Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.
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.
Dept of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, CA, 92093 USA.
Peter
P
Lönn
Dept of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, CA, 92093 USA.
Apollo D
AD
Kacsinta
Dept of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, CA, 92093 USA.
Xian-Shu
XS
Cui
Dept of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, CA, 92093 USA.
Alexander S
AS
Hamil
Dept of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, CA, 92093 USA.
Manuel
M
Kaulich
Dept of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, CA, 92093 USA.
Khirud
K
Gogoi
Dept of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, CA, 92093 USA.
Steven F
SF
Dowdy
D001665:Binding Sites / Q000235:genetics; D001688:Biological Products / Q000008:administration & dosage / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000235:genetics / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D046911:Macromolecular Substances / Q000008:administration & dosage / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods
D001688:Biological Products; D057846:Cell-Penetrating Peptides; D046911:Macromolecular Substances; D010455:Peptides; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins