Detection of the Entry of Nonlabeled Transportan 10 into Single Vesicles.

The entry of cell-penetrating peptides (CPPs) into live cells and lipid vesicles has been monitored using probe (e.g., fluorescent dye)-labeled CPPs. However, probe labeling may alter the interaction of CPPs with membranes. We have developed a new method to detect the entry of nonlabeled CPPs into the lumens of single giant unilamellar vesicles (GUVs) without pore formation in the GUV membrane. The GUVs contain large unilamellar vesicles (LUVs) whose lumens contain a high (self-quenching) concentration of the fluorescent dye calcein. If the CPPs enter the GUV lumen and interact with these LUVs to induce calcein leakage, the fluorescence intensity (FI) due to calcein in the GUV lumen increases. The lipid compositions of the LUVs and GUVs allow leakage from LUVs but not from the GUVs. We applied this method to detect the entry of transportan 10 (TP10) into single GUVs comprising dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine and examined the interaction of low concentrations of nonlabeled TP10 with single GUVs whose lumens contain Alexa Fluor 647 hydrazide (AF647) and the LUVs mentioned above. The FI of the GUV lumen due to calcein increased continuously with time without leakage of AF647, indicating that TP10 entered the GUV without pore formation in the GUV membrane. The lumen intensity due to calcein increased with TP10 concentration, indicating that the rate of entry of TP10 into the GUV lumen increased. We estimated the minimum TP10 concentration in a GUV lumen detected by this method. We discuss the entry of nonlabeled TP10 and the characteristics of this method.

Location of Peptide-Induced Submicron Discontinuities in the Membranes of Vesicles Using ImageJ.

Cell penetrating peptide transportan 10 and antimicrobial peptide melittin formed submicron pores in the lipid membranes of vesicles which are explained by the leakage of water-soluble fluorescent probes from the inside of vesicles to the outside. It is hypothesized that these submicron pores induce submicron discontinuities in the membranes. Considering this hypothesis, a technique has developed to locate the submicron discontinuities in the membranes of giant unilamellar vesicles (GUVs) using ImageJ. In this technique, at first the edges of membrane of a 'single GUV' are detected and then these edges are used to locate the submicron discontinuities. Two continuous rings are observed after applying the ImageJ in GUVs which indicated the edges of membrane. In contrast, the submicron discontinuations are detected at the edges of transportan 10 and melittin induced pore formed membranes. This investigation might be helpful for the elucidation of mechanism of the peptide-induced pore formation in the membranes of vesicles.

Cell Penetration Profiling Using the Chloroalkane Penetration Assay.

Biotherapeutics are a promising class of molecules in drug discovery, but they are often limited to extracellular targets due to their poor cell penetration. High-throughput cell penetration assays are required for the optimization of biotherapeutics for enhanced cell penetration. We developed a HaloTag-based assay called the chloroalkane penetration assay (CAPA), which is quantitative, high-throughput, and compartment-specific. We demonstrate the ability of CAPA to profile extent of cytosolic penetration with respect to concentration, presence of serum, temperature, and time. We also used CAPA to investigate structure-penetration relationships for bioactive stapled peptides and peptides fused to cell-penetrating sequences. CAPA is not only limited to measuring cytosolic penetration. Using a cell line where HaloTag is localized to the nucleus, we show quantitative measurement of nuclear penetration. Going forward, CAPA will be a valuable method for measuring and optimizing the cell penetration of biotherapeutics.

Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.

Renal Clearable Peptide Functionalized NaGdF4 Nanodots for High-Efficiency Tracking Orthotopic Colorectal Tumor in Mouse.

