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Cy5 fluorescence dye was encapsulated into liposomes prepared with E. coli polar lipids and L-α-phosphatidylcholine 3:1 ratio (w/w) (Avanti Polar Lipids). Liposomes were diluted to 12.5 mg mL−1 final lipid concentration with M-buffer (20 mM MOPS, 100 mM NaCl, and 1 mM TCEP pH = 7). Cy5 was added to a final concentration of 200 nM and encapsulated to the liposome lumen by freeze–thaw membrane fracture followed by extrusion through PC membranes with decreasing pore sizes (1 µm, 400 nm, and 200 nm), similar to the liposome preparation protocol. Excess Cy5 was removed by three successive ultracentrifugation and resuspension steps with M-buffer at 160,000 × g at 4 °C in a Sorvall mX120+ Micro-Ultracentrifuge for 45 min. The Cy5-encapsulated liposome mixture was finally resuspended to the initial volume with the M-buffer for ZIF immobilization
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