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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.

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Optimizing design of endosomal escape domain (EED).(a) Structures of EEDs. (b–e) Dose-dependent comparison of GFPβ1-10 H1299-c#G3 cells treated with GFPβ11-(S-S)-TAT-(X) peptides containing a PEG6-spaced aromatic ring hydrophobic endosomal escape domain (EED), as indicated, to parental GFPβ11-(S-S)-TAT peptide and control GFPβ11-(S-S)-TAT-PEG6-GG peptide analyzed by FACS for GFP fluorescence (b,c), cell viability (d), and cellular morphology (e). The table (b) displays mean values from triplicate samples and the graphs (c–e) show the same mean values with S.D.
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f4: Optimizing design of endosomal escape domain (EED).(a) Structures of EEDs. (b–e) Dose-dependent comparison of GFPβ1-10 H1299-c#G3 cells treated with GFPβ11-(S-S)-TAT-(X) peptides containing a PEG6-spaced aromatic ring hydrophobic endosomal escape domain (EED), as indicated, to parental GFPβ11-(S-S)-TAT peptide and control GFPβ11-(S-S)-TAT-PEG6-GG peptide analyzed by FACS for GFP fluorescence (b,c), cell viability (d), and cellular morphology (e). The table (b) displays mean values from triplicate samples and the graphs (c–e) show the same mean values with S.D.

Mentions: Both Trp (W) and Phe (F) residues are both known to destabilize cellular membranes by burying their hydrophobic R groups into the lipid bilayer2728. To optimize the EED, we systematically synthesized C-terminal hydrophobic EEDs with various combinations of Trp and Phe residues that included the optimal six PEG unit (P6) spacer. After initial analyses, we focused our efforts on seven different hydrophobic EED motifs: -GFFG, -GWG, -GFWG, -GFWFG, -GWWG, -GWGGWG, and -GWWWG and a control –GG motif (Fig. 4a). Addition of aromatic rings from either two Phe residues, GFPβ11-(S-S)-TAT-P6-GFFG, or one Trp residue, GFPβ11-(S- S)-TAT-P6-GWG, to the C-terminus, had no net effect on delivery compared to the parental GFPβ11-(S-S)-TAT peptide (Fig. 4b–e). However, addition of aromatic rings from both a Phe and Trp, GFPβ11-(S-S)-TAT-P6-GFWG, showed a two-fold increase in GFP fluorescence compared to the parental GFPβ11-(S-S)-TAT peptide with no signs of cytotoxicity. Moreover, increasing hydrophobicity by inclusion of either Phe-Trp-Phe residues, GFPβ11-(S-S)-TAT-P6-GFWFG, or two Trp residues, GFPβ11-(S-S)-TAT-P6-GWWG, to the C-terminus, resulted in a five-fold increase in GFP fluorescence in the absence of cytotoxicity (Fig. 4b–e). However, addition of six aromatic rings by inclusion of three Trp residues, GFPβ11-(S-S)-TAT-P6-GWWWG, resulted in a dramatic increase in cytotoxicity that hampered uptake (Fig. 4b–e).


Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics
Optimizing design of endosomal escape domain (EED).(a) Structures of EEDs. (b–e) Dose-dependent comparison of GFPβ1-10 H1299-c#G3 cells treated with GFPβ11-(S-S)-TAT-(X) peptides containing a PEG6-spaced aromatic ring hydrophobic endosomal escape domain (EED), as indicated, to parental GFPβ11-(S-S)-TAT peptide and control GFPβ11-(S-S)-TAT-PEG6-GG peptide analyzed by FACS for GFP fluorescence (b,c), cell viability (d), and cellular morphology (e). The table (b) displays mean values from triplicate samples and the graphs (c–e) show the same mean values with S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Optimizing design of endosomal escape domain (EED).(a) Structures of EEDs. (b–e) Dose-dependent comparison of GFPβ1-10 H1299-c#G3 cells treated with GFPβ11-(S-S)-TAT-(X) peptides containing a PEG6-spaced aromatic ring hydrophobic endosomal escape domain (EED), as indicated, to parental GFPβ11-(S-S)-TAT peptide and control GFPβ11-(S-S)-TAT-PEG6-GG peptide analyzed by FACS for GFP fluorescence (b,c), cell viability (d), and cellular morphology (e). The table (b) displays mean values from triplicate samples and the graphs (c–e) show the same mean values with S.D.
Mentions: Both Trp (W) and Phe (F) residues are both known to destabilize cellular membranes by burying their hydrophobic R groups into the lipid bilayer2728. To optimize the EED, we systematically synthesized C-terminal hydrophobic EEDs with various combinations of Trp and Phe residues that included the optimal six PEG unit (P6) spacer. After initial analyses, we focused our efforts on seven different hydrophobic EED motifs: -GFFG, -GWG, -GFWG, -GFWFG, -GWWG, -GWGGWG, and -GWWWG and a control –GG motif (Fig. 4a). Addition of aromatic rings from either two Phe residues, GFPβ11-(S-S)-TAT-P6-GFFG, or one Trp residue, GFPβ11-(S- S)-TAT-P6-GWG, to the C-terminus, had no net effect on delivery compared to the parental GFPβ11-(S-S)-TAT peptide (Fig. 4b–e). However, addition of aromatic rings from both a Phe and Trp, GFPβ11-(S-S)-TAT-P6-GFWG, showed a two-fold increase in GFP fluorescence compared to the parental GFPβ11-(S-S)-TAT peptide with no signs of cytotoxicity. Moreover, increasing hydrophobicity by inclusion of either Phe-Trp-Phe residues, GFPβ11-(S-S)-TAT-P6-GFWFG, or two Trp residues, GFPβ11-(S-S)-TAT-P6-GWWG, to the C-terminus, resulted in a five-fold increase in GFP fluorescence in the absence of cytotoxicity (Fig. 4b–e). However, addition of six aromatic rings by inclusion of three Trp residues, GFPβ11-(S-S)-TAT-P6-GWWWG, resulted in a dramatic increase in cytotoxicity that hampered uptake (Fig. 4b–e).

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.


Related in: MedlinePlus