Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides

Abstract

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.

Intracellular Delivery of Active Proteins by Polyphosphazene Polymers

“…Thus, cytosolic delivery of the Bax-BH3 peptide should enable this function. However, this 20-amino acid peptide is impermeable to cells, for which its effective delivery requires direct injection into this compartment [ 43 , 44 , 45 ]. Hence, this example can also serve to verify whether active cargo can be delivered to the cytosol by PZ polymers.…”

“…Following entry into cells by endocytosis, protein delivery systems are often retained in the endo-lysosomal compartment and cannot escape into the cytosol to traffic to or reach other intracellular targets [ 2 , 5 , 8 , 24 , 45 ]. With regard to the subsequent endo-lysosomal trafficking of internalized PZ complexes, we again saw a difference in the kinetics of the process between the two polymers, with the PZ-PYR/FITC-avidin trafficking rapidly to the lysosomal compartment, as evidenced by a low colocalization with endosomes at 1 h or 5 h, while their localization with lysosomes was slightly higher compared to endosomes and sustained over this time ( Figure 5 A,B).…”

Chemical Manipulation of the Endosome Trafficking Machinery: Implications for Oligonucleotide Delivery

“…Thus, the OEC seems to act at a site in the trafficking pathway that is after early endosomes, but before lysosomes, probably within the key MVB/LE compartments that are the natural sites of productive oligonucleotide release to the cytosol [ 4 , 33 ]. Additional reinforcement regarding the site of action of OEC UNC7938 comes from a recent study that demonstrated that this compound binds preferentially to lysobisphosphatidic acid, a lipid found predominately in MVBs [ 71 ]. Thus, the UNC7938 and UNC2383 OECs have many interesting and valuable characteristics as modulators of oligonucleotide release from endosomes and are currently being explored for possible therapeutic use.…”

Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules

“…The conjugates were tested in a luciferase reporter gene assay with the well-known splice switching ON sequence SSO 623 [ 176 ] in HeLa Luc 704 cells. Unfortunately, none of the novel conjugates afforded an improvement in splice-switching activity over the dual use of SSO 623 with UNC7938 as a widely used endosome-destabilizing agent [ 344 , 346 ] or even SSO 623 alone. The reasons for this have not been elucidated yet, but ongoing research attempts to use more potent analogues of Retro-1 [ 344 , 347 ] or variations of spacer units to overcome possible impairments with the splice-switching machinery.…”