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

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.

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Unravelling cytosolic delivery of endosomal escape peptides with a quantitative endosomal escape assay (SLEEQ)

10.1101/2020.08.20.258350 ◽

2020 ◽

Author(s):

Serena L.Y. Teo ◽

Joshua J. Rennick ◽

Daniel Yuen ◽

Hareth Al-Wassiti ◽

Angus P.R. Johnston ◽

...

Keyword(s):

Endosomal Escape ◽

Current Understanding ◽

Specific Cell ◽

Essential Requirement ◽

Intracellular Drug Delivery ◽

Cytosolic Delivery ◽

Efficient Delivery ◽

Highly Sensitive ◽

Therapeutic Peptides ◽

Low Sensitivity

AbstractEndosomal escape is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of endosomal escape mechanisms remains limited due to significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we developed a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We applied SLEEQ to evaluate the endosomal escape of a range of widely studied putative endosomal escape peptides (EEPs). We demonstrated that positively-charged EEPs enhanced 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 EEPs 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.

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Efficient Delivery of Macromolecules into Human Cells by Improving the Endosomal Escape Activity of Cell-Penetrating Peptides: Lessons Learned from dfTAT and its Analogs

Biomolecules ◽

10.3390/biom8030050 ◽

2018 ◽

Vol 8 (3) ◽

pp. 50 ◽

Cited By ~ 16

Author(s):

Jason Allen ◽

Dakota Brock ◽

Helena Kondow-McConaghy ◽

Jean-Philippe Pellois

Keyword(s):

Cell Penetrating Peptides ◽

Human Cells ◽

Lessons Learned ◽

Endosomal Escape ◽

Efficient Delivery ◽

Cell Penetrating

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

ACS Chemical Biology ◽

10.1021/acschembio.9b00585 ◽

2019 ◽

Vol 14 (12) ◽

pp. 2641-2651 ◽

Cited By ~ 10

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

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Arf6-mediated macropinocytosis enhanced suicide gene therapy of C16TAB-condensed Tat/pDNA nanoparticles in ovarian cancer

Nanoscale ◽

10.1039/d1nr03974a ◽

2021 ◽

Author(s):

Zhe Sun ◽

Jinhai Huang ◽

Linjia Su ◽

Jing Li ◽

Fangzheng Qi ◽

...

Keyword(s):

Ovarian Cancer ◽

Gene Therapy ◽

Cancer Treatment ◽

Plasmid Dna ◽

Suicide Gene ◽

Cell Penetrating Peptides ◽

Therapeutic Gene ◽

Hiv Tat ◽

Efficient Delivery ◽

Cell Penetrating

Using cell-penetrating peptides (CPPs), typically HIV-Tat, to deliver the therapeutic gene for cancer treatment has being hampered by low efficient delivery and complicated uptake route of plasmid DNA (pDNA). On...

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

BioMolecular Concepts ◽

10.1515/bmc-2017-0023 ◽

2017 ◽

Vol 8 (3-4) ◽

pp. 131-141 ◽

Cited By ~ 34

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.

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Leaky Fusion Between Intraluminal Vesicles Of Late Endosomes That Have An Unusual Lipid Composition As A Mechanism Of Endosomal Escape By Cell-penetrating Peptides

Biophysical Journal ◽

10.1016/j.bpj.2008.12.1815 ◽

2009 ◽

Vol 96 (3) ◽

pp. 359a

Author(s):

Sung-Tae Yang

Keyword(s):

Lipid Composition ◽

Cell Penetrating Peptides ◽

Endosomal Escape ◽

Late Endosomes ◽

Cell Penetrating

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

Biomolecules ◽

10.3390/biom8030051 ◽

2018 ◽

Vol 8 (3) ◽

pp. 51 ◽

Cited By ~ 5

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.

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