scholarly journals Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides

2019 ◽  
Vol 14 (12) ◽  
pp. 2641-2651 ◽  
Author(s):  
Jason Allen ◽  
Kristina Najjar ◽  
Alfredo Erazo-Oliveras ◽  
Helena M. Kondow-McConaghy ◽  
Dakota J. Brock ◽  
...  
Keyword(s):  
Small Molecule ◽  
Cell Penetrating Peptides ◽  
Human Cells ◽  
Endosomal Escape ◽  
Cytosolic Delivery ◽  
Cell Penetrating

scholarly journals Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Serena L. Y. Teo ◽  
Joshua J. Rennick ◽  
Daniel Yuen ◽  
Hareth Al-Wassiti ◽  
Angus P. R. Johnston ◽  
...  
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Current Understanding ◽  
Specific Cell ◽  
Intracellular Drug Delivery ◽  
Cytosolic Delivery ◽  
Efficient Delivery ◽  
Cell Penetrating ◽  
Model Protein ◽  
Low Sensitivity

AbstractCytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

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 β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

Biomolecules ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 51 ◽  
Author(s):  
Shane Stone ◽  
Tatjana Heinrich ◽  
Suzy Juraja ◽  
Jiulia Satiaputra ◽  
Clinton Hall ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Cell Penetrating Peptides ◽  
Stable Cell Line ◽  
Intracellular Space ◽  
Biologically Relevant ◽  
Cell Internalization ◽  
Cytosolic Delivery ◽  
Cell Penetrating ◽  
Split Protein

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

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 The Telomerase-Derived Anticancer Peptide Vaccine GV1001 as an Extracellular Heat Shock Protein-Mediated Cell-Penetrating Peptide

2016 ◽  
Author(s):  
Hong Kim ◽  
Eun-Hye Seo ◽  
Seung-Hyun Lee ◽  
Bum-Joon Kim
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Mhc Class Ii ◽  
Biological Effects ◽  
Peptide Vaccine ◽  
Heat Shock Protein 90 ◽  
Cell Penetrating Peptides ◽  
Small Peptides ◽  
Cytosolic Delivery ◽  
Cell Penetrating

Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting the transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered macromolecules. GV1001, a peptide derived from a reverse-transcriptase subunit of telomerase (hTERT) and developed as a vaccine against various cancers, reportedly has unexpected CPP properties. Unlike typical CPPs, such as the HIV-1 TAT peptide, GV1001 enabled the cytosolic delivery of macromolecules such as proteins, DNA and siRNA via extracellular heat shock protein 90 (eHSP90) and 70 (eHSP70) complexes. The eHSP-GV1001 interaction may have biological effects in addition to its cytosolic delivery function. GV1001 was originally designed as a MHC class II-binding cancer epitope, but its CPP properties may contribute to its strong anti-cancer immune response relative to other telomerase peptide-based vaccines. Cell signaling via eHSP-GV1001 binding may lead to unexpected biological effects, such as direct anticancer or antiviral effects. In this review, we focus on the CPP effects of GV1001 bound to eHSP90 and ehsp70.

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.

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