The role of glycocalyx-protein corona interactions in the cell type-specific uptake of mRNA nanocarriers

Non-viral nanocarriers for oligonucleotide delivery have enormous therapeutic potential, but the factors determining cell type-specific uptake remain incompletely understood. One major cell surface component that delivery vectors encounter is the glycocalyx. While membrane glycans are established interactors for many viruses, their impact on non-viral particles has remained underexplored. Here, we show that the role of membrane glycosaminoglycans (GAGs) in mRNA delivery differs across nanocarrier categories and cell types. We employed novel glycosylation inhibitors as well as enzymatic strategies to modulate the glycocalyx, and measured their effects on mRNA delivery efficiency. Metabolic labelling enabled colocalization analysis of glycans with mRNA. Whereas lipoplexes and lipid nanoparticles (LNPs) require an intact glycocalyx for uptake by HeLa cervical cancer cells, a cationic peptide-based formulation does not. Heparan sulfate (HS) is the most important GAG for LNP uptake. Further experiments demonstrate that this interaction is mediated through HS-binding proteins of the protein corona, such as ApoE and vitronectin. HS-dependence varied across cell types: whereas HeLa and HCT116 tumor cells strongly depend on HS for LNP uptake, primary dendritic cells, normal human dermal fibroblasts, and MCF-7 breast cancer cells did not. HepG2 cells showed mild HS-dependence. Across all cell types, HS-dependence directly correlated with LNP uptake and expression, providing new mechanistic insight into why mRNA nanoparticles display cell-selective uptake.