*3.2. Transfection Experiments*

We studied the effects of liposomal formulation (ICG and lipid concentrations) and light exposure (intensity, duration) on transfection efficacy. Moreover, transfection efficacy at different time points was measured. Successful transfection was seen as increased EGFP fluorescence in the cell cytoplasm (Figure 2). Treatment with ICG liposomes encapsulated with the negative control oligonucleotide induced no detectable transfection (Figure S2). Light triggering was shown not to affect the activity of the SSO by comparing transfection efficacy of Lipofectamin-transfected cells with and without light exposure (Figure S3).

**Figure 2.** Confocal images of HeLa S3 IVS2-654 enhanced green fluorescent protein (EGFP) cells. Action of SSO restores EGFP expression. The cells were administered with (**A**) 0, (**B**) 0.5, (**C**) 0.7, and (**D**) 1.4 mM of SSO-encapsulated ICG liposomes and exposed to 808 nm light with the intensity of 370 mW/cm<sup>2</sup> for 2 min. The green color represents EGFP fluorescence. The concentrations refer to lipid concentrations of the liposome dispersions. The liposomes contained ICG at a molar ratio of 1/50 ICG to lipid. Scale bar = 100 μm.

## 3.2.1. Liposome Concentration

Liposome concentration had a clear effect on transfection efficacy (Figure 2). Increasing liposome concentrations led to higher transfection percentages: when the concentration was approximately tripled (from 0.5 mM to 1.4 mM), the transfection percentage increased roughly 3-fold (from 20% to 60% and from 30% to 80%) (Figure 3A,B). An increased transfection was detected in the cells treated with liposomes and exposed to light, as well as in the cells that were incubated with liposomes without light exposure. This was especially evident for liposomes having a higher concentration of ICG; with 1.4 mM liposomes of 1/25 ICG-to-lipid ratio, 14% of the cells were transfected without light exposure (Figure 3B). Yet, the transfection efficacy was considerably higher (78%) with light exposure than without light. In general, the transfection efficacies obtained with the light-triggered liposomes were in the same range as with Lipofectamine®3000 that was used as a positive control. However, compared to Lipofectamine, the results with the light-triggered delivery were much more consistent (Figure S4).

**Figure 3.** Effect of (**A**,**B**) liposome concentration and (**C**–**F**) ICG concentration of the liposomes on the transfection efficacy. The HeLa S3 IVS2-654 EGFP cells were incubated with liposomes having the following lipid concentrations and ICG-to-lipid molar ratios: (**A**) 0.5 mM–1.4 mM, 1/50 ICG/lipid, (**B**) 0.5 mM–1.4 mM, 1/25 ICG/lipid, (**C**) 0.7 mM, 1/25–1/50 ICG/lipid, (**D**) 1.4 mM, 1/25–1/50 ICG/lipid, (**E**) 0.7 mM–2.8 mM, 1/50–1/200 ICG/lipid, and (**F**) 0.7 mM–2.8 mM, 1/25–1/100 ICG/lipid. In (**E**) and (**F**), all the treatments had the same ICG concentration, 14 μM in (**E**) and 28 μM in (**F**). After incubation, the cells were exposed to 808 nm light (370 mW/cm2 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.

#### 3.2.2. ICG Concentration

Increasing the ICG concentration that was added to the liposomes led to an increased transfection efficacy. The higher the ICG concentration of the liposomes, the higher the percentage of transfected cells (Figure 3C,D). Instead, cells treated with liposomes having different lipid concentrations but same ICG concentration had equal transfection percentages (Figure 3E,F). As a conclusion, efficacy of the transfection process is mainly dependent on the ICG concentration, and both lipid and SSO concentrations have a smaller impact. Moreover, the liposomes with a higher ICG concentration induced transfection in the absence of light as well. At the highest ICG concentration, about 20% of the cells were EGFP positive without light exposure (Figure 3D,F).

#### 3.2.3. Illumination Time

In general, the transfection efficacy was not affected when the illumination time was increased to longer than 1 min (Figure 4A,B). Effects of illumination times shorter than 1 min were dependent on other variables, the major determinant being ICG concentration. At a higher ICG concentration, changes in transfection efficacy with illumination times of 15 s, 30 s, and 1 min were more prominent than those observed for lower ICG concentrations. In addition, at very high ICG concentrations, some increase in transfection efficacy could be seen between 1 min and 2 min light exposures (data not shown). Consequently, the illumination time of 2 min was chosen for further studies.

**Figure 4.** Effect of (**A**,**B**) illumination time and (**C**,**D**) light intensity on transfection efficacy. The HeLa S3 IVS2-654 EGFP cells were incubated with 0.7 mM liposomes having (**A**,**C**) 1/50 and (**B**,**D**) 1/25 ICG-to-lipid molar ratio. After incubation, the cells were exposed to 808 nm light with (**A**,**B**) the intensity of 370 mW/cm<sup>2</sup> for 15 s to 8 min or (**C,D**) the intensities of 370–1500 mW/cm2 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.
