scholarly journals Impact of the Endosomal Escape Activity of Cell-Penetrating Peptides on the Endocytic Pathway

2020 ◽  
Vol 15 (9) ◽  
pp. 2355-2363 ◽  
Author(s):  
Helena M. Kondow-McConaghy ◽  
Nandhini Muthukrishnan ◽  
Alfredo Erazo-Oliveras ◽  
Kristina Najjar ◽  
Rudolph L. Juliano ◽  
...  
Keyword(s):  
Cell Penetrating Peptides ◽  
Endocytic Pathway ◽  
Endosomal Escape ◽  
Cell Penetrating

scholarly journals Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem

2017 ◽  
Vol 8 (3-4) ◽  
pp. 131-141 ◽  
Author(s):  
Julia C. LeCher ◽  
Scott J. Nowak ◽  
Jonathan L. McMurry
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Great Promise ◽  
Delivery Methods ◽  
Oligomeric Proteins ◽  
Cell Penetrating ◽  
History Of ◽  
A Cell ◽  
Escape Problem ◽  
Primary Focus

AbstractCell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This ‘endosomal escape problem’ has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.

scholarly journals HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

2018 ◽  
Author(s):  
Angela Steinauer ◽  
Jonathan R. LaRochelle ◽  
Rebecca Wissner ◽  
Samuel Berry ◽  
Alanna Schepartz
Keyword(s):  
Cell Penetrating Peptides ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Endosomal Escape ◽  
Stapled Peptides ◽  
Critical Determinant ◽  
Small Proteins ◽  
Primary Means ◽  
The Difference

AbstractProtein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered that cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine motif traffic readily to the cytosol and nucleus with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using a broad spectrum of techniques, including an RNA interference (RNAi) screen, fluorescence correlation spectroscopy (FCS), and confocal imaging, we identify VPS39—a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex—as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. Subsequent multi-color confocal imaging studies identify CPMPs within the endosomal lumen, particularly within the intraluminal vesicles (ILVs) of Rab7+ and Lamp1+ endosomes that are the products of HOPS-mediated fusion. These results suggest that CPMPs require HOPS to reach ILVs—an environment that serves as a prerequisite for efficient endosomal escape.

Decoding the Entry of Two Novel Cell-Penetrating Peptides in HeLa Cells:  Lipid Raft-Mediated Endocytosis and Endosomal Escape†

Biochemistry ◽  
2005 ◽  
Vol 44 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Christina Foerg ◽  
Urs Ziegler ◽  
Jimena Fernandez-Carneado ◽  
Ernest Giralt ◽  
Robert Rennert ◽  
...  
Keyword(s):  
Lipid Raft ◽  
Hela Cells ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Cell Penetrating

scholarly journals A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

2021 ◽  
Author(s):  
Jonathan L. McMurry ◽  
Schuyler B. Gentry ◽  
Scott J. Nowak ◽  
Xuelei Ni ◽  
Stephanie A. Hill ◽  
...  
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
Flow Chamber ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Calmodulin Binding ◽  
Distance Analysis ◽  
Cell Penetrating ◽  
Broad Array ◽  
Escape Problem

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called ‘endosomal escape problem,’ i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, “TAT-CaM,” evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio . Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat ( Heterocephalus glaber ) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

scholarly journals Conjugation of oligo-His peptides to magnetic γ-Fe2O3@SiO2 core-shell nanoparticles promotes their access to the cytosol

2021 ◽  
Author(s):  
Mathilde Le Jeune ◽  
Emilie Secret ◽  
Michaël Trichet ◽  
Aude Michel ◽  
Delphine Ravault ◽  
...  
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Core Shell ◽  
Shell Nanoparticles ◽  
Heating Efficiency ◽  
Multiple Substrates ◽  
Transmission Electron ◽  
Cell Penetrating ◽  
Calcein Leakage ◽  
Core Shell Nanoparticles

The endosomal entrapment of functional nanoparticles is a severe limitation to their use for biomedical applications. In the case of magnetic nanoparticles (MNPs), this entrapment leads to poor heating efficiency for magnetic hyperthermia and suppresses the possibility to manipulate them in the cytosol. Current strategies to limit their entrapment are based on their functionalization with cell-penetrating peptides in order to promote their translocation directly across the cell membrane or their endosomal escape. However, these strategies suffer from potential release of free peptides in cell and to the best of our knowledge there is currently a lack of effective methods for the cytosolic delivery of MNPs after incubation with cells. Herein, we report the conjugation of fluorescently labelled cationic peptides to γ-Fe2O3@SiO2 core-shell nanoparticles by click chemistry to improve MNP access to the cytosol. We compare the effect of Arg9 and His4 peptides. On one hand, Arg9 is a classical cell-penetrating peptide, able to enter cells by direct translocation and on the other hand, it has been demonstrated that sequences rich in histidine residues promote endosomal escape, most probably by the proton sponge effect. The methodology developed allows to have a high co-localization of the peptides and core-shell nanoparticles in cells and to attest that the grafting onto nanoparticles of peptides rich in histidine promotes NP access to the cytosol. The endosomal escape was confirmed by a calcein leakage assay and by ultrastructural analysis in transmission electron microscopy. No toxicity of the nanoparticles functionalized with peptides was found. We show that our conjugation strategy is compatible with the addition of multiple substrates and can thus be used for the delivery of cytoplasm-targeted therapeutics.

scholarly journals Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport across Phospholipid Membranes Induced by a Proton Gradient

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Fatemeh Madani ◽  
Alex Perálvarez-Marín ◽  
Astrid Gräslund
Keyword(s):  
Cell Penetrating Peptides ◽  
Proton Pumping ◽  
Model Systems ◽  
Endosomal Escape ◽  
Vesicle Membrane ◽  
Large Unilamellar Vesicles ◽  
Octyl Glucoside ◽  
Cell Penetrating ◽  
Pumping Activity ◽  
Extrusion Method

Detergent-mediated reconstitution of bacteriorhodopsin (BR) into large unilamellar vesicles (LUVs) was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG) as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

scholarly journals Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges

2012 ◽  
Vol 5 (11) ◽  
pp. 1177-1209 ◽  
Author(s):  
Alfredo Erazo-Oliveras ◽  
Nandhini Muthukrishnan ◽  
Ryan Baker ◽  
Ting-Yi Wang ◽  
Jean-Philippe Pellois
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Cell Penetrating
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