Programmable Lipid Functionalization of Nucleic Acid Nanoparticles Modulates Liver Cell-Type Targeting

Nucleic acid nanoparticles
(NANPs) fabricated by using DNA origami
are an emerging delivery vector for nucleic acid therapeutics. Despite
their advantages over other nanomaterials that include controlled
spatial presentation of targeting ligands such as lipids and sugars,
understanding their cell targeting and uptake mechanisms remains limited.
Here, we investigated NANP cellular targeting, uptake, and delivery
of small interfering RNAs (siRNAs) to liver and neuronal cell models in vitro. Using a rational design approach, we targeted
NANPs to two clinically validated receptors, the asialoglycoprotein
receptor (ASGPR) and the low-density lipoprotein receptor (LDLR),
respectively, using GalNAc and lipidation. We systematically evaluated
how the ligand valency, interligand spacing, linker length, and ligand
chemistry affected NANP association with on- and off-target liver
cell types, revealing the relative roles of the biomolecular corona,
receptor engagement, and endocytosis in these targeting strategies.
We found that lipidation enhanced NANP uptake into HepG2 cells, a
model cell line for hepatocytes, by promoting apolipoprotein recruitment,
LDLR engagement, and clathrin-mediated endocytosis and also increased
association with nonparenchymal cells. HepG2 uptake was further improved
by conjugating NANPs to lipids with higher valency provided that lipids
were adequately displayed away from the surface of NANP edges with
more lipophilic lipids yielding greater cell association. We then
benchmarked the potential for NANPs to deliver siRNAs to HepG2 cells
in comparison with lipid nanoparticle and conjugate technologies and
explored lipid functionalization as a strategy for nonhepatic NANP
targeting to model neuronal cells. Overall, this study advances the
foundational understanding of how clinically relevant targeting ligands
mediate NANP interactions with both on- and off-target liver cell
types in vitro, offering insights into potential
design criteria for nucleic acid therapeutic delivery.