Amphiphilic Polyaspartamide Derivatives with Cholesterol Introduction Enhanced Ex Vivo mRNA Transfection Efficiency to Natural Killer Cells

Abstract

Engineered natural killer (NK) cells eliminate cancer cells by overexpressing a chimeric antigen receptor, producing highly efficient and safe NK cell therapies. This study investigated the polyplex formulation for the fusion protein GreenLantern-natural killer group 2D (NKG2D) mRNA to evaluate its ex vivo delivery efficacy into NK cells, wherein NKG2D on the surface of NK cells recognized its counterpart NKG2D ligands on cancer cells. Amphiphilic polyaspartamide derivatives Chol-PAsp(DET/CHE) were prepared by adding cyclohexylethylamine (CHE) and diethylenetriamine (DET) in the side chains and cholesterol (Chol) at the alpha-terminus to enhance endosomal escapability and optimize hydrophobicity. Chol-PAsp(DET/CHE) significantly improved mRNA delivery efficacy into NK-92mi cells, explained by increased polyplex stability and improved cellular uptake of mRNA. The NKG2D-overexpressing NK-92mi cells exhibited high anticancer efficacy against human colon cancer cells without affecting the viability of fibroblasts. Therefore, Chol-PAsp(DET/CHE) could be a promising mRNA delivery carrier for the ex vivo engineering of NK cells.