| dc.description.abstract | Anchoring lipids are functionalized lipids that can immobilize biomolecules or organic molecules on lipid membranes. They are used to enhance the functionality of liposomes, such as conjugating peptides to the surface for targeting or triggering drug release. Commercially available liposomal drugs primarily utilize formulations with saturated lipids due to their higher mechanical strength. However, when choosing between saturated and unsaturated versions of anchoring lipids, there is a lack of guidelines. In this study, we used the antimicrobial peptide Magainin 2 to conjugate to saturated or unsaturated anchoring lipids using thiol-maleimide Michael addition chemistry, which allowed us to anchor the peptide to liposomes and form peptidyl liposomes. We encapsulated doxorubicin in the peptidyl liposomes and observed the drug release to estimate the effect of different anchoring lipids on the membrane-disrupting activity. We found that when using unsaturated anchoring lipids, the drug release rate was close to 100%, whereas when using saturated anchoring lipids, the drug release rate was nearly zero. To further demonstrate that the effect is due to the unsaturation of the anchoring lipids rather than the unsaturation of the liposomes themselves, we added an equal concentration of non-reactive unsaturated lipid to the peptide liposomes containing saturated anchoring lipids. We found that the liposomes still did not release the drug, which indicates that it is not the unsaturated lipids weakening the mechanical strength of the liposomes and causing successful triggering of drug release. Instead, it is the membrane-disrupting ability of the synergistic effect of Magainin 2 peptide conjugated to unsaturated anchoring lipids that plays a role. This suggests that there should be more than simple peptide-membrane interaction involved. Through circular dichroism (CD) analysis, we also observed that liposomes containing Magainin 2-18:1 lipopeptide exhibited a higher extent of α-helical structure, while Magainin 2-18:0 lipopeptide showed a lower extent of α-helical structure. This further shows that peptides interact with membrane differently when using anchoring lipid with/without saturation. To compare the differences in the effects of anchoring lipid saturation on the membrane, we used Giant Unilamellar Vesicles (GUVs) for visual (fluorescent) observation to determine whether the lipid phase separation in liposomes is caused by general lipid saturation or the coupling of the peptide to anchoring lipids leading to phase separation and effective peptide aggregation for membrane disruption. Since GUVs have an average diameter of over 10 micrometers, the observation of the membrane′s condition can be conducted by adding appropriate fluorescent probes during the vesicle formation process. The observation results showed that only when Magainin 2 was conjugated to unsaturated anchoring lipids, lipid phase separation occurred. This indicates the crucial importance of lipid phase separation in this system for the membrane-disrupting activity of the anchored peptide. These findings contribute to the selection of appropriate anchoring lipids in drug delivery systems and membrane protein functional reconstruction systems. | en_US |