A Copper‐Complexing Lipid Unmasks the Ethanol Hidden Within Liposomes and Highlights the Relevance of Fabrication Method on the Performance of Lipophilic Functional Molecules

Liposomes are traditionally produced batch‐wise using laborious bulk methods. In contrast, microfluidics ( μ F) enables operator‐independent, continuous, and scalable production of liposomes with narrow size distributions, aided by advanced chip technology. However, μ F fabrication may leave traces of the water‐miscible organic solvent used to dissolve lipids. Thereby, benign solvents such as ethanol are employed, and post‐processing ensures acceptable limits of residues. Nonetheless, residual ethanol can influence liposome structure, stability, and the performance of component functional molecules. These effects are typically investigated by microscopy, scattering, and diffraction techniques, not suited for high‐throughput applications. Here, the impact of liposome fabrication method on the incorporation of 2‐(hydroxyimino)‐3‐octyltridecanal (HIOTD) and on its performance as a Cu(II) chelator is assessed using UV–vis spectrometry. DMPC liposomes and a commercial μ F micromixer chip are employed as a representative case, with solvent‐free thin film hydration (TF) as a benchmark for comparison. Combined DLS, calorimetry, and fluorescence anisotropy analyses reveal physicochemical differences between μ F and TF liposomes, while UV–vis spectroscopy highlights variations in incorporation and metal‐binding performance of HIOTD. These findings demonstrate that the liposome fabrication route critically affects the functional behavior of embedded molecules and propose a convenient UV–vis approach for comparative evaluation of fabrication methods, in view of their clinical translation.