Calcein leakage test using the LUV

The LUV was synthesized on the surface of agarose film according to previous reports (49, 80). Briefly, melted low-melting temperature agarose [1% (w/w); Sigma-Aldrich, A9414] was added dropwise to the surface of a tilted microscope slide to allow the slide to be covered with a thin layer of agarose. The slide was placed on a hot plate (40°C) for solidification. Afterward, 100 μl of egg phosphatidylcholine (3.75 mg ml⁻¹; Sigma-Aldrich Y0001905) in chloroform-methanol (9:1, v/v) was added dropwise to the surface of agarose-coated slide (1 inch × 3 inches), and the residual organic solvent was removed in a vacuum overnight to form a thin lipid film on the surface of agarose layer. Then, the slide was immersed for 12 hours in 15 ml of calcein (1 mg ml⁻¹) dissolved in PBS (pH 7.4). In such a way, LUV encapsulating fluorescent calcein was generated on the slide surface. After removal of the slide, LUV in the resulting solution was harvested through standard dialysis (molecular weight cutoff: 1000 kDa) for 36 hours in PBS solution. For staining of the liposome membrane, FM 4-64 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"F34653","term_id":"4820279","term_text":"F34653"}}F34653) was added to LUV solution to a final concentration of 10 μg ml⁻¹ at 4°C for 10 min. TNA (10 mg) or R-TNA (10 mg) was dissolved in 200 μl of DMSO and subsequently diluted to a concentration of 2 mM with PBS. TNA or R-TNA solutions were added to the hydrated liposomes to a final concentration of 1 mM and monitored using fluorescence imaging (LSM800, Zeiss, Germany). TNA and R-TNA nanoparticles can be adsorbed on the surface of LUV by electrostatic interaction. The disruption of LUV is affected by molecular diffusion from supramolecular materials (49). As a control, the addition of PBS instead of TNA/R-TNA caused no notable leakage of calcein, confirming that osmotic pressure does not play a dominant role in calcein release.