Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity

2018 ◽  
Vol 475 (10) ◽  
pp. 1773-1788 ◽  
Author(s):  
Ditlev Birch ◽  
Malene V. Christensen ◽  
Dan Staerk ◽  
Henrik Franzyk ◽  
Hanne Mørck Nielsen
Keyword(s):  
Amino Acids ◽  
Cell Viability ◽  
Mammalian Cells ◽  
Membrane Integrity ◽  
Cell Penetrating Peptides ◽  
Proliferating Cells ◽  
Epithelial Integrity ◽  
Cell Penetrating ◽  
Culture Models ◽  
Well Differentiated

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Owing to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of l-amino acids with the corresponding d-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of l-Penetratin (l-PEN) and its corresponding all-d analog (d-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, a rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to d-PEN. At equimolar concentrations, these observations were less pronounced or even absent for cells exposed to l-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), although d-PEN exhibited a longer half-life when compared with that of l-PEN when exposed to well-differentiated cell monolayers (cultured for 18–20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.

scholarly journals Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules

2005 ◽  
Vol 390 (2) ◽  
pp. 407-418 ◽  
Author(s):  
Catherine de Coupade ◽  
Antonio Fittipaldi ◽  
Vanessa Chagnas ◽  
Matthieu Michel ◽  
Sophie Carlier ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Mammalian Cells ◽  
Heparan Sulphate ◽  
Intracellular Delivery ◽  
Membrane Lipid ◽  
Cell Penetrating Peptides ◽  
Heparin Binding ◽  
Independent Manner ◽  
Cell Penetrating

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell® peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell® peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell® peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

scholarly journals Enhanced delivery of protein fused to cell penetrating peptides to mammalian cells

BMB Reports ◽  
2019 ◽  
Vol 52 (5) ◽  
pp. 324-329 ◽  
Author(s):  
Jung-Il Moon ◽  
Min-Joon Han ◽  
Shin-Hye Yu ◽  
Eun-Hye Lee ◽  
Sang-Mi Kim ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating

scholarly journals Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

2015 ◽  
Vol 1 (10) ◽  
pp. e1500821 ◽  
Author(s):  
Hong-Bo Pang ◽  
Gary B. Braun ◽  
Erkki Ruoslahti
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Vascular Permeability ◽  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Proteolytic Processing ◽  
Cell Surface Receptor ◽  
Neuropilin 1 ◽  
Cell Penetrating ◽  
Surface Receptor

Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery.

Development of helix-stabilized cell-penetrating peptides containing cationic α,α-disubstituted amino acids as helical promoters

2017 ◽  
Vol 25 (6) ◽  
pp. 1846-1851 ◽  
Author(s):  
Hiroko Yamashita ◽  
Takashi Misawa ◽  
Makoto Oba ◽  
Masakazu Tanaka ◽  
Mikihiko Naito ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating ◽  
Disubstituted Amino Acids

Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids

2016 ◽  
Vol 24 (12) ◽  
pp. 2681-2687 ◽  
Author(s):  
Takuma Kato ◽  
Hiroko Yamashita ◽  
Takashi Misawa ◽  
Koyo Nishida ◽  
Masaaki Kurihara ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasmid Dna ◽  
Unnatural Amino Acids ◽  
Dna Delivery ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating

scholarly journals Covalent attachment of enzyme as a membrane-label for viable eucaryotic cells.

1979 ◽  
Vol 83 (2) ◽  
pp. 511-515 ◽  
Author(s):  
N M Hogg ◽  
B Rotman
Keyword(s):  
Cell Surface ◽  
Cell Viability ◽  
Mammalian Cells ◽  
Membrane Integrity ◽  
Covalent Attachment ◽  
Histological Evidence ◽  
Coupling Procedure ◽  
Specific Affinity ◽  
Enzyme Label ◽  
Membrane Components

An enzyme, beta-D-galactosidase, was covalently coupled to mammalian cells by means of a bifunctional reagent. The coupling procedure did not cause appreciable loss of cell viability (less than 6%) as measured by plating efficiently and membrane integrity. After 24 h in culture, the cells exhibited an average of 2.6 x 10(4) molecules of beta-D-galactosidase per cell. Histological evidence indicated that the enzyme was localized on the cell surface and distributed uniformly among the cell population. Considerations for choosing enzyme-label include sensitivity of assay by enzymatic, immunologic and histochemical methods, and the possibility of isolating labeled membrane components by enzyme-specific affinity chromatography.

scholarly journals Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study

2007 ◽  
Vol 407 (2) ◽  
pp. 285-292 ◽  
Author(s):  
Samir El-Andaloussi ◽  
Peter Järver ◽  
Henrik J. Johansson ◽  
Ülo Langel
Keyword(s):  
Membrane Integrity ◽  
Cell Penetrating Peptides ◽  
Bioactive Molecules ◽  
Cargo Delivery ◽  
Wide Range ◽  
Cell Penetrating ◽  
Coupling Strategy ◽  
Transactivator Of Transcription

