**4. Discussion**

The light-triggered liposome formulation used in this work is based on our previous studies on ICG liposomes [16,17], but ICG was now incorporated into the liposome dispersion. This approach avoided any interference of ICG on oligonucleotide encapsulation. As an amphiphilic compound, ICG can associate with the hydrophilic surface layer of liposomes or penetrate the hydrophobic lipid bilayer. The post-inserted ICG may be partly dissolved in the lipid bilayer, because the *Tm* values of liposomes with ICG were slightly lower than the *Tm* values of liposomes without ICG (42.6–42.7 ◦C and 41.9–42.3 ◦C for liposomes without and with ICG, respectively, Table 2). Based on molecular modeling simulations [16,31], ICG molecules can also bind to the PEG chains on the surface of the lipid bilayers. Thus, ICG is likely to be partly solubilized in the lipid bilayer and interacts with its more polar regions with surface-grafted PEG.

Most studies on liposomal oligonucleotide delivery systems have utilized either cationic or ionizable lipids. We chose non-cationic lipids for two reasons. Firstly, we wanted to specifically investigate the light-activated delivery. Since cationic as well as ionizable lipids were known to interact with cell membranes and in this way induced intracellular delivery and release [32–34], we preferred to study the light-activated process with neutral lipids and exclude the possible effects of charged lipids. Secondly, compared to cationic liposomes, neutral liposomes were less toxic, more stable, and showed improved pharmacokinetics [35–37]. However, neutral liposomes were generally less efficient in intracellular delivery and transfection, and their endosomal escape and intracellular release properties were poor [38,39]. We aimed to improve the drawbacks of neutral liposomes with light-induced release of oligonucleotides.

We demonstrated effective light-induced release and cell transfection with the ICG liposomes. ICG was required for the light-induced contents' release from the liposomes (Figure S5), and the ICG dose was the major factor influencing the transfection efficacy (Figure 3A–D). The oligonucleotide and lipid concentrations (Figure 3E,F) and extent of light exposure (Figure 4) were less important. The liposomes efficiently released both the encapsulated SSO (*MW* ~ 7 kDa) and siRNA (*MW* ~ 14 kDa) upon light activation (Figure 1), suggesting that the system was functional with oligonucleotides of different sizes. Light exposure did not affect the activity of the SSO (Figure S3).

Interestingly, free ICG and SSO in solution also led to transfection upon light activation (Figure 5), indicating that the intracellular delivery of ICG and light was not dependent on the liposomal components. However, light triggering without ICG did not lead to any detectable transfection. Hence, ICG is a necessary component for the light-triggered delivery, while the liposomes are optional. The use of non-liposomal, free ICG and oligonucleotides can be applied as a simple delivery system to certain tissues. For example, in the treatment of eye diseases, ICG and oligonucleotides may be injected into the vitreous (to treat posterior eye diseases) or instilled topically (to treat corneal diseases). The small size of naked oligonucleotides and ICG compared to liposomes may be beneficial for penetration into certain targets. The combination of ICG and oligonucleotides to a liposomal formulation has, however, certain advantages. Firstly, this approach avoids problems related to differences in clearance kinetics of oligonucleotides versus ICG. Secondly, it ensures that ICG and the oligonucleotides are taken up by the same cell, which is a requirement for the light-induced transfection. Thirdly, liposomes stabilize ICG in vivo [40,41]. Fourthly, liposomes can be targeted to selected cells by attaching suitable ligands on their surface [42]. Dual targeting with ligands and light triggering could maximize the therapeutic effect in target cells and minimize the exposure of non-target cells.

In general, oligonucleotides should cross at least two cellular membranes to reach their sites of action inside cells in the cytosol or in the nucleus: firstly, they should permeate the cellular membrane surrounding the cell (cellular uptake) and secondly, the endosomal membrane after cellular uptake (endosomal escape). As large and anionic molecules, naked oligonucleotides are not easily taken up by negatively charged cells. Further, the endocytosed oligonucleotides are not released from endosomes, but transferred to lysosomes, where they are degraded enzymatically [43–45]. Cationic and ionizable lipids interact with negatively charged cellular membranes inducing cellular uptake and endosomal escape [32,46]. With ICG and light triggering, the mechanism must be different, as transfection takes place without cationic lipids or even without any delivered lipids (free ICG, Figure 5). A possible mechanism for the increased cellular uptake and endosomal escape induced by ICG and light is partial fluidization of the cellular membranes, allowing oligonucleotides to permeate and cross the

cellular lipid bilayers. This hypothesis is supported by the ability of free ICG without lipids to induce transfection, while in the absence of ICG, no transfection takes place (Figure 5B).

