Egg pc

Powder form of Egg-PC (Sigma Aldrich), L-α-phosphatidylethanolamine (Sigma Aldrich), 16:0 Cardiolipin (Avanti Polar Lipids), 3-sn-PA (Sigma Aldrich), Brain PS (Avanti Polar Lipids) was dissolved in chloroform. Next, 270 μM PC, 145 μM phosphatidylethanolamine, 65 μM PIP4, 5 μM CL, 5 μM PA, 10 μM PS were mixed and kept for overnight evaporation. The thin lipid film obtained the following day after removal of residual chloroform was dissolved in 500 μl of 20 mM Tris pH 7.5 and 150 mM NaCl with gentle pipetting. The solution was next incubated at 55 °C for 30 min. The resultant colloidal heated solution was subjected to extrusion as mentioned above. Finally, the model membrane liposomes comprising a mixture of PC, phosphatidylethanolamine, PIP4, CL, PA, and PS were used for downstream experiments and the remaining was stored at 4 °C for not more than 3 days.

Powder form of Egg-PC (Sigma Aldrich) and cholesterol (Sigma Aldrich) was dissolved in chloroform. Next, 250 μM chloroform–solubilized PC was mixed with 250 μM chloroform–solubilized cholesterol and kept for overnight evaporation. The thin lipid film of mixed lipids obtained after removal of residual chloroform was dissolved in 500 μl of 20 mM Tris pH 7.5 and 150 mM NaCl with gentle pipetting. The solution was next incubated at 55 °C for 30 min. This colloidal heated solution was subjected to extrusion as described above. The PC-Chol unilamellar vesicles were used for downstream experiments and the remaining was stored at 4 °C for not more than 3 days.

For aqueous magnetoliposome (AML) preparation, where magnetic nanoparticles are encapsulated in liposomes, an ethanolic solution of egg yolk–phosphatidylcholine (Egg–PC, from Sigma-Aldrich, St. Louis, MO, USA), was injected, under vortex, onto an aqueous solution containing 0.1 mM of nanoparticles, to a final lipid concentration of 1 mM (ethanolic injection method [58 (link),59 (link)]). Then, the ferrofluid was washed with water by magnetic decantation to eliminate the nonencapsulated nanoparticles. Curcumin was incorporated into AMLs by the coinjection method, as previously described [22 (link)].
The preparation method of solid magnetoliposomes (SMLs), consisting in a lipid bilayer surrounding a cluster of magnetic nanoparticles, was previously developed by us and the formation of the liposomal structure has been confirmed in previous works [22 (link),24 (link)], using the lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, from Sigma-Aldrich, St. Louis, MO, USA). First, 10 μL of a solution of magnetic nanoparticles (0.02 mg/mL) were ultrasonicated for one minute at 189 W, and 3 mL of chloroform was added to the solution, resulting in the formation of nanoparticle clusters. To form the first lipid layer, the solution containing the clusters was heated up to 55 °C (above the melting temperature of DPPC, 41 °C [46 (link)]) and 150 µL of a lipid methanolic solution (20 mM) was injected under vortexing. Magnetic decantation and several washing steps with ultrapure water were performed for purification, removing lipid aggregates and liposomes without a magnetic core. For the second lipid layer formation, an aqueous solution of the purified systems (3 mL) was heated up to 55 °C and 150 µL of a lipid methanolic solution (20 mM) was injected under vortexing. The resulting solid magnetoliposomes were then washed and purified with ultrapure water by centrifugation. Curcumin was incorporated by injection of an ethanolic solution, under vortexing, right before the formation of the second lipid layer.
Giant Unilamellar Vesicles (GUVs) were used as models of cell membranes and were prepared using a procedure previously described [44 (link),45 (link)], using a 1 mM solution of L-α-phosphatidylcholine (Soybean lecithin, from Sigma-Aldrich, St. Louis, MO, USA).