# POx-Lipids as an Alternative to PEG-Lipids? Multimethod Assessment of Chemistry and Structure

## TL;DR

This paper explores PEtOx-lipids as a potential alternative to PEG-lipids in pharmaceutical formulations, comparing their chemical and structural properties using multiple analytical methods.

## Contribution

The study introduces a multimethod approach to assess PEtOx-lipids as a novel alternative to PEG-lipids in lipid nanoparticles.

## Key findings

- PEtOx-lipids show similar hydrodynamic size and hydration to PEG-lipids despite lower aggregation numbers.
- High sample purity is essential for accurate hydrodynamic analysis of polymer conformation and hydration.
- Hydration in PEtOx-lipid micelles is due to water between extended polymer chains, not hydrogen bonding.

## Abstract

The characterization of polymer–lipid conjugates
is essential
for their successful application in pharmaceutical formulations, particularly
in lipid nanoparticles (LNPs). While poly­(ethylene glycol) (PEG) lipids
are widely used in LNPs, growing concerns over PEG immunogenicity
have prompted the search for alternatives. Here, we investigate a
series of poly­(2-ethyl-2-oxazoline) (PEtOx) lipids as promising PEG
substitutes featuring biocompatibility, tunable hydrophilicity, and
stealth-like properties. We apply a comprehensive, multimethod approach
utilizing liquid chromatography (LC) and matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS) to assess end group
homogeneity, sample purity, and information about overall hydrophobicity/hydrophilicity
of PEtOx-lipids compared to benchmark PEG-lipids used in COVID vaccines.
We also demonstrate that under conditions of high sample purity only,
hydrodynamic techniques such as analytical ultracentrifugation (AUC)
become robust tools for analyzing conformational properties, aggregation
behavior, and hydration of the polymers in aqueous environments. Though
PEtOx-lipid analogues form micelles with lower aggregation numbers
than PEG-lipids, they feature the same hydrodynamic size and values
of hydration. The large levels of hydration of PEtOx-lipid micelles
cannot be explained by hydrogen bonded water. Water present in the
micelles constitutes a major part of their overall hydrodynamic volume
and is located between the extended chain conformation of assembled
polymers. Our study, with a unique multimethod approach, shows the
importance of sample purity in high-end hydrodynamic analysis and
paves the way for a quantitative replacement of PEG-based lipid conjugates
by PEtOx-based lipid conjugates through a distinct set of properties
to be considered for a particular application.