*2.4. Light-Activated Liposomes*

ICG liposomes were prepared via the thin film hydration method followed by an extrusion and purification, as described previously with a few modifications [20,29]. Three formulations were prepared, uncoated (F1), PEG-coated (F2), and HA-coated (F4) liposomes with a basic lipid composition of DPPC/DSPC/(18:0)LysoPC at molar ratios of 75/15/10, supplemented with either 4 mol DSPE, 4 mol DSPE-PEG2000, or 1 mol DSPE–HA. The molar ratio of the DSPE–HA was chosen to correspond with the coating polymer mass of traditional PEG-coated liposomes. The thin lipid layer was hydrated with 0.322 mg/mL of ICG in 500 µL of HEPES buffer or calcein solution. In the case of the HA-coated liposomes, DSPE–HA was added during the hydration step instead of the initial mixture in chloroform. Prior to the thin film hydration step, the hydration solution with DSPE–HA was vortexed and heated to 64 ◦C until the lipid conjugate dissolved. The liposomes were extruded 11 times at 60 ◦C through a polycarbonate membrane with 100 nm diameter pores (unless otherwise specified) with a syringe-type extrusion device (Avanti Polar Lipids). Thereafter, the liposomes were quickly cooled and stored in a refrigerator. The unencapsulated calcein and ICG were removed by gel filtration through a Sephadex G-50 (Sigma-Aldrich) column with HEPES buffer elution. The final lipid and ICG concentration of the purified samples was 1.5 mM and 30 µM, respectively. For protein corona studies, similar liposomes with PEG (F3) and HA coating (F5), but without ICG, were also prepared. Additionally, anionic 50 nm liposomes with a lipid composition of DPPC/DSPC/DSPG/(18:0)LysoPC/DSPE (F6) or DSPE-PEG2000 (F7) at molar ratios of 75/5/10/10/4 were prepared as described by Tavakoli et al. [29].
