Unveiling the power of liquid chromatography in examining a library of degradable poly(2-oxazoline)s in nanomedicine applications.

A library of degradable poly(2-alkyl-2-oxazoline) analogues (dPOx) with different length of the alkyl substituents was characterized in detail by gradient elution liquid chromatography. The hydrophobicity increased with increased side chain length as confirmed by a hydrophobicity row, established by reversed-phase liquid chromatography. Those dPOx were cytocompatible and formed colloidally stable nanoparticle (NP) formulations with positive zeta potential. Dynamic light scattering (DLS) revealed that dPOx with increased hydrophobicity tended to form NPs with increased sizes. NPs created from the most hydrophobic polymer, degradable poly(2-nonyl-2-oxazoline) (dPNonOx), showed tendency for aggregation at pH 5.0, and in the presence of protease in solution, in particular for NPs formulated without surfactant. Liquid chromatography revealed enzymatic degradation of dPNonOx NPs, clearly demonstrating the disappearance of polymer signals and the appearance of hydrophilic degradation products eluting close to the chromatographic void time. The degradation process was confirmed by ¹H NMR spectroscopy. dPNonOx NPs containing the anti-inflammatory drug BRP-201 as payload reduced 5-lipoxygenase activity in human neutrophils. Thereby, composition analysis of the resultant NPs, including drug quantification, was also enabled by liquid chromatography. The results indicate the importance of a detailed analysis of the final polymer-based NP formulations by a multimethod approach, including, next to standard applied techniques such as DLS/ELS, the underexplored potential of liquid chromatography. The latter is demonstrated to resolve a fine structure of solution composition, together with an assessment of possible degradation pathways and is versatile in determining hydrophobicity/hydrophilicity of polymer materials. Our study underscores the power of liquid chromatography for characterization of soft matter drug carriers.