We suggest that the increased fluidity of cell membranes and consequent increase in cellular permeability by this delivery system is dependent on two factors: Firstly, the ability of ICG to diffuse into cellular membranes, and this way, to create leakiness. Secondly, released heat of ICG after light exposure causes changes in the organization of lipid bilayers, leading to fluidization of the membranes. The first factor is supported by the ability of free ICG and ICG liposomes to induce some transfection in the absence of light (20% transfection efficacy without light exposure in Figure 3D,F). Yet, the major effect on cellular membrane permeability and successful transfection is most likely the effect of increased temperature after light triggering. We have previously shown using gold nanoparticle-containing liposomes that light activation increases endosomal escape and intracellular release [47]. It is, therefore, likely that a similar mechanism is involved in the endosomal release of oligonucleotides with ICG liposomes and light.

The light exposure or liposome administration separately did not affect cell viability (Figure 7A,B). However, the combination of these treatments reduced cell numbers indicating cytotoxicity (Figure 7C). In general, the observed toxic effects were mostly dependent on the ICG dose. Yet, the effects on cell viability were transient, as the cells reverted to normal state in 4 days (Figure 7C). Apparently, the transfection process causes stress for the cells, leading to slower growth and decreased viability for a certain period. Importantly, the percentage of transfected cells also increased over time (Figure 6). This proves that the observed increase in cell numbers was not solely dependent on growth of the cells that were non-transfected, but also on the transfected cells as they remained viable. A likely mechanism for the cytotoxicity is the local heat production by ICG after light exposure, causing toxic hyperthermia for the cells. However, no increased temperature in the medium surrounding the cells could be detected after treatment with ICG liposomes and light triggering (data not shown). We assume that the quick and transient increase in temperature produced by ICG [18] leads to highly localized temperature effects not detectable in the bulk surroundings.

As such, this light-activated delivery system is potential for the local delivery of oligonucleotides into tissues that can be exposed to light, including the eye, skin, lungs, the gastrointestinal tract, and tumours. The system is excited with NIR light of 800 nm that penetrates deep into tissues [28]. Moreover, utilization of fiberoptic technologies enables light exposure of tissues that cannot be reached by superficial illumination. For example, the posterior segment of the eye is an interesting target. Retinal disorders are the leading causes of impaired vision and blindness, but drug delivery to this tissue is challenging [48,49]. Since transparent ocular tissues allow straightforward light exposure of the posterior eye and neutral nanoparticles diffuse in the vitreous without aggregation, the light-activated liposomes might be suitable for the delivery of therapeutic oligonucleotides in the posterior eye tissues via intravitreal injection [17,50,51]. Another potential application is cancer therapy, where the site- and time-controlled delivery could be used to reduce the off-target effects of toxic anti-cancer drugs. The treatments with ICG-containing liposomes can be further optimized by utilizing the detection of ICG fluorescence in vivo, allowing imaging-guided drug delivery.

#### **5. Conclusions**

The present study describes a light-induced method for the cellular delivery of oligonucleotides. The light-triggered ICG liposomes released and delivered oligonucleotides into cultured cells. Interestingly, free ICG also facilitated oligonucleotide delivery to cells in the presence of low intensity NIR light. This method of oligonucleotide delivery avoids the need for cationic lipids that are often accompanied with the activation of innate immune pathways, and at high doses even hepatotoxicity. As such, the light-triggered liposomes might be a good system for local delivery of therapeutic oligonucleotides at places where light penetration is not limited.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/2/90/s1, Table S1: Intensity outputs of the laser with different power settings, Figure S1: Analysis file exported from

Columbus software used to determine the transfection percentages of HeLa S3 IVS2-654 EGFP cells, Figure S2: Transfection efficacy of the negative control oligonucleotide in HeLa S3 IVS2-654 EGFP cells, Figure S3: Effect of light exposure on transfection efficacy of Lipofectamine-SSO complexes in HeLa S3 IVS2-654 EGFP cells, Figure S4: Transfection efficacy and cytotoxicity of Lipofectamine-SSO complexes in HeLa S3 IVS2-654 EGFP cells, Figure S5: Light-triggered release of SSO and siRNA from temperature-sensitive liposomes without ICG.

**Author Contributions:** Conceptualization, L.-S.K., A.U. and E.M.; methodology, L.-S.K. and E.M.; validation, L.-S.K.; formal analysis, L.-S.K.; investigation, L.-S.K. and J.v.d.D.; data curation, L.-S.K.; writing—original draft preparation, L.-S.K.; writing—review and editing, A.U., W.E.H., and E.M.; visualization, L.-S.K.; supervision, E.M. and W.E.H.; funding acquisition, L.-S.K., A.U., W.E.H., and E.M.

**Funding:** This research was funded by the Academy of Finland, projects 294315 and 307088.

**Acknowledgments:** Arto Urtti acknowledges the support by the Government of Russian Federation Mega-Grant 14.W03.031.0025 "Biohybrid technologies for modern biomedicine".

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