The effective delivery of bioimaging probes to a selected cancerous tissue has extensive significance for biological studies and clinical investigations. Herein, the peptide functionalized NaGdF4 nanodots (termed as, pPeptide-NaGdF4 nanodots) have been prepared for highly efficient magnetic resonance imaging (MRI) of tumor by formation of Gd-phosphonate coordinate bonds among hydrophobic NaGdF4 nanodots (4.2 nm in diameter) with mixed phosphorylated peptide ligands including a tumor targeting phosphopeptide and a cell penetrating phosphopeptide. The tumor targeting pPeptide-NaGdF4 nanodots have paramagnetic property with ultrasmall hydrodynamic diameter (HD, c.a., 7.3 nm) which greatly improves their MRI contrast ability of tumor and facilitates renal clearance. In detail, the capability of the pPeptide-NaGdF4 nanodots as high efficient contrast agent for in vivo MRI is evaluated successfully through tracking small drug induced orthotopic colorectal tumor (c.a., 195 mm3 in volume) in mouse.

The function of activatable cell-penetrating peptides in human intrahepatic bile duct epithelial cells.

This study aimed to investigate the function of Activatable Cell-Penetrating Peptides (ACPP) in detecting the changes of human intrahepatic bile duct epithelial cell(hIBDEC). ACPP, which target matrix metalloproteinases, were constructed. All were labeled with FITC and Gd-DTPA at the N-terminal. Fluorescence microscopy was used to observe the fluorescence intensity inside hIBDEC after stimulating with different concentrations of LPS and incubating with different concentrations of ACPP to determine the optimal concentration range for LPS stimulation and the optimal concentration for FITC-ACPP effect. Flow cytometry and magnetic resonance imaging were used to detect fluorescence signal intensity and nuclear magnetic resonance signal intensity, respectively, after stimulating with different concentrations of LPS. LPS stimulation time and ACPP incubation time were also evaluated, and variance analysis was conducted to analyze intracellular signal change characteristics for every group. Activatable Cell-Penetrating Peptides (ACPP), which were marked with FITC and Gd-DTPA had target-penetrating activity. The intracellular signal intensity gradually increased with the increase in LPS stimulation time and ACPP incubation time within a certain range; however, it did not increase with the increase of LPS concentration. ACPP can be used for imaging hIBDEC with epithelial-mesenchymal transition.

Translocation mechanism(s) of cell-penetrating peptides: biophysical studies using artificial membrane bilayers.

The ability of cell-penetrating peptides (CPPs) to cross cell membranes has found numerous applications in the delivery of bioactive compounds to the cytosol of living cells. Their internalization mechanisms have been questioned many times, and after 20 years of intense debate, it is now widely accepted that both energy-dependent and energy-independent mechanisms account for their penetration properties. However, the energy-independent mechanisms, named "direct translocation", occurring without the requirement of the cell internalization machinery, remain to be fully rationalized at the molecular level. Using artificial membrane bilayers, recent progress has been made toward the comprehension of the direct translocation event. This review summarizes our current understanding of the translocation process, starting from the adsorption of the CPP on the membrane to the membrane crossing itself. We describe the different key steps occurring before direct translocation, because each of them can promote and/or hamper translocation of the CPP through the membrane. We then dissect the modification to the membranes induced by the presence of the CPPs. Finally, we focus on the latest studies describing the direct translocation mechanisms. These results provide an important framework within which to design new CPPs and to rationalize an eventual selectivity of CPPs in their penetration ability.

Detecting Cytosolic Peptide Delivery with the GFP Complementation Assay in the Low Micromolar Range.

Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as a fusion protein with mCherry.

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα.

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.

Solution NMR studies on the orientation of membrane-bound peptides and proteins by paramagnetic probes.

Many peptides and proteins are attached to or immersed in a biological membrane. In order to understand their function not only the structure but also their topology in the membrane is important. Solution NMR spectroscopy is one of the most often used approaches to determine the orientation and localization of membrane-bound peptides and proteins. Here we give an application-oriented overview on the use of paramagnetic probes for the investigation of membrane-bound peptides and proteins. The examples discussed range from the large pool of antimicrobial peptides, bacterial toxins, cell penetrating peptides to domains of larger proteins or the calcium regulating protein phospholamban. Topological information is obtained in all these examples by the use of either attached or freely mobile paramagnetic tags. For some examples information obtained from the paramagnetic probes was included in the structure determination.

