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

Area of Science:

• Biotechnology and Nanomedicine
• Molecular Biology and Genetics
• Drug Delivery Systems

Background:

• Nucleic acid nanoparticles (NANPs) are promising vectors for nucleic acid therapeutics, offering precise ligand presentation.
• Understanding the cellular targeting and uptake mechanisms of NANPs is crucial for optimizing their therapeutic potential.
• Current knowledge on how NANPs interact with cells and deliver cargo remains limited.

Purpose of the Study:

• To investigate the cellular targeting, uptake, and small interfering RNA (siRNA) delivery mechanisms of NANPs in liver and neuronal cell models.
• To evaluate the impact of ligand design (valency, spacing, linker length, chemistry) on NANP interactions with liver cells and their receptors (ASGPR, LDLR).
• To explore lipid functionalization as a strategy for targeting non-hepatic cells, specifically neuronal models.

Main Methods:

• Fabrication of NANPs using DNA origami with targeted ligands (GalNAc for ASGPR, lipids for LDLR).
• Systematic evaluation of ligand parameters (valency, spacing, linker length, lipophilicity) on NANP association with liver cell models (HepG2).
• Assessment of NANP uptake pathways (clathrin-mediated endocytosis) and biomolecular corona formation.
• Benchmarking NANP siRNA delivery efficiency against lipid nanoparticles and conjugate technologies.

Main Results:

• Lipidation significantly enhanced NANP uptake in HepG2 cells by promoting apolipoprotein recruitment, LDLR engagement, and clathrin-mediated endocytosis.
• Higher lipid valency improved HepG2 uptake when lipids were adequately spaced from the NANP surface; more lipophilic lipids increased cell association.
• NANP lipidation also increased association with non-parenchymal liver cells, indicating potential off-target effects.
• NANPs demonstrated potential for siRNA delivery to HepG2 cells and exploration of lipid functionalization for neuronal targeting.

Conclusions:

• Ligand design, particularly lipidation and valency, critically influences NANP targeting and uptake in liver cells via specific receptor-mediated endocytosis.
• Lipid functionalization is a viable strategy for enhancing NANP delivery to hepatocytes and potentially targeting non-hepatic cells like neurons.
• This research provides foundational insights into NANP-ligand interactions, informing the design criteria for effective nucleic acid therapeutic delivery systems.