Fusogenic intracellular delivery of CRISPR ribonucleoproteins using lipid-silica hybrid nanoparticles

Abstract

CRISPR/Cas9 and other engineered CRISPR systems hold great promise for therapeutic applications, offering the potential to correct disease-causing mutations. However, their translation into effective treatments for human patients is hindered by the lack of efficient delivery systems. Here, we present a cell membrane fusion-inspired delivery vehicle for CRISPR ribonucleoproteins (RNPs). Mesoporous silica nanoparticles are eligible to securely load the RNPs while fusogenic lipid layer is readily coated on the nanoparticles. Conventional nanoparticles are typically transported into the cells via endocytosis which poses the risk of endosomal degradation before reaching the nucleus. In contrast, our nanoparticles facilitate directed delivery into the cytosol by fusing with the cellular membrane, thereby bypassing the risk of endosomal degradation. To enhance the release of RNP payloads from the mesoporous cavity, we also incorporated a set of ionizable lipids on the nanoparticles. Those lipid exhibits a cationic charge in the acidic tumor microenvironment, facilitating the release of RNPs into the cytosol, while remaining neutral at the physiological pH of the cytosolic side. This strategy enabled efficient cytosolic delivery of CRISPR RNPs. Furthermore, by adjusting the ratio between cationic and ionizable lipids, we could selectively target tumors by presenting a positive charge for membrane fusion specifically in the acidic tumor microenvironment. To demonstrate the therapeutic potential of this delivery system, we delivered CRISPR/Cas9 RNPs, which induce DNA double-strand breaks at specific genomic loci in cancer cells. The fusogenic nanoparticle-based platform offers a universal approach for CRISPR RNP delivery, enabling precision medicine by delivering tailored CRISPR systems to target specific genomic sequences, thereby advancing personalized cancer therapy.