Protein Delivery to Cytosol by Cell-Penetrating Peptide Bearing Tandem Repeat Penetration-Accelerating Sequence.

Pas2r12 is comprised of a repeat of the penetration-accelerating sequence (Pas) (Pas2: FFLIG-FFLIG) and D-form dodeca-arginine (r12), a cell-penetrating peptide. Pas2r12 significantly enhances cytosolic delivery of cargo proteins, including enhanced green fluorescent protein and immunoglobulin G. Simply incubating Pas2r12 with cargo leads to their cytosolic tranlsocation. Cytosolic delivery of cargo by Pas2r12 involves caveolae-mediated endocytosis. In this chapter, we describe methods of cytosolic delivery of cargo using Pas2r12 and provide methods for investigating the cellular uptake pathway of cargo by Pas2r12.

A Single GUV Method for Revealing the Action of Cell-Penetrating Peptides in Biomembranes.

The mechanism of entry of cell-penetrating peptides (CPPs) into the cytosol of various cells has been studied by examining the interaction of CPPs with lipid bilayers and their entry into lipid vesicle lumens using various methods. Here we describe a single giant unilamellar vesicle (GUV) method to study CPPs. In this new method, we use GUVs containing small GUVs in the mother GUV lumen or GUVs containing large unilamellar vesicles (LUVs) in the GUV lumen and investigate the interaction of fluorescent probe-labeled CPPs with single GUVs in real time using confocal laser scanning microscopy. This method can detect CPPs in the GUV lumen with high sensitivity, allowing immediate measurement of the time course of entry of CPPs into the vesicle lumen. This method allows simultaneous measurement of the entry of CPPs and of CPP-induced pore formation, allowing the relationship between the two events to be determined. One can also simultaneously measure the entry of CPPs and the CPP concentration in the GUV membrane. The rate of entry of CPPs into a single GUV lumen can be estimated by obtaining the fraction of GUVs into which CPPs entered before a specific time t without pore formation among all examined GUVs (i.e., the fraction of entry) and the lumen intensity due to LUVs with bound CPPs. This method is therefore useful for elucidating the mechanism of entry of CPPs into lipid vesicles.

Biological Membrane-Penetrating Peptides: Computational Prediction and Applications.

Peptides comprise a versatile class of biomolecules that present a unique chemical space with diverse physicochemical and structural properties. Some classes of peptides are able to naturally cross the biological membranes, such as cell membrane and blood-brain barrier (BBB). Cell-penetrating peptides (CPPs) and blood-brain barrier-penetrating peptides (B3PPs) have been explored by the biotechnological and pharmaceutical industries to develop new therapeutic molecules and carrier systems. The computational prediction of peptides' penetration into biological membranes has been emerged as an interesting strategy due to their high throughput and low-cost screening of large chemical libraries. Structure- and sequence-based information of peptides, as well as atomistic biophysical models, have been explored in computer-assisted discovery strategies to classify and identify new structures with pharmacokinetic properties related to the translocation through biomembranes. Computational strategies to predict the permeability into biomembranes include cheminformatic filters, molecular dynamics simulations, artificial intelligence algorithms, and statistical models, and the choice of the most adequate method depends on the purposes of the computational investigation. Here, we exhibit and discuss some principles and applications of these computational methods widely used to predict the permeability of peptides into biomembranes, exhibiting some of their pharmaceutical and biotechnological applications.

Protein Delivery by PTDs/CPPs.

