2.3.3. Phase Transition Temperature (*Tm*)

Differential scanning calorimetry (DSC) (Discovery DSC, TA instruments, New Castle, DE, USA) was used to determine the *Tm* values of the liposomes. Liposome sample with a concentration of 50 mM and a reference sample (20 mM HEPES with 140 mM NaCl, pH 7.4) were placed in hermetically sealed aluminum pans, and heated from 20 to 60 ◦C at a rate of 0.5 ◦C/min. *Tm* represents the peak temperature of the endotherm recorded during the heating scan.

#### 2.3.4. Encapsulation Efficiency

The amount of SSO or siRNA encapsulated inside the liposomes was determined using Quant-iT™ RiboGreen®RNA Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The measurement was performed for non-treated liposomes and for liposomes disrupted with 0.5% Triton-X 100. As the Ribogreen reagent does not penetrate liposomal membranes, the signal measured after treatment with Triton-X 100 represents the oligonucleotide concentration entrapped inside the liposomes. Concentrations of the samples were calculated based on calibration curves prepared with (1) oligonucleotides in the presence of empty liposomes and (2) oligonucleotides in the presence of empty liposomes and Triton-X 100. The encapsulation efficiency was calculated using the formula: % encapsulation = (ONt − ON0)/ONi × 100, where ONt = oligonucleotide concentration of Triton-X 100 treated liposomes, ON0 = oligonucleotide concentration of non-treated liposomes, and ONi = initial oligonucleotide concentration. Measurements were done at wavelengths of 480/520 nm.

#### 2.3.5. Light-Induced Oligonucleotide Release

The light-induced release of SSO or siRNA from the liposomes was determined by measuring the concentrations of non-treated liposomes, liposomes after light exposure, and liposomes treated with 0.5% Triton-X 100. The liposomes were diluted to 1 mM (lipid concentration) in buffer, heated to 37 ◦C on a thermomixer heating device and exposed to 808 nm light with the intensity of 370 mW/cm2 for 1 min. Control samples were kept at similar conditions, but were shielded from the light. Non-treated samples at +4 ◦C represented the background signal and Triton-X 100 treated samples were set at 100% release. The oligonucleotide concentrations were measured immediately after the light exposure using Quant-iT RiboGreen RNA Assay Kit as described in the previous section, and the release percentage was calculated using the formula: % released = (ONl − ON0)/(ONt − ON0) × 100, where ONl = oligonucleotide concentration of light exposed liposomes, ON0 = oligonucleotide concentration of non-treated liposomes, and ONt = oligonucleotide concentration of Triton-X 100 treated liposomes.

Both the SSO- and siRNA-encapsulated ICG liposomes were prepared and characterized as described above, while cell studies were performed using only the SSO-encapsulated liposomes. The purpose of the siRNA-encapsulated liposomes was to investigate if the light-triggered release of siRNA (*MW* ~ 14 kDa) differed from the release of SSO (*MW* ~ 7 kDa).

#### *2.4. Cell Studies*

#### 2.4.1. Cell Line

To study the ability of the light-activated liposomes to deliver oligonucleotides intracellularly, an EGFP-based splicing assay with HeLa S3 cells was used [29]. The assay was based on a construct where a C-to-T mutation at nucleotide 654 of the human β-globin intron-2 was inserted in the EGFP cDNA (IVS2-654), preventing correct translation of EGFP. Delivery of SSO (antisense oligonucleotide directed to position 654) blocked the aberrant splice site and restored the correct splicing of the EGFP precursor mRNA, generating properly translated EGFP. In this approach, antisense activity of SSO was directly proportional to up-regulation of EGFP in cells transfected with the IVS2-654 EGFP construct, thus providing a positive, quantitative readout.

#### 2.4.2. Cell Culture

HeLa S3 IVS2-654 EGFP cells were cultured in Dulbecco's Modified Eagle's Medium with high glucose supplemented with 10% (*v*/*v*) fetal bovine serum and 400 μg/mL G418 at 37 ◦C under a humidified atmosphere containing 5% CO2. Cells were routinely passaged twice a week and used for the experiments at passages 5–20. Cells were regularly tested for mycoplasma and found to be negative.

