Significant research efforts have been dedicated to developing effective intracellular delivery systems for biomacromolecules. Our laboratory has engineered a class of peptides in which glutamic acid (Glu) residues are incorporated into cationic membrane-lytic sequences—such as L17E and HAad [1,2]—to modulate hydrophobic interactions with cellular membranes. This approach allows precise control of membrane-disruptive activity, facilitating intracellular delivery. When cells were exposed to large macromolecules, including immunoglobulin G (IgG, ~150 kDa), in the presence of these peptides, efficient cytosolic dispersion was achieved.
Notably, particle-like liquid droplets spontaneously formed upon mixing Alexa Fluor 488-labeled IgG (Alexa488-IgG) with a conjugate comprising an Fc region-binding peptide and a trimeric L17E [FcB(L17E)₃] [3]. Upon contact with the cellular membrane, these condensates rapidly infused into cells, leading to broad intracellular distribution of Alexa488-IgG. This process occurred within minutes and was accompanied by dynamic reorganization of F-actin and membrane structures.
A comparable infusion behavior was observed using droplets generated from Alexa488-IgG and L17E conjugated to pullulan [4,5]. These findings suggest that the delivery mechanism is generalizable and may involve previously uncharacterized membrane translocation pathways.
Reference
[1] Akishiba et al. Nature Chem. 2017, 9, 751–761.
[2] Sakamoto et al. Angew. Chem. Int. Ed. 2020, 59, 19990-19998.
[3] Iwata et al. Angew. Chem. Int. Ed. 2021, 60, 19804–19812.
[4] Michibata et al. Bioconjug. Chem. 2024, 35, 1888–1899.
[5] Michibata et al. Bioconjug. Chem. in press; DOI: 10.1021/acs.bioconjchem.5c00176.