholesterol content and tighter packing of the membrane predominantly reduces accumulation of transportan, TP10 and the model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions3. Our circular dichroism results show a change in the secondary structure of the peptides upon interaction with model membranes. In addition, fluorescence spectroscopy studies have demonstrated changes in the interaction of peptides with LUV ternary mixtures of DMPC/DMPG/CHO as a function of the cholesterol content.Acknowledgements: We thank Project FONDECYT 1140800 for funding and CONICYT for the PhD scholarship for VSS.1.Trabulo S., Cardoso A.L., Mano M., y Pedroso de Lima M.C., Pharmaceuticals 3, 961-993 (2010).2.Arsov1 Z., Nemec1 M., Schara1 M., Johansson H., Langel U. y Zorko M., Journal of Peptide Science 14(12), 1303-1308 (2008).3.Paea J., Saalikb P., Liivamagia L., Lubenetsa D., Arukuuskc P., Langelc U. y Pooga M. Journal of Controlled Release 192, 103-113 (2014).

Role of arginine and lysine in the antimicrobial mechanism of histone-derived antimicrobial peptides

Translocation of cell-penetrating peptides is often promoted by increased content of arginine or other guanidinium groups. However, relatively little research has considered the role of these functional groups on antimicrobial peptide activity. This study compared the activity of three histone-derived antimicrobial peptides—buforin II, DesHDAP1, and parasin—with variants that contain only lysine or arginine cationic residues. These peptides operate via different mechanisms as parasin causes membrane permeabilization while buforin II and DesHDAP1 translocate into bacteria. For all peptides, antibacterial activity increased with increased arginine content. Higher arginine content increased permeabilization for parasin while it improved translocation for buforin II and DesHDAP1. These observations provide insight into the relative importance of arginine and lysine in these antimicrobial peptides.

Accumulation of cell-penetrating peptides in large unilamellar vesicles: A straightforward screening assay for investigating the internalization mechanism.

The internalization of cell-penetrating peptides (CPPs) into liposomes (large unilamellar vesicles, LUVs) was studied with a rapid and robust procedure based on the quenching of a small fluorescent probe, 7-nitrobenz-2-oxa-1,3-diazole (NBD). Quenching can be achieved by reduction with dithionite or by pH jump. LUVs with different compositions of phospholipids (PLs) were used to screen the efficacy of different CPPs. In order to "validate" the composition of the membrane models, a control cationic peptide, which does not enter eukaryotic cells, was included in the study. It was found that pure DOPG or DOPG within ternary mixtures with cholesterol are the most appropriate models for studying CPP translocation. An anionic lipid, such as DOPG, is required for the adsorption of the basic peptides on the surface of LUVs. In addition, it acts as transfer agent through the lipid bilayer. A fluid phase and/or the presence of phase defects also appear mandatory for the internalization to occur. The neutralization of charges within an inverted micelle demonstrated in the case of DOPG and also proposed for a ternary mixture of PLs might not be the only mechanism for the CPP translocation. Finally, it is shown that oleic acid facilitates the entry inside LUVs in gel phase of a series of cationic peptides including CPPs and also the negative control peptide PKCi.

Assessing the Translocation of CPPs using Force Measurement, Single Molecules and Emulsions

Gene therapy and inhibition of malign molecules inside abnormal cells rely on an efficient and specific delivery of drugs into the targeted cells. To do so, disruptive methods such as electroporation or partial destruction of the cell membrane through chemical reagent are currently used.An alternative technique is the use of carriers that will help the drugs to cross the membrane, without introducing deleterious effects due to the membrane disruption. A family of such carriers is known as Cell Penetrating Peptides (CPPs). Those peptides are short, about ten amino acids, and often cationic. They are able to translocate through the membrane with different cargos and deliver them into the cytosol. However the mechanisms are still unknown.We use three different techniques to gain insights into the mechanism leading to the translocation of a CPP. One method use a force measuring tool adapted to living cells: the Biomembrane Force Probe. It gives us the partners that can be involved in such process. A second technique is based on a TIRF microscope where the reflection is made on a suspended bilayer due to a high optical index differences between each side of the membrane. This allows us to follow single peptides close to a suspended bilayer. We managed to observe single CPPs that are in suspension or bound to the membrane. A third method relies on a water-oil emulsion. Two water droplets, one containing fluorescent CPPs and the other only buffer, are generated in an oil-lipid mixture, so that a lipid bilayer is formed at their interface. We follow the translocation of the fluorescent CPPs between the two droplets.So far we were able to identify sugars as binding partner of the CPPs and to image CPP translocation.

Molecular mechanism of translocation and design of cell penetrating peptides based on molecular dynamics simulation

The lack of cell specificity of cell penetrating peptides (CPPs) remains a major issue for their clinical application. To successfully design disease targeting CPPs, it is necessary to improve our understanding of the molecular mechanism of membrane translocation of CPPs and locate the key factors that affect this mechanism. Molecular dynamics simulations have been proven to be a valuable tool to study the molecular mechanism of membrane penetration by small peptides. Molecular dynamics simulations have mainly been used to study the translocation process, including structural properties of transition states, free energy profiles, and the effect of cell membrane peptide sequence/structure variation on possible molecular mechanisms and delivery efficiency. With the development of enhanced sampling techniques, it is possible to obtain translocation free energy and possible transition pathways from one continuous simulation. Hence, molecular dynamics simulations will provide in depth mechanistic understanding for designing cell targeting CPPs that are critical for reducing side effects and enhancing the efficient delivery of drugs into target cells.

Translocation of cell-penetrating peptides across the plasma membrane is controlled by cholesterol and microenvironment created by membranous proteins.

Despite the extensive research in the field of CPPs' cell entry the exact mechanisms underlying their cellular uptake and the role of involved cell surface molecules in the internalization process have remained controversial. The present study focused on the interactions between CPPs and plasma membrane compounds using giant plasma membrane vesicles (GPMVs). GPMVs have shown to be a suitable model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis. Our results show that higher cholesterol content and tighter packing of membrane predominantly reduce the accumulation of transportan, TP10 and model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions. The partial digestion of membrane proteins from GPMVs' surface, on the other hand, drastically reduced the accumulation of nona-arginine and Tat peptide into vesicles, suggesting that proteins play a crucial role in the uptake of arginine-rich CPPs.