Development of Self-Assembled Polymeric Polyplexes for MicroRNA Delivery: Toward Treatment of Alcohol-Associated Liver Disease

Self-assembled polymeric polyplexes have the potential to serve as an RNA interference (RNAi) delivery platform for the combined inhibition of CXCR4 and miR-155 in the treatment of alcohol-associated liver disease (AALD). A central challenge in RNA delivery is balancing the extracellular stability of polyplexes, cellular uptake, and intracellular release of RNA cargo. We developed polymeric polyplexes using the CXCR4-antagonist polymer PAMD, designed to target CXCR4 on activated hepatic stellate cells (HSCs) and deliver therapeutic miRNA to activated Kupffer cells (KCs) in fibrotic liver. We improved the polyplexes by cholesterol modification of PAMD and by PEGylation of PAMD-Ch. The cholesterol modification of PAMD reduced the hydrodynamic size of the polyplexes and improved their colloidal stability through hydrophobic core compaction, facilitating passage through liver sinusoidal fenestrae. This modification also partially neutralized the surface charge of the polyplexes, reducing their cytotoxicity and improving the stability in heparin and plasma. Furthermore, cholesterol modification increased the hydrophobicity of the polyplexes, resulting in enhanced cellular uptake in macrophages. PEGylation of PAMD-Ch further improved polyplex resistance to heparin and blood plasma (especially at moderate PEG content) through charge neutralization and the introduction of stealth properties. Our formulated polyplexes achieved high RNA transfection efficiency in vitro. Overall, this makes the novel PEG-PAMD-Ch/miR a promising miRNA delivery platform for AALD therapy.