3.2.4. Light Intensity

The intensity of 808 nm light used to trigger the liposomes did not affect the transfection efficacy in the range of 370–1500 mW/cm<sup>2</sup> (Figure 4C,D). The laser instrument used in this study limited the intensities to 370 mW/cm2 and higher levels. Intensities higher than 1500 mW/cm2 led to variability in results between repeats, and in general did not significantly improve the transfection efficacy (data not shown). As a conclusion, light intensities in the range of 370–1500 mW/cm<sup>2</sup> were suitable for induction of the oligonucleotide release.

## 3.2.5. Free ICG and SSO

During the studies, we observed that ICG and SSO can transfect cells upon light triggering without incorporation into liposomes. Therefore, experiments with free ICG or empty ICG liposomes and free SSO were performed. The transfection efficacies of free ICG and SSO, and SSO-encapsulated ICG liposomes were equal (Figure 5). In addition, a mixture of empty ICG liposomes and free-SSO transfected cells, but less effectively compared to free ICG and SSO or the SSO-encapsulated ICG liposomes (transfection efficacies of 19% versus 40% with 14 μM ICG; 45 versus 60% with 35 μM ICG, respectively). In the absence of ICG, treatment with SSO did not result in detectable levels of transfected cells.

**Figure 5.** Free ICG- and SSO-induced transfection after light exposure. The HeLa S3 IVS2-654 EGFP cells were incubated with free ICG or empty ICG liposomes and SSO corresponding to the following concentrations of SSO-encapsulated ICG liposomes: (**A**) 0.7 mM, 1/50 ICG-to-lipid ratio (240 nM SSO, 14 μM ICG) and (**B**) 1.4 mM, 1/40 ICG-to-lipid ratio (480 nM SSO, 35 μM ICG). After incubation, the cells were exposed to 808 nm light (370 mW/cm<sup>2</sup> for 2 min). The columns represent average values of EGFP-positive cells (*n* = 3) with error bars as standard deviation. Cells without treatment had less than 1% of EGFP-positive cells.

#### 3.2.6. Time after Transfection and Light Triggering

At higher liposome concentrations of 0.7 and 1.4 mM, transfection efficacies increased from 48 to 96 h after light triggering: transfection percentages increased from ~35% to 50% with 0.7 mM liposomes and from ~55% to 70% with 1.4 mM liposomes (Figure 6). At the lower liposome concentrations of 0.25 and 0.5 mM, differences between the time points were smaller and a slight increase in the transfection percentages could be seen only between 48 and 72 h (from 10% to 14% with 0.25 mM liposomes; from 22% to 27% with 0.5 mM liposomes). After 4 days, the HeLa cells grew over-confluent making quantitative analysis of the results impossible.

**Figure 6.** Effect of time after transfection and light triggering on transfection efficacy. The HeLa S3 IVS2-654 EGFP cells were incubated with 4 different liposome concentrations having 1/50 ICG-to-lipid molar ratio. Light exposure was performed at 808 nm (370 mW/cm<sup>2</sup> for 2 min), and transfection efficacy was measured at 48, 72, and 96 h after light triggering. The columns represent average values of EGFP-positive cells (*n* = 3) with error bars as standard deviation. Cells without treatment had less than 1% of EGFP-positive cells.

#### *3.3. Cytotoxicity*

The light exposure or liposome incubation separately showed no toxic effects (Figure 7A,B). Slightly lower cell numbers were seen after incubation with 1.0 and 1.4 mM liposomes: cell numbers

were 92%–96% of the control cell numbers at the first time point, 48 h after liposome incubation. At 72 and 96 h, cell numbers were similar in all the groups. The combination of liposomes and light exposure resulted in moderate toxic effects (Figure 7C). Especially, 48 h after light triggering, the cell numbers were lower than in the control group: 75% in cells treated with 0.5 and 0.7 mM liposomes and 65% in cells treated with 1.0 and 1.4 mM liposomes. However, cell numbers in the treated groups had increased at 72 h after light triggering and at 96 h, the cell numbers did not differ from the non-treated control group.

**Figure 7.** Effect of (**A**) light exposure, (**B**) the ICG liposome concentration, or (**C**) light exposure and the ICG liposome concentration on cell growth. Cells were (**A**) exposed to 808 nm light with the intensities of 370–1500 mW/cm<sup>2</sup> for 2 min or (**B**) incubated with 0.5–1.4 mM liposomes having 1/50 ICG-to-lipid molar ratio overnight. In (**C**), light exposure was performed after overnight liposome incubation. The columns represent average values of cell numbers (*n* = 3) calculated as percentage of non-treated control cells with error bars as standard deviation.
