Fig. 1: Preparation and characterization of liposomal ICG/DOX (LID).

a Schematic showing the preparation of LID. Blank liposomes were prepared with 250 mM (NH4)₂SO₄, followed by removal of external (NH4)₂SO₄ using size exclusion chromatography to establish the transmembrane gradient. DOX was incubated with the blank liposomes at 55°C to enable drug loading. When NH₃ escaped liposomes, one H⁺ was produced and retained in the liposome, resulting in an acidic core. When DOX diffused into the liposome, it became protonated and trapped within the liposome. As DOX loading into the liposome transiently increased the internal pH, it further increased the level of ammonia and created more H⁺, allowing more DOX to be loaded into the liposome. Ultimately, DOX forms a crystalline precipitate due to the presence of sulfate anions inside the liposome. ICG was covalently attached to DOPE before incubation with DOX-loaded liposomes such that DOPE could anchor ICG onto liposomes. b The absorption spectrum of LID. c representative size distribution of LID measured by dynamic laser scattering (DLS). d Cryo-electron microscopy (cryo-EM) of LID, scale bar = 100 nm. White arrows indicate low-dose DOX nanocrystals. The data are representative of two independent experiments (b–d). Source data are provided as a Source Data file.