Cyf Ramos-Colon Ph.D. Senior Scientist Gyros Protein Technologies

Macromolecules such as peptides, proteins and siRNAs show great promise as therapeutic agents in precision medicine, but their large size complicates delivery into the cytoplasm or nucleus. Adding peptide/protein transduction domains (PTDs) or cell-penetrating peptides (CPPs) can promote cellular uptake of macromolecules through endocytosis, but escape from endosomes into the cytoplasm remains a challenge. Using an elegantly simple complementation assay, Peter Lönn and his colleagues at UCSD School of Medicine, La Jolla, USA have investigated how covalent attachment of the hydrophobic motifs of designer peptides—endosomal escape domains (EEDs)—to a PTD/CPP can overcome the challenge of ensuring efficient release of a macromolecular drug from the endosome into the cytoplasm with minimum cytotoxicity. 

Cell-Penetrating Peptides Help Deliver the Cargo

Successfully delivering a macromolecule to a target in the cytoplasm involves cell association, stimulation of endocytosis, and escape from the endosome. Stimulating a cell to take up macromolecules of 1 kDa or more can be difficult, but CPPs are one solution. These peptides stimulate, for example, endocytosis-mediated entry and can be used to transfer a so-called “cargo”, such as a macromolecular therapeutic drug, into the cell. CPPs can have sequences that are highly positively charged, alternating polar/nonpolar or apolar/hydrophobic. The first CPP discovered was the trans-activating transcriptional activator (TAT) from human immunodeficiency virus 1 (HIV-1) that can be efficiently taken up by numerous cell types in culture.

Peptide Sequences Stimulate Endosomal Cargo Release

Peter Lönn and his colleagues employed the TAT CPP and synthesized a macromolecule—a TAT peptide hybrid. While the peptide hybrid did undergo endocytosis, the bottleneck of release from the endosome remained. They therefore tested if adding additional hydrophobic sequences, or EEDs, would stimulate the release of the cargo from the endosome into the cytoplasm.

To do this, the research team developed a detection method to screen EED sequences that was both elegant and simple. The assay built on the principle that green fluorescent protein (GFP) lacking the sixteen-residue β-strand #11 (GFPβ11) does not fluoresce but can be induced to fluoresce if co-incubated with the missing GFPβ11 peptide. The idea was that test cells would contain the non-fluorescent GFPβ1–10, and the cargo to be delivered to the cytoplasm would be the GFPβ11 required to complement with GFPβ1–10 to enable fluorescence. The result was a real-time flow-cytometry (FACS) assay with zero false positives and direct 1:1 quantification. They also monitored cytotoxicity by measuring any negative effects of the peptides on cell viability and morphology.

Peptide design for uptake and escape

The overall design of the peptide was:


A disulfide bond was included to enable the cell to easily free the cargo from the TAT–EED delivery domain, thus avoiding steric hindrance during the complementation process. The researchers also included a PEG linker designed to reduce cytotoxicity by distancing the EED from the charged TAT delivery domain. The peptides were synthesized by fmoc solid-phase peptide synthesis using a Symphony Quartet peptide synthesizer, purified using prep-scale RP-HPLC, and analyzed by mass spectrometry using α-CHCA matrix.

Optimizing Escape From the Endosome

Initial tests using constructs with various linker lengths showed that a PEG6 linker gave the best balance between high delivery and low cytotoxicity. Following up on this, the researchers tested a range of EED sequences with the PEG6 linker arm and found a number of promising candidates that enabled release of the GFPβ11 cargo into the cytoplasm without leading to cytotoxicity, such as peptide 6 shown in the figure.

While further biophysical work is needed to understand the mechanisms better, the TAT-EED peptide hybrid concept promises to improve endosomal escape and thereby increase the likelihood of success of macromolecular therapeutics for precision medicine.

Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics. Lönn P et al. Sci Rep. 2016 Sep 8;6:32301. doi: 10.1038/srep32301.

Cyf Ramos-Colon, Ph.D. ([email protected]), is a senior scientist at Gyros Protein Technologies.

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