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

Nature protocols (2022-02-06)
Xiangang Huang, Chuang Liu, Na Kong, Yufen Xiao, Arif Yurdagul, Ira Tabas, Wei Tao
ABSTRACT

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 γ (CaMKIIγ) 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 CaMKIIγ in lesional macrophages holds great promise for the treatment of advanced atherosclerosis. We recently developed a siRNA NP platform that can selectively silence CaMKIIγ 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
牛血清白蛋白 来源于牛血清, lyophilized powder, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
山羊血清
Sigma-Aldrich
4',6-二脒基-2-苯基吲哚 二盐酸盐, powder, BioReagent, suitable for cell culture, ≥98% (HPLC and TLC), suitable for fluorescence