Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages

Macrophages in atherosclerotic lesions promote plaque progression and are an attractive therapeutic target in cardiovascular research. Here we present a protocol for synthesis of small interfering RNA (siRNA) nanoparticles (NP) that target lesional macrophages as a potential treatment for atherosclerosis. Ca2+/calmodulin-dependent protein kinase gamma (CaMKII gamma) activity in macrophages of advanced human and mouse atherosclerotic plaques drives necrosis by downregulating the expression of the efferocytosis receptor MerTK. Therefore, selective inhibition of CaMKII gamma in lesional macrophages holds great promise for the treatment of advanced atherosclerosis. We recently developed a siRNA NP platform that can selectively silence CaMKII gamma in macrophages, resulting in increased plaque stability. We provide a detailed protocol for the synthesis of NP components, the preparation and characterization (physicochemical and in vitro) of siRNA NPs, and the evaluation of in vivo therapeutic effects of siRNA NPs and their biocompatibility in atherosclerotic mice. Our siRNA-loaded polymer-lipid hybrid NPs are constructed via a robust self-assembly method, exhibiting excellent in vivo features for systemic siRNA delivery. Following this protocol, it takes 3-5 d to prepare the siRNA NPs, 8-10 d to characterize the NPs and 4-5 weeks to evaluate their therapeutic effects in established atherosclerotic mice. By changing the RNA molecules loaded in the NPs, lesional macrophages can be targeted for the exploration and validation of new targets/pathways in atherosclerosis. Tao et al. present the synthesis and characterization of siRNA-loaded polymer-lipid hybrid nanoparticles for targeted delivery to macrophages. The nanoparticles are used to silence CaMKII gamma activity in a mouse model of atherosclerosis.

Automated summary

Macrophages play a role in promoting plaque progression in atherosclerosis and are an attractive therapeutic target. Researchers have developed siRNA nanoparticles that can silence CaMKII γ activity in macrophages for the treatment of atherosclerosis.