#### 2.4.3. Transfection Studies

HeLa S3 IVS2-654 EGFP cells were seeded on white μView clear-bottom 96-well plates (Greiner Bio-One B.V., Alphen aan de Rijn, The Netherlands) at a density of 9000 cells/well. After attachment (5–6 h after seeding), liposomes diluted in growth medium were added to the cells for overnight incubation. Next day, the liposomes were removed, the cells were washed with phosphate buffered saline, and the light triggering was performed. In the light triggering set-up, the cell culture plate was placed on a thermomixer heater to keep the temperature at 37 ◦C and the cells were exposed to 808 nm light, while control samples on the same plate were shielded from the light. After light exposure, the cells were transferred back to the culture incubator (37 ◦C under a humidified atmosphere containing 5% CO2). Transfection was typically measured either 24 or 48 h after light exposure (in some experiments, also the time points of 72 and 96 h were used). Detection was done by confocal imaging using a Cell Voyager CV7000s high-content confocal imager (Yokogawa, Tokyo, Japan). The nuclei were stained by incubating the cells in 2 μg/mL Hoechst 33342 (Life Technologies, Bleiswijk, The Netherlands) for 15 min at 37 ◦C. Imaging was done by acquiring 20–30 images with 3–4 z-stacks from each well using a 20× objective.

The effect of the following variables on transfection efficacy was investigated: (1) liposome concentration, (2) ICG concentration of the liposomes, (3) intensity of light, and (4) duration of light exposure. In addition, possible changes in transfection efficacy were followed for 48, 72, and 96 h after transfection and light triggering. Finally, the ability of free ICG or empty ICG liposomes in combination with free SSO to induce transfection upon light triggering was studied. Lipofectamine®3000 (Thermo Fisher Scientific, Waltham, MA, USA) was used as a positive control according to the manufacturer's protocol. As a negative control, an antisense oligonucleotide directed to position 705 of the cDNA was used [30]. This is an oligonucleotide of the same length as the active SSO, but complementary to a different position and will thus not result in altered splicing.

## 2.4.4. Cytotoxicity

Cytotoxicity of light exposure and/or the liposome formulation were measured by calculating Hoechs-labeled nuclei (2 μg/mL Hoechst 33342 for 15 min at 37 ◦C) from confocal images. Numbers of the treated cells were compared to numbers of cells without any treatment and reported as percentage values (cells without treatment set as 100%). Effect of light intensity (370–1500 mW/cm2), liposome concentration (0.5–1.4 mM, overnight incubation), and the combination of these were studied, and the cell numbers were measured at 48, 72, and 96 h after treatment.

#### 2.4.5. Image Analysis

Customized image analysis protocols were developed with Columbus Software (version 2.7.1; PerkinElmer Inc., Waltham, MA, USA). The determination of transfection efficacy was based on the intensity of EGFP fluorescence signal in cell cytoplasm. The percentage of transfected cells was obtained by setting a threshold value for the EGFP intensity and by separating the cell population using this value into transfected (intensity above the threshold) and non-transfected (intensity below the threshold) cells. The threshold was set to give less than 1 as the transfection percentage (<1% transfection efficacy) for the non-treated control cells. Details of the analysis can be

found from supplementary information (Figure S1). Cell numbers were determined by calculating Hoechst-labeled nuclei.

#### **3. Results**

#### *3.1. Characterization of the Oligonucleotide-Encapsulated ICG Liposomes*

The mean diameter of the liposomes was ~150 nm with a PDI < 0.15. The incorporation of ICG did not significantly affect the liposome size. Zeta-potential values of the liposomes without ICG were negative (~ −8 mW), and inclusion of ICG further lowered the zeta-potentials. The *Tm* values were 42.6–42.7 ◦C, and the incorporation of ICG lowered these values by 0.4–0.8 ◦C. Encapsulation efficiency of oligonucleotides was 5%–8%. Light-induced release of oligonucleotides was effective; approximately 90% of the SSO and siRNA were released after exposure to 808 nm light with the intensity of 370 mW/cm<sup>2</sup> for 1 min. Results on the characterization of the liposomes are shown in Table 1; Table 2 and in Figure 1. In general, there were no significant differences in the characteristics between the liposomes encapsulated with the SSO or siRNA or empty liposomes.

**Table 1.** Size, PDI, and encapsulation efficiency of the liposomes. SD, standard deviation (n = 3) and PDI, polydispersity index.


**Table 2.** Zeta-potential and transition temperature (Tm) of the liposomes. SD, standard deviation (*n* = 3).


**Figure 1.** Light-triggered release of splice switching antisense oligonucleotide (SSO) and siRNA from the indocyanine green (ICG) liposomes. The liposomes were kept at 37 ◦C and exposed to 808 nm light with the intensity of 370 mW/cm<sup>2</sup> for 1 min (light-triggered samples, orange columns) or shielded from the light (control samples, blue columns). The columns represent average values of released SSO or siRNA (*n* = 3) with error bars as standard deviation.
