Mulberry Leaf Lipid Nanoparticles: a Naturally Targeted CRISPR/Cas9 Oral Delivery Platform for Alleviation of Colon Diseases

Abstract Oral treatment of colon diseases with the CRISPR/Cas9 system has been hampered by the lack of a safe and efficient delivery platform. Overexpressed CD98 plays a crucial role in the progression of ulcerative colitis (UC) and colitis‐associated colorectal cancer (CAC). In this study, lipid nanoparticles (LNPs) derived from mulberry leaves are functionalized with Pluronic copolymers and optimized to deliver the CRISPR/Cas gene editing machinery for CD98 knockdown. The obtained LNPs possessed a hydrodynamic diameter of 267.2 nm, a narrow size distribution, and a negative surface charge (−25.6 mV). Incorporating Pluronic F127 into LNPs improved their stability in the gastrointestinal tract and facilitated their penetration through the colonic mucus barrier. The galactose end groups promoted endocytosis of the LNPs by macrophages via asialoglycoprotein receptor‐mediated endocytosis, with a transfection efficiency of 2.2‐fold higher than Lipofectamine 6000. The LNPs significantly decreased CD98 expression, down‐regulated pro‐inflammatory cytokines (TNF‐ α and IL‐6), up‐regulated anti‐inflammatory factors (IL‐10), and polarized macrophages to M2 phenotype. Oral administration of LNPs mitigated UC and CAC by alleviating inflammation, restoring the colonic barrier, and modulating intestinal microbiota. As the first oral CRISPR/Cas9 delivery LNP, this system offers a precise and efficient platform for the oral treatment of colon diseases.