Fig. 3: Tightly sealed 3DFLBs in sucrose and physiological ionic solutions.

a Schematics of the tight sealing mechanism via hydraulic pressure in sucrose (non-ionic) and physiological (ionic) solutions. Outside molecules move into the unsealed 3DFLBs fabricated without hydraulic pressure through nanocapillaries. Hydraulic pressure promotes tight sealing of the 3DFLBs even under physiological ionic conditions, in which the electrical repulsion between lipid membranes is enhanced. b, c Morphological and hermetical phase diagrams of the 3DFLBs formed at different AC frequencies and hydraulic pressures in 10 mM sucrose solution (b) and KCl solution (140 mM KCl, 10 mM HEPES; 300 mOsml/L) (c). d–f Representative 3D reconstructed and cross-sectional confocal fluorescence microscopy images of 3DFLBs corresponding to each zone in (c) and the corresponding sealing test: multilayer/multistructure formed at 500 Hz frequency and 0.59 kPa hydraulic pressure (Zone 1) (d), leaky 3DFLBs formed at 10 kHz and 0.82 kPa (Zone 2) (e) and tightly sealed 3DFLBs formed at 10 kHz and 1.52 kPa (Zone 3) (f) in KCl solution. Lipid composition: DOPC with 1 mol% Rhod-PE. g, h Representative cross-sectional confocal fluorescence microscopy images and the corresponding sealing tests of the tightly sealed 3DFLBs generated in NaCl solution (140 mM NaCl, 10 mM HEPES) (g) and 150 mM DPBS solution (both solutions are physiologically ionic at 300 mOsml/L) (h) at 10 kHz frequency and 1.52 kPa hydraulic pressure. Lipid composition: DOPC with 1 mol% Rhod-PE. i–l Representative cross-sectional confocal fluorescence microscopy images and corresponding tight-sealing tests of 3DFLBs generated in KCl solution with different lipid compositions as SOPC (i), POPE (j), a mixture of DOPC (50 mol%) and DOPE (50 mol%) (k), and a mixture of DOPC (50 mol%), DOPE (20 mol%), and DOPS (30 mol%) (l). All the lipids are labeled with 1 mol% Rhod-PE. The 3DFLBs with various lipids were generated under the same conditions as 10 kHz frequency and a hydraulic pressure of 1.52 kPa. All the representative 3D reconstructed and cross-sectional confocal fluorescence microscopy images of 3DFLBs were obtained from repeated experiments more than 4 times and their scale bar is 5 µm.