Endosome-disruptive peptides for improving cytosolic delivery of bioactive macromolecules

Biopolymers ◽  
2010 ◽  
Vol 94 (6) ◽  
pp. 763-770 ◽  
Author(s):  
Ikuhiko Nakase ◽  
Sachiko Kobayashi ◽  
Shiroh Futaki
Keyword(s):  
Cytosolic Delivery

scholarly journals Cytosolic Delivery of Argininosuccinate Synthetase Using a Cell-Permeant Miniature Protein

2021 ◽  
Vol 7 (4) ◽  
pp. 641-649
Author(s):  
Susan L. Knox ◽  
Rebecca Wissner ◽  
Samantha Piszkiewicz ◽  
Alanna Schepartz
Keyword(s):  
Argininosuccinate Synthetase ◽  
Cytosolic Delivery ◽  
A Cell

scholarly journals Efficacy and Selectivity of FGF2-Saporin Cytosolically Delivered by PCI in Cells Overexpressing FGFR1

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1476
Author(s):  
Aurora K. Vikan ◽  
Michal Kostas ◽  
Ellen Margrethe Haugsten ◽  
Pål K. Selbo ◽  
Jørgen Wesche
Keyword(s):  
Treatment Options ◽  
Current Treatment ◽  
Fibroblast Growth Factor Receptors ◽  
Ribosome Inactivating Protein ◽  
Cytosolic Delivery ◽  
Low Levels ◽  
U2os Cells ◽  
Targeting Effect ◽  
Target Effects ◽  
In Cells

Fibroblast growth factor receptors (FGFRs) have become an attractive target in cancer research and therapy due to their implication in several cancers. Limitations of current treatment options require a need for additional, more specific and potent strategies to overcome cancers driven by FGFRs. Photochemical internalization (PCI) is a light-controlled method for cytosolic delivery of drugs that are entrapped in endosomes and lysosomes. We here evaluated the efficacy and selectivity of PCI of FGF2-saporin (FGF-SAP) in cells overexpressing FGFR1. FGF-SAP is a conjugate of FGF2 and the highly cytotoxic ribosome-inactivating protein (RIP) saporin, which is used as payload to eliminate cancer cells. Evaluation of the targeting effect of PCI of FGF-SAP was done by comparing the cytotoxic response in osteosarcoma cells with very low levels of FGFR1 (U2OS) to cells overexpressing FGFR1 (U2OS-R1). We demonstrate that PCI greatly enhances cytotoxicity of the drug showing efficient cell killing at pM concentrations of the drug in U2OS-R1 cells. However, U2OS cells were also sensitive to the toxin after PCI. Binding experiments using confocal microscopy and Western blotting techniques indicate that FGF-SAP is taken up by cells through heparan sulfate proteoglycans (HSPGs) in U2OS cells. We further show that the cytotoxicity of FGF-SAP in U2OS cells was reduced when cells were co-treated with heparin to compete out binding to HSPG, demonstrating that the cytotoxic effect was due to internalization by HSPGs. We conclude that to prevent off-target effects of FGF-based toxins, it will be necessary to circumvent binding to HSPGs, for example by mutating the binding site of FGF2 to HSPGs.

scholarly journals Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins

2018 ◽  
Vol 4 (10) ◽  
pp. 1379-1393 ◽  
Author(s):  
Rebecca F. Wissner ◽  
Angela Steinauer ◽  
Susan L. Knox ◽  
Alexander D. Thompson ◽  
Alanna Schepartz
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Miniature Proteins ◽  
Cytosolic Delivery

scholarly journals RetargetingClostridium difficileToxin B to Neuronal Cells as a Potential Vehicle for Cytosolic Delivery of Therapeutic Biomolecules to Treat Botulism

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Greice Krautz-Peterson ◽  
Yongrong Zhang ◽  
Kevin Chen ◽  
George A. Oyler ◽  
Hanping Feng ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Neuronal Cells ◽  
Vero Cells ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Delivery Vehicle ◽  
Toxin B ◽  
Cytosolic Delivery ◽  
Botulinum Neurotoxins ◽  
Neuronal Delivery

Botulinum neurotoxins (BoNTs) deliver a protease to neurons which can cause a flaccid paralysis called botulism. Development of botulism antidotes will require neuronal delivery of agents that inhibit or destroy the BoNT protease. Here, we investigated the potential of engineeringClostridium difficiletoxin B (TcdB) as a neuronal delivery vehicle by testing two recombinant TcdB chimeras. For AGT-TcdB chimera, an alkyltransferase (AGT) was appended to the N-terminal glucosyltransferase (GT) of TcdB. Recombinant AGT-TcdB had alkyltransferase activity, and the chimera was nearly as toxic to Vero cells as wild-type TcdB, suggesting efficient cytosolic delivery of the AGT/GT fusion. For AGT-TcdB-BoNT/A-Hc, the receptor-binding domain (RBD) of TcdB was replaced by the equivalent RBD from BoNT/A (BoNT/A-Hc). AGT-TcdB-BoNT/A-Hc was >25-fold more toxic to neuronal cells and >25-fold less toxic to Vero cells than AGT-TcdB. Thus, TcdB can be engineered for cytosolic delivery of biomolecules and improved targeting of neuronal cells.

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.

scholarly journals Diselenolane‐Mediated Cellular Uptake: Efficient Cytosolic Delivery of Probes, Peptides, Proteins, Artificial Metalloenzymes and Protein‐Coated Quantum Dots

2019 ◽  
Vol 25 (16) ◽  
pp. 4047-4051 ◽  
Author(s):  
Eline Bartolami ◽  
Dimitris Basagiannis ◽  
Lili Zong ◽  
Rémi Martinent ◽  
Yasunori Okamoto ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Cellular Uptake ◽  
Cytosolic Delivery ◽  
Artificial Metalloenzymes

scholarly journals A boronic acid–rich dendrimer with robust and unprecedented efficiency for cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (6) ◽  
pp. eaaw8922 ◽  
Author(s):  
Chongyi Liu ◽  
Tao Wan ◽  
Hui Wang ◽  
Song Zhang ◽  
Yuan Ping ◽  
...  
Keyword(s):  
Boronic Acid ◽  
Protein Delivery ◽  
High Efficiency ◽  
Intracellular Delivery ◽  
Cytosolic Protein ◽  
Native Proteins ◽  
Cytosolic Delivery ◽  
Cas9 Protein ◽  
Cargo Proteins ◽  
Positively Charged

Cytosolic protein delivery is of central importance for the development of protein-based biotechnologies and therapeutics; however, efficient intracellular delivery of native proteins remains a challenge. Here, we reported a boronic acid–rich dendrimer with unprecedented efficiency for cytosolic delivery of native proteins. The dendrimer could bind with both negatively and positively charged proteins and efficiently delivered 13 cargo proteins into the cytosol of living cells. All the delivered proteins kept their bioactivities after cytosolic delivery. The dendrimer ensures efficient intracellular delivery of Cas9 protein into various cell lines and showed high efficiency in CRISPR-Cas9 genome editing. The rationally designed boronic acid–rich dendrimer permits the development of an efficient platform with high generality for the delivery of native proteins.

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