Limits...
Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics

View Article: PubMed Central - PubMed

ABSTRACT

Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.

No MeSH data available.


The study concept.PTD/CPP binds to negatively charged molecules on the cell surface and stimulates macropinocytotic uptake and endosomal escape of GFPβ11-PTD/CPP peptide into the cytoplasm. When concentrated with the PTD/CPP in the endosomes, the hydrophobic EED motif buries itself into the lipid bilayer membrane which leads to a localized membrane destabilization that enhances endosomal escape into the cytoplasm. Binding of GFPβ11 peptide to non-fluorescent GFPβ1-10 protein fragment in the cytoplasm induces chemical formation of the GFP fluorescent chromophore.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5015074&req=5

f1: The study concept.PTD/CPP binds to negatively charged molecules on the cell surface and stimulates macropinocytotic uptake and endosomal escape of GFPβ11-PTD/CPP peptide into the cytoplasm. When concentrated with the PTD/CPP in the endosomes, the hydrophobic EED motif buries itself into the lipid bilayer membrane which leads to a localized membrane destabilization that enhances endosomal escape into the cytoplasm. Binding of GFPβ11 peptide to non-fluorescent GFPβ1-10 protein fragment in the cytoplasm induces chemical formation of the GFP fluorescent chromophore.

Mentions: Using the split-GFP complementation assay with TAT-PTD/CPP,, we investigated an array of endosomal escape domains (EEDs) with various hydrophobic features (Fig. 1a). Hydrophobic residues have previously been described to play important roles in endosomal escape of viruses and are also known to modulate PTD/CPP uptake1122232425. Furthermore, we also investigated the effect of the position of EEDs in relation to the TAT PTD/CPP. We found that the addition of EEDs with specific hydrophobic patches, containing either two aromatic indole rings or one indole ring and two aromatic phenyl groups, at a fixed distance of six polyethylene glycol (PEG) units from the PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs are an exciting addition that opens up new potential for intracellular delivery of new macromolecular biologic therapeutics.


Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics
The study concept.PTD/CPP binds to negatively charged molecules on the cell surface and stimulates macropinocytotic uptake and endosomal escape of GFPβ11-PTD/CPP peptide into the cytoplasm. When concentrated with the PTD/CPP in the endosomes, the hydrophobic EED motif buries itself into the lipid bilayer membrane which leads to a localized membrane destabilization that enhances endosomal escape into the cytoplasm. Binding of GFPβ11 peptide to non-fluorescent GFPβ1-10 protein fragment in the cytoplasm induces chemical formation of the GFP fluorescent chromophore.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015074&req=5

f1: The study concept.PTD/CPP binds to negatively charged molecules on the cell surface and stimulates macropinocytotic uptake and endosomal escape of GFPβ11-PTD/CPP peptide into the cytoplasm. When concentrated with the PTD/CPP in the endosomes, the hydrophobic EED motif buries itself into the lipid bilayer membrane which leads to a localized membrane destabilization that enhances endosomal escape into the cytoplasm. Binding of GFPβ11 peptide to non-fluorescent GFPβ1-10 protein fragment in the cytoplasm induces chemical formation of the GFP fluorescent chromophore.
Mentions: Using the split-GFP complementation assay with TAT-PTD/CPP,, we investigated an array of endosomal escape domains (EEDs) with various hydrophobic features (Fig. 1a). Hydrophobic residues have previously been described to play important roles in endosomal escape of viruses and are also known to modulate PTD/CPP uptake1122232425. Furthermore, we also investigated the effect of the position of EEDs in relation to the TAT PTD/CPP. We found that the addition of EEDs with specific hydrophobic patches, containing either two aromatic indole rings or one indole ring and two aromatic phenyl groups, at a fixed distance of six polyethylene glycol (PEG) units from the PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs are an exciting addition that opens up new potential for intracellular delivery of new macromolecular biologic therapeutics.

View Article: PubMed Central - PubMed

ABSTRACT

Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.

No MeSH data available.