The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.

scholarly journals Membrane Active Peptides and Their Biophysical Characterization

Biomolecules ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 77 ◽  
Author(s):  
Fatma Gizem Avci ◽  
Berna Sariyar Akbulut ◽  
Elif Ozkirimli
Keyword(s):  
Antimicrobial Peptides ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Membrane Integrity ◽  
Cell Penetrating Peptides ◽  
Single Molecule Level ◽  
Active Peptides ◽  
Cell Penetrating ◽  
Biophysical Techniques ◽  
Membrane Active Peptides

In the last 20 years, an increasing number of studies have been reported on membrane active peptides. These peptides exert their biological activity by interacting with the cell membrane, either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. Membrane active peptides are attractive alternatives to currently used pharmaceuticals and the number of antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery in the drug pipeline is increasing. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). Antimicrobial peptides are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus, and fungi. Cell penetrating peptides are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity. Biophysical techniques shed light on peptide–membrane interactions at higher resolution due to the advances in optics, image processing, and computational resources. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Live imaging techniques allow examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provide clues on the peptide–lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.

scholarly journals Transportan Peptide Stimulates the Nanomaterial Internalization into Mammalian Cells in the Bystander Manner through Macropinocytosis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 552
Author(s):  
Yue-Xuan Li ◽  
Yushuang Wei ◽  
Rui Zhong ◽  
Ling Li ◽  
Hong-Bo Pang
Keyword(s):  
Mammalian Cells ◽  
Ex Vivo ◽  
Cell Penetrating Peptides ◽  
Cell Entry ◽  
Physical Interaction ◽  
Chemical Modifications ◽  
Cell Penetrating ◽  
Common Strategy ◽  
Entry Mechanism

Covalent coupling with cell-penetrating peptides (CPPs) has been a common strategy to facilitate the cell entry of nanomaterial and other macromolecules. Though efficient, this strategy requires chemical modifications on nanomaterials, which is not always desired for their applications. Recent studies on a few cationic CPPs have revealed that they can stimulate the cellular uptake of nanoparticles (NPs) simply via co-administration (bystander manner), which bypasses the requirement of chemical modification. In this study, we investigated the other classes of CPPs and discovered that transportan (TP) peptide, an amphiphilic CPP, also exhibited such bystander activities. When simply co-administered, TP peptide enabled the cells to engulf a variety of NPs, as well as common solute tracers, while these payloads had little or no ability to enter the cells by themselves. This result was validated in vitro and ex vivo, and TP peptide showed no physical interaction with co-administered NPs (bystander cargo). We further explored the cell entry mechanism for TP peptide and its bystander cargo, and showed that it was mediated by a receptor-dependent macropinocytosis process. Together, our findings improve the understanding of TP-assisted cell entry, and open up a new avenue to apply this peptide for nanomaterial delivery.

scholarly journals Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops

2020 ◽  
Author(s):  
Kuangyu Chen ◽  
Dehua Pei
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Tyrosine Phosphatase ◽  
Structural Features ◽  
Cell Penetrating Peptides ◽  
Biologically Active ◽  
Enzyme Replacement ◽  
Loop Region ◽  
Wide Range ◽  
Cell Penetrating

ABSTRACTEffective delivery of proteins into the cytosol and nucleus of mammalian cells would open the door to a wide range of applications including treatment of many currently intractable diseases. However, despite great efforts from numerous investigators and the development of a variety of innovative methods, effective protein delivery in a clinical setting is yet to be accomplished. Herein we report a potentially general approach to engineering cell-permeable proteins by genetically grafting a short cell-penetrating peptide to an exposed loop region of a protein of interest. The grafted peptide is conformationally constrained by the protein structure, sharing the structural features of cyclic cell-penetrating peptides and exhibiting enhanced proteolytic stability and cellular entry efficiency. Insertion of an amphipathic motif, Arg-Arg-Arg-Arg-Trp-Trp-Trp, into the loop regions of enhanced green fluorescent protein (EGFP), protein-tyrosine phosphatase 1B (PTP1B), and purine nucleoside phosphorylase (PNP) rendered all three proteins cell-permeable and biologically active in cellular assays. When added into growth medium, the engineered PTP1B dose-dependently reduced the phosphotyrosine levels of intracellular proteins, while the modified PNP protected PNP-deficient mouse T lymphocytes (NSU-1) against toxic levels of deoxyguanosine, providing a potential enzyme replacement therapy for a rare genetic disease.

Sign in / Sign up

Export Citation Format

Share Document