An Amphiphilic Cell-Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA, and CRISPR Cas9

The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self-assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell-penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self-assembling into micellar-like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion. [+] S. Q. and I. A. B. contributed equally to this work.A cell penetrating macrocycle (CPM) with flexible amine linkages and amphiphilic nature assembles into a micellar-like structure that can passively permeate through the cell membrane. CPM can efficiently transfer fully functioning proteins and genetic materials into the cytosol, independent of size, charge, or complexity. image