SEC-based isolation of phosphatidylserine-liposomes from biological samples for preclinical studies

Autoimmune diseases represent a significant clinical challenge due to their high prevalence, chronic progression, and profound impact on patients' quality of life. Despite advances in understanding their pathophysiological mechanisms, in most cases, only palliative treatments are available. In this regard, a novel therapeutic approach is under development, using phosphatidylserine-liposomes (PS-Liposomes) encapsulating specific antigenic peptides that trigger the autoimmune attack. By varying the encapsulated antigen, this method offers the potential to treat different autoimmune diseases. The development of robust bioanalytical methods to characterize PS-Liposomes in biological fluids is of great importance to evaluate their efficacy and safety in preclinical trials. This presents a significant challenge due to the presence of extracellular vesicles and proteins in blood with similar size and composition to PS-Liposomes. In this study, size exclusion chromatography (SEC) was assessed for the isolation of PS-Liposomes encapsulating acetylcholine receptor peptide (AChR), intended for myasthenia gravis treatment, from minipig serum samples. A home-packed column filled with Sepharose 2B-CL resin and two commercial columns with agarose resin (qEV 35 nm and qEV 70 nm) were tested by using fluorescently labelled PS-Liposomes. Fractions containing PS-Liposomes were monitored by flow cytometry and qEV columns were selected for further experiments. For PS-liposome preconcentration of the obtained fractions, two different methodologies were also evaluated (Centrisart filters and qEV kit). Lipid and antigenic peptide recoveries obtained for each method were determined by HPLC-ELSD and HPLC-MS, and the optimum isolation and preconcentration approach was selected. Finally, the established methodology was successfully applied to spiked minipig serum with unlabelled PS-Liposomes, and the preservation of structural integrity after isolation was demonstrated by nanoparticle tracking analysis (NTA) and scanning electron microscopy (SEM).