The ability to deliver or transduce proteins into cells allows for the manipulation of cell biology in culture, preclinical models, and potentially human disease. Fusion proteins containing the TAT peptide transduction domain (PTD), also known as cell-penetrating peptide (CPP), allow for delivery of a wide variety of proteins, including enzymes, transcription factors, tumor suppressor proteins, and many more. TAT-fusion proteins are generated cloning in-frame into the pTAT-HA plasmid, then transformed into E. coli for expression, and purified by the 6-His affinity tag over Ni-NTA column, followed by a final IEX FPLC purification step.

MALDI-TOF mass spectrometry: a powerful tool to study the internalization of cell-penetrating peptides.

This review summarizes the contribution of MALDI-TOF mass spectrometry in the study of cell-penetrating peptide (CPP) internalization in eukaryote cells. This technique was used to measure the efficiency of cell-penetrating peptide cellular uptake and cargo delivery and to analyze carrier and cargo intracellular degradation. The impact of thiol-containing membrane proteins on the internalization of CPP-cargo disulfide conjugates was also evaluated by combining MALDI-TOF MS with simple thiol-specific reactions. This highlighted the formation of cross-linked species to cell-surface proteins that either remained trapped in the cell membrane or led to intracellular delivery. MALDI-TOF MS is thus a powerful tool to dissect CPP internalization mechanisms.

Development of novel peptides for mitochondrial drug delivery: amino acids featuring delocalized lipophilic cations.

PURPOSE: To create a new class of mitochondria-penetrating peptides (MPPs) that would facilitate drug delivery into the organelle through the inclusion of delocalized lipophilic cations (DLCs) in the peptide sequence. METHODS: We synthesized two novel amino acids featuring DLCs and incorporated them into peptides. Systematic studies were conducted to compare peptides containing these residues to those with natural cationic amino acids. Diastereomers were compared to determine the most advantageous arrangement for these peptides. Peptide lipophilicity, cellular uptake and mitochondrial specificity were compared for a variety of peptides. RESULTS: Synthetic DLC residues were found to increase mitochondrial localization of MPPs due to higher overall hydrophobicity. MPP stereochemistry was important for cellular uptake rather than subcellular localization. This study reaffirmed the importance of uniform overall charge distribution for mitochondrial specificity. CONCLUSIONS: DLCs can be incorporated into synthetic peptides and facilitate mitochondrial drug delivery. Lipophilicity and charge distribution must be carefully balanced to ensure localization within mitochondria.

Dimeric cationic amphiphilic polyproline helices for mitochondrial targeting.

PURPOSE: Efficient delivery of therapeutic biopolymers across cell membranes remains a daunting challenge. The development of cell-penetrating peptides (CPPs) has been useful; however, many CPPs are found trapped within endosomes, limiting their use as delivery agents. We optimize a class of CPPs, cationic amphiphilic polyproline helices (CAPHs), for direct transport into cells with mitochondrial localization through dimerization. METHODS: The CAPH P11LRR used for this study has been found to enter cells by two distinct pathways: an endocytotic pathway was favored at low concentrations; internalization by direct transport was observed at higher concentrations. CAPH was dimerized to probe if direct transport within cells may be enhanced through increased association of CAPH with the membrane and through the association of individual peptides within the membrane. RESULTS: The dimerization of the CAPH was found to significantly increase cellular uptake over its monomeric counterpart, with a concomitant lowering of the concentration threshold favoring direct transport. Evidence for direct transport within cells and mitochondrial localization was observed. CONCLUSIONS: CAPH cellular uptake efficiency can be significantly enhanced through peptide dimerization while favoring cell entry via direct transport at low concentration with low cell toxicity.

Cleavage of the vaspin N-terminus releases cell-penetrating peptides that affect early stages of adipogenesis and inhibit lipolysis in mature adipocytes.

