Considerable research interest has been devoted to the development of effective intracellular delivery systems for biomacromolecules. Our laboratory has developed a class of peptides in which Glu residues are inserted into cationic membrane-lytic peptides, such as L17E and HAad [1,2], to modulate hydrophobic interactions with cell membranes and thereby regulate cell surface lytic activity for intracellular delivery. When cells were exposed to large macromolecules, including IgG (~150 kDa), in the presence of these peptides, efficient cytosolic distribution was observed.
Importantly, particle-like liquid droplets were formed by mixing Alexa Fluor 488-labeled IgG (Alexa488-IgG) with a conjugate of an Fc region-binding peptide and a trimer of L17E [FcB(L17E)₃] [3]. Contact between these droplets and the cell membrane led to immediate infusion and widespread intracellular distribution of Alexa488-IgG. This process was completed within a few minutes and was accompanied by dynamic rearrangement of cytoskeletal F-actin and membrane structures.
A similar mode of IgG infusion was observed with droplets formed from Alexa488-IgG and L17E conjugated to pullulan [4,5], suggesting both the generality of this delivery concept and the involvement of novel, non-canonical pathways in membrane translocation. Our ongoing efforts include mRNA delivery using this microcondensate-based system, as well as the application of engineered IgGs in place of Alexa488-IgG, which will also be discussed.
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.