Direct Cytosolic Delivery of Proteins and CRISPR-Cas9 Genome Editing by Gemini Amphiphiles via Non-Endocytic Translocation Pathways

A robust gemini amphiphile-based carrierplatform for facilitatingdirect cytosolic delivery of broad-spectrum proteins, including Cas9ribonucleoproteins, through the lipid raft-dependent membrane fusionmechanism. Intracellular delivery of therapeutic biomacromoleculesis oftenchallenged by the poor transmembrane and limited endosomal escape.Here, we establish a combinatorial library composed of 150 molecularweight-defined gemini amphiphiles (GAs) to identify the vehicles thatfacilitate robust cytosolic delivery of proteins in vitro and in vivo.These GAs display similar skeletal structures but differential amphiphilicityby adjusting the length of alkyl tails, type of ionizable cationicheads, and hydrophobicity or hydrophilicity of a spacer. The top candidateis highly efficient in translocating a broad spectrum of proteinswith various molecular weights and isoelectric points into the cytosol.Particularly, we notice that the entry mechanism is predominantlymediated via the lipid raft-dependent membrane fusion, bypassing theclassical endocytic pathway that limits the cytosolic delivery efficiencyof many presently available carriers. Remarkably, the top GA candidateis capable of delivering hard-to-deliver Cas9 ribonucleoprotein invivo, disrupting KRAS mutation in the tumor-bearingmice to inhibit tumor growth and extend their survival. Our studyreveals a GA-based small-molecule carrier platform for the directcytosolic delivery of various types of proteins for therapeutic purposes.

Automated summary

This study establishes a combinatorial library composed of 150 molecular weight-defined gemini amphiphiles (GAs) to identify carriers that facilitate robust cytosolic delivery of proteins. The top candidate efficiently translocates a broad spectrum of proteins into the cytosol, primarily through lipid raft-dependent membrane fusion, bypassing the classical endocytic pathway. This study reveals a GA-based small-molecule carrier platform for the direct cytosolic delivery of various types of proteins for therapeutic purposes.