Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics

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.

Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

“…28,58–60 Reinforcement of the peptide secondary structure or addition of cationic membrane-penetrating amino acids could theoretically facilitate cytoplasmic trafficking. 15,59–61 Future studies aim to measure p53 (14–29) movement within treated cells and enhance endosomal escape and organelle trafficking.…”

Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking

“…However, when GFP11 is in the same cellular compartment as GFP1–10, GFP is reconstituted, generating a fluorescent signal. This approach was used to demonstrate cytoplasmic delivery of soluble fusion proteins linked to CPPs (Lonn et al, 2016; Milech et al, 2015). …”

Guiding Drugs to Target‐Harboring Organelles: Stretching Drug‐Delivery to a Higher Level of Resolution

“…Endosomes are pleomorphic membrane compartments that transport material from the cell surface to other organelles. [84][85][86] One strategy involves the escape of the drug from the endosome to the cytosol to prevent it from reaching the lysosome where it would be degraded. [84] Targeting of the endosome/lysosome system involves two main strategies.…”