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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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Related in: MedlinePlus

Evaluation of GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide in multiple cell types.Dose-dependent analysis of GFPβ1-10 expressing HaCaT keratinocytes, MDA-MB-231 and MCF7 breast carcinoma cells treated with GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide and parental GFPβ11-(S-S)-TAT peptide by FACS for GFP complementation fluorescence (a–f), cellular morphology (g–i) and cell viability (j–l), respectively. Graphs display mean values of triplicate samples with S.D.
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f5: Evaluation of GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide in multiple cell types.Dose-dependent analysis of GFPβ1-10 expressing HaCaT keratinocytes, MDA-MB-231 and MCF7 breast carcinoma cells treated with GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide and parental GFPβ11-(S-S)-TAT peptide by FACS for GFP complementation fluorescence (a–f), cellular morphology (g–i) and cell viability (j–l), respectively. Graphs display mean values of triplicate samples with S.D.

Mentions: Increasing the spacing between the two Trp residues by insertion of two Gly residues, -GWGGWG in GFPβ11-(S-S)-TAT-P6-GWGGWG, decreased the enhancement significantly compared to the -GWWG motif, suggesting that a concentrated hydrophobic patch is required for the enhanced endosomal escape. We also noted that addition of a control C-terminal PEG6-GG tail, GFPβ11-(S-S)-TAT-P6-GG, resulted in a lower uptake compared to parental GFPβ11-(S-S)-TAT peptide, suggesting that a free PEG polymer tail alone reduced uptake. Surprisingly, while both the -GWWG and -GFWFG domains enhanced cytoplasmic escape compared to parental GFPβ11-(S-S)-TAT peptide, inclusion of four consecutive aromatic ring Phe residues, -GFFFFG, resulted in adverse cytotoxic effects on cells, causing gross morphological changes and cell death (Fig. S6), suggesting that too long of a hydrophobic patch results in cell membrane destabilization leading to cytotoxicity. TAT peptides are taken up into cells by stimulating macropinocytosis1112. Using 70 kDa neutral dextran-Texas Red as a marker of macropinocytosis11, we determined that the control TAT, TAT-P6-GFWFG and TAT-P6-GWWG peptides all stimulate macropinocytosis to a similar extent (Fig. S7), arguing that the increased GFP fluorescence by GFPβ11-(S-S)-TAT-P6-GFWFG and GFPβ11-(S-S)-TAT-P6-GWWG peptides is indeed due to enhanced endosomal escape and not merely an increased stimulation of macropinocytosis. Lastly, we confirmed that the optimized GFPβ11-(S-S)-TAT-P6-GFWFG peptide significantly enhanced endosomal escape in three additional human cell lines in a non-cytotoxic fashion compared to the parental GFPβ11-(S-S)-TAT peptide (Fig. 5), suggesting that EEDs universally enhance endosomal escape.


Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics
Evaluation of GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide in multiple cell types.Dose-dependent analysis of GFPβ1-10 expressing HaCaT keratinocytes, MDA-MB-231 and MCF7 breast carcinoma cells treated with GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide and parental GFPβ11-(S-S)-TAT peptide by FACS for GFP complementation fluorescence (a–f), cellular morphology (g–i) and cell viability (j–l), respectively. Graphs display mean values of triplicate samples with S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Evaluation of GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide in multiple cell types.Dose-dependent analysis of GFPβ1-10 expressing HaCaT keratinocytes, MDA-MB-231 and MCF7 breast carcinoma cells treated with GFPβ11-(S-S)-TAT-PEG6-GFWFG peptide and parental GFPβ11-(S-S)-TAT peptide by FACS for GFP complementation fluorescence (a–f), cellular morphology (g–i) and cell viability (j–l), respectively. Graphs display mean values of triplicate samples with S.D.
Mentions: Increasing the spacing between the two Trp residues by insertion of two Gly residues, -GWGGWG in GFPβ11-(S-S)-TAT-P6-GWGGWG, decreased the enhancement significantly compared to the -GWWG motif, suggesting that a concentrated hydrophobic patch is required for the enhanced endosomal escape. We also noted that addition of a control C-terminal PEG6-GG tail, GFPβ11-(S-S)-TAT-P6-GG, resulted in a lower uptake compared to parental GFPβ11-(S-S)-TAT peptide, suggesting that a free PEG polymer tail alone reduced uptake. Surprisingly, while both the -GWWG and -GFWFG domains enhanced cytoplasmic escape compared to parental GFPβ11-(S-S)-TAT peptide, inclusion of four consecutive aromatic ring Phe residues, -GFFFFG, resulted in adverse cytotoxic effects on cells, causing gross morphological changes and cell death (Fig. S6), suggesting that too long of a hydrophobic patch results in cell membrane destabilization leading to cytotoxicity. TAT peptides are taken up into cells by stimulating macropinocytosis1112. Using 70 kDa neutral dextran-Texas Red as a marker of macropinocytosis11, we determined that the control TAT, TAT-P6-GFWFG and TAT-P6-GWWG peptides all stimulate macropinocytosis to a similar extent (Fig. S7), arguing that the increased GFP fluorescence by GFPβ11-(S-S)-TAT-P6-GFWFG and GFPβ11-(S-S)-TAT-P6-GWWG peptides is indeed due to enhanced endosomal escape and not merely an increased stimulation of macropinocytosis. Lastly, we confirmed that the optimized GFPβ11-(S-S)-TAT-P6-GFWFG peptide significantly enhanced endosomal escape in three additional human cell lines in a non-cytotoxic fashion compared to the parental GFPβ11-(S-S)-TAT peptide (Fig. 5), suggesting that EEDs universally enhance endosomal escape.

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