*2.2. Liposome Preparation*

Liposomes were prepared by a lipid film hydration method using a composition of DPPC/DSPC/Lyso PC/DSPE-PEG at a molar ratio of 75:15:10:4, respectively. The lipids dissolved in chloroform were mixed and dried to a thin lipid film by rotary evaporation. Residual chloroform was removed under a nitrogen flow for 30 min. The film was hydrated at 55 ◦C for 1 h with 2.5 mg/mL oligonucleotides dissolved in 20 mM HEPES buffer, pH 7.4. To promote encapsulation of oligonucleotides, a high lipid concentration of 100 mM (typically, 50 μmol of total lipids and 0.5 mL of hydration solution) was used. The formed liposomes were extruded 5 times through a track-etched polycarbonate membrane (Whatman Nuclepore, GE Healthcare, Chicago, IL, USA) with 100-nm pore size using a syringe extrusion device (Avanti Polar Lipids Inc., Alabaster, AL, USA). The free, unencapsulated oligonucleotides were separated by ultracentrifugation for 1 h at 55,000 rpm at 4 ◦C for 2–3 times, and the liposomes were dispersed in 20 mM HEPES, 140 mM NaCl, pH 7.4.

For ICG incorporation, the liposomes were incubated in ICG solution of 1 mg/mL (in 20 mM HEPES, 140 mM NaCl, pH 7.4) for 1 h in rotation at room temperature. The volumes of the incubated liposomes and ICG solution were adjusted to molar ratios in the range of 1/25–1/200 ICG to lipid. The amount of ICG in the liposomes was determined by separating the free ICG by ultracentrifugation (1 h at 55,000 rpm at 4 ◦C) after incubation. The amount of free ICG in the supernatant was determined based on ICG fluorescence that was measured at 770/810 nm (excitation/emission wavelengths), and the concentration was determined using a calibration curve. As the percentage of non-incorporated ICG was less than 10% even at the highest ICG concentration, the separation step by ultracentrifugation was not considered necessary and was not carried out in further experiments. The addition of ICG to the liposomes was done always immediately before the experiments.

#### *2.3. Liposome Characterization*

#### 2.3.1. Size

The mean particle size and polydispersity index (PDI) were measured by dynamic light scattering (DLS) with a Malvern CGS-3 multiangle goniometer with He–Ne laser source (λ = 632.8 nm, 22 mW output power) using an angle of 90◦ (Malvern Instruments, Malvern, UK). For the measurement, liposome samples were diluted to 0.25 mM (lipid concentration) in 20 mM HEPES with 140 mM NaCl, pH 7.4.

#### 2.3.2. Zeta-Potential

The zeta-potential of the liposomes was measured on a Zetasizer Nano-Z (Malvern Instruments) with samples diluted to 0.25 mM (lipid concentration) in 20 mM HEPES, pH 7.4.
