Delivery of MicroRNAs by Chitosan Nanoparticles to Functionally Alter Macrophage Cholesterol Efflux in Vitro and in Vivo.

The prevention and treatment of cardiovascular diseases (CVD) has largely focused on lowering circulating LDL cholesterol, yet a significant burden of atherosclerotic disease remains even when LDL is low. Recently, microRNAs (miRNAs) have emerged as exciting therapeutic targets for cardiovascular disease. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs. A number of miRNAs have been found to modulate all stages of atherosclerosis, particularly those that promote the efflux of excess cholesterol from lipid-laden macrophages in the vessel wall to the liver. However, one of the major challenges of miRNA-based therapy is to achieve tissue-specific, efficient, and safe delivery of miRNAs in vivo. We sought to develop chitosan nanoparticles (chNPs) that can deliver functional miRNA mimics to macrophages and to determine if these nanoparticles can alter cholesterol efflux and reverse cholesterol transport in vivo. We developed chNPs with a size range of 150-200 nm via the ionic gelation method using tripolyphosphate (TPP) as a cross-linker. In this method, negatively charged miRNAs were encapsulated in the nanoparticles by ionic interactions with polymeric components. We then optimized the efficiency of intracellular delivery of different formulations of chitosan/TPP/miRNA to mouse macrophages. Using a well-defined miRNA with roles in macrophage cholesterol metabolism, we tested whether chNPs could deliver functional miRNAs to macrophages. We find chNPs can transfer exogenous miR-33 to naïve macrophages and reduce the expression of ABCA1, a potent miR-33 target gene, both in vitro and in vivo, confirming that miRNAs delivered via nanoparticles can escape the endosomal system and function in the RISC complex. Because miR-33 and ABCA1 play a key role in regulating the efflux of cholesterol from macrophages, we also confirmed that macrophages treated with miR-33-loaded chNPs exhibited reduced cholesterol efflux to apolipoprotein A1, further confirming functional delivery of the miRNA. In vivo, mice treated with miR33-chNPs showed decreased reverse cholesterol transport (RCT) to the plasma, liver, and feces. In contrast, when efflux-promoting miRNAs were delivered via chNPs, ABCA1 expression and cholesterol efflux into the RCT pathway were improved. Over all, miRNAs can be efficiently delivered to macrophages via nanoparticles, where they can function to regulate ABCA1 expression and cholesterol efflux, suggesting that these miRNA nanoparticles can be used in vivo to target atherosclerotic lesions.