PEG chain length modulates hepatic targeting of GalNAc liposomes via ligand exposure and optimal glycocluster conformation

This study optimized polyethylene glycol (PEG) chain length in cholesterol-GalNAc conjugates (CHS-PEG n -6-GalNAc, n = 2–5) to enhance hepatic targeting of liposomes by balancing glycan exposure and glycocluster conformation. The conjugates were synthesized via enzymatic catalysis and incorporated into liposomes (LP-PEG n -6-GalNAc). Systems were evaluated through in vitro HepG2 cell uptake, in vivo murine biodistribution, and molecular dynamics (MD) simulations. LP-PEG n -6-GalNAc showed significantly higher in vitro uptake ( P  < 0.01 vs unmodified liposomes) and superior in vivo hepatic accumulation ( P  < 0.01). ASGPR-mediated targeting was confirmed by competitive inhibition (GalNAc in vitro , asialofetuin in vivo ; both P  < 0.01). Targeting efficiency exhibited identical PEG-length dependency: PEG 3  > PEG 4  > PEG 5  > PEG 2 . MD simulations revealed a dual mechanism: Shorter chains (PEG 2 ) maintained higher glycocluster stability (frequency = 40.8 %; RMSF = 0.39 Å) but limited GalNAc accessibility (SASA = 49.9 %). Longer chains (PEG 4-5 ) increased SASA (73.38–86.08 %) but reduced stability (frequency = 23.0–35.0 %; RMSF = 1.05–1.30 Å). PEG 3 balanced SASA (61.7 %) and stability (36.2 %), synergistically enhancing uptake. This provides the first evidence that PEG length coordinates glycan accessibility and glycocluster stability to improve hepatic targeting.