Vaspin expression and function is related to metabolic disorders and comorbidities of obesity. In various cellular and animal models of obesity, diabetes and atherosclerosis vaspin has shown beneficial, protective and/or compensatory action. While testing proteases for inhibition by vaspin, we noticed specific cleavage within the vaspin N-terminus and sequence analysis predicted cell-penetrating activity for the released peptides. These findings raised the question whether these proteolytic peptides exhibit biological activity.We synthesized various N-terminal vaspin peptides to investigate cell-penetrating activity and analyse uptake mechanisms. Focusing on adipocytes, we performed microarray analysis and functional assays to elucidate biological activities of the vaspin-derived peptide, which is released by KLK7 cleavage (vaspin residues 21-30; VaspinN). Our study provides first evidence that proteolytic processing of the vaspin N-terminus releases cell-penetrating and bioactive peptides with effects on adipocyte biology. The VaspinN peptide increased preadipocyte proliferation, interfered with clonal expansion during the early stage of adipogenesis and blunted adrenergic cAMP-signalling, downstream lipolysis as well as insulin signalling in mature adipocytes.Protease-mediated release of functional N-terminal peptides presents an additional facet of vaspin action. Future studies will address the mechanisms underlying the biological activities and clarify, if vaspin-derived peptides may have potential as therapeutic agents for the treatment of metabolic diseases.

Quantitation of Super Basic Peptides in Biological Matrices by a Generic Perfluoropentanoic Acid-Based Liquid Chromatography-Mass Spectrometry Method.

Peptides represent a promising modality for the design of novel therapeutics that can potentially modulate traditionally non-druggable targets. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two large families that are being explored extensively as drug delivery vehicles, imaging reagents, or therapeutic treatments for various diseases. Many CPPs and AMPs are cationic among which a significant portion is extremely basic and hydrophilic (e.g., nona-arginine). Despite their attractive therapeutic potential, it remains challenging to directly analyze and quantify these super cationic peptides from biological matrices due to their poor chromatographic behavior and MS response. Herein, we describe a generic method that combines solid phase extraction and LC-MS/MS for analysis of these peptides. As demonstrated, using a dozen strongly basic peptides, low µM concentration of perfluoropentanoic acid (PFPeA) in the mobile phase enabled excellent compound chromatographic retention, thus avoiding co-elution with solvent front ion suppressants. PFPeA also had a charge reduction effect that allowed the selection of parent/ion fragment pairs in the higher m/z region to further reduce potential low molecular weight interferences. When the method was coupled to the optimized sample extraction process, we routinely achieved low digit ng/ml sensitivity for peptides in plasma/tissue. The method allowed an efficient evaluation of plasma stability of CPPs/AMPs without fluorescence derivatization or other tagging methods. Importantly, using the widely studied HIV-TAT CPP as an example, the method enabled us to directly assess its pharmacokinetics and tissue distribution in preclinical animal models.

Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus.

Cell-penetrating peptides (CPPs) are used for various applications, especially in the biomedical field. Recently, CPPs have been used as a part of carrier to deliver proteins and/or genes into plant cells and tissues; hence, these peptides are attractive tools for plant biotechnological and agricultural applications, but require more efficient delivery rates and optimization by species before wide-scale use can be achieved. Here, we developed a library containing 55 CPPs to determine the optimal CPP characteristics for penetration of BY-2 cells and leaves of Nicotiana benthamiana, Arabidopsis thaliana, tomato (Solanum lycopersicum), poplar (hybrid aspen Populus tremula × tremuloides line T89), and rice (Oryza sativa). By investigating the cell penetration efficiency of CPPs in the library, we identified several efficient CPPs for all the plants studied except rice leaf. In the case of rice, several CPPs showed efficient penetration into rice callus. Furthermore, we examined the relationship between cell penetration efficiency and CPP secondary structural characteristics. The cell penetration efficiency of Lys-containing CPPs was relatively greater in plant than in animal cells, which could be due to differences in lipid composition and surface charge of the cell membranes. The variation in optimal CPPs across the plants studied here suggests that CPPs must be optimized for each plant species and target tissues of interest.