Steric Stabilization of Lipid Nanoparticles by Sequence-Defined Polymer−Lipids Enhances Gene Expression

Abstract

Polyethylene glycol (PEG) conjugated lipids are frequently used for the design of lipid nanoparticles (LNPs) in the field of gene therapy. However, conventional PEG−lipids contain a distribution of polymer chain lengths. To overcome this limitation, we have prepared a structurally defined PEG-alternative polymer-lipid platform synthesized via solid-phase peptide synthesis (SPPS), enabling precise control over polymer chain length and lipid anchor identity. A library of chain-length-defined poly(succinamide−4,7,10-trioxatridecane)n−lipid (poly(SA-TTD)n-lipid) conjugates, with hydrophilic polymer segments ranging from 923 to 2133 g/mol and varied lipid tails, was synthesized and formulated into DNA-loaded LNPs using microfluidic mixing. Systematic variation of polymer length and lipid tail structure revealed their distinct influence on NP size, colloidal stability, and DNA encapsulation efficiency. All formulations produced particles with narrow size distributions (PDI <0.2) with efficient DNA association. In vitro studies demonstrated that selected PEG-alternative LNPs significantly enhanced transfection efficiency compared to PEG2000-containing controls, achieving up to 18-fold higher transgene expression. Notably, these formulations also exhibited an approximately fourfold higher endosomal escape-associated signal. In vivo evaluation in zebrafish larvae (ZFL) further confirmed efficient systemic circulation and superior transgene expression without apparent toxicity. In summary, this work establishes a modular, sequence-defined polymer-lipid platform that provides a promising PEG alternative for LNP-mediated gene delivery.