Speaker
Description
The method employed for liposome preparation plays a critical role in defining their physicochemical properties, which in turn strongly influence their stability, functionality, and suitability for downstream applications. Conventional bulk techniques, such as thin film hydration followed by extrusion, are widely used and well established; however, they rely on multi-step, time-consuming procedures and may suffer from limited batch-to-batch reproducibility.1 These drawbacks can represent a significant limitation, particularly in view of large-scale production and clinical translation. In recent years, microfluidic technologies have emerged as a promising alternative for liposome fabrication, offering continuous, automated, and potentially scalable production with precise control over size and narrow size distributions.2 Despite these advantages, microfluidic liposome preparation typically involves the controlled mixing of aqueous and organic phases, most commonly using ethanol as the organic solvent. While this approach enables efficient vesicle formation, it also raises concerns regarding the partial incorporation of solvent molecules into the lipid bilayer.3 Although purification strategies are routinely applied to remove the organic solvent, residual ethanol traces often persist and their impact on liposome structure and functionality is frequently overlooked. In this study, a systematic comparison between thin film hydration and microfluidic preparation of DMPC liposomes was carried out to evaluate the influence of the production method on vesicle properties. The physicochemical characteristics of the resulting liposomes, including size distribution and thermal behavior, as well as membrane fluidity, were investigated using dynamic light scattering (DLS), differential scanning calorimetry (DSC), and fluorescence anisotropy measurements. These complementary techniques allowed a detailed assessment of the effects induced by residual ethanol within the lipid bilayer, highlighting differences in membrane organization and phase behavior between the two preparation methods. Beyond basic characterization, particular attention was devoted to evaluating the functional performance of liposomes as carriers for active molecules. To this end, the insertion efficiency and Cu(II) binding capability of 2-(hydroxyamino)-3-octyltridecanal were investigated. This synthetic amphiphilic compound, endowed with long alkyl chains, is specifically designed to favor stable insertion into lipid bilayers while retaining metal-chelating functionality. The incorporation of the molecule and its ability to complex Cu(II) ions were studied using UV–Vis spectroscopy and fluorescence quenching experiments. The comparative analysis reveals that residual solvent traces associated with microfluidic preparation significantly influence not only the physicochemical properties of liposomes but also the functional behavior of the embedded chelating agent. In particular, changes in membrane fluidity and organization were found to directly affect the efficiency of molecular insertion and metal complexation. Overall, this work underscores the critical importance of solvent selection, purification efficiency, and a thorough understanding of the effects of trace solvent contamination when designing liposomal formulations. These considerations are especially relevant for the development and clinical translation of microfluidics-fabricated liposomes intended for biomedical applications.
[1] Colloid. Surface A. 617, (2021). https://doi.org/10.1016/j.colsurfa.2021.126321.
[2]. Method. Enzymol. 465, (2009). https://doi.org/10.1016/S0076-6879(09)65007-2.
[3] J. Colloid. Interface Sci. 578, (2020). https://doi.org/10.1016/j.jcis.2020.05.114.