*3.7. Cellular Uptake of Three Dodecyl-Containing Conjugates*

We also examined the cellular accumulation of FAM-labeled lipophilic oligonucleotide conjugates bearing three dodecyl groups (FAM-D-17PG) or their duplex (D-17PG/FAM-17- ). The concentration of oligonucleotides was determined by CAC to ensure that oligonucleotide derivatives present in an aggregated state in the transfection solution. As shown above, the addition of BSA or FBS to the DOC aggregates leads to the protein binding of the lipophilic conjugate (Figure 5). To define the influence of BSA or other serum proteins on cellular internalization of DOCs, the transfection of HepG2 cells was performed with 5 μM concentrations of conjugated oligonucleotide in DMEM, either alone or supplemented with 10% FBS or 30 μM BSA. Prior to transfection procedure, we verified the stability of FAM-D-17PG conjugate and D-17PG/FAM-17 duplex in 10% FBS (Figure S22). The efficacy of transfection was evaluated using flow cytofluorometry. The results demonstrated that DOCs penetrate the human cells with different efficacy, depending on the medium (Figure 7).

**Figure 7.** Cellular uptake of fluorescently labelled DOC (FAM-D-17PG) and its duplex (D-17PG/FAM-17- ) estimated by flow cytometry after 4 h incubation of HepG2 cells with conjugates and additional followed 14 h without it: (**a**) percentage of FAM-positive HepG2 cells in the population; (**b**) normalized median value of the cell fluorescence to the autofluorescence of control cells. The highest transfection efficacy was achieved for cells treated with FAM-D-17PG in DMEM (Figure 7a,b). All cells were transfected (Figure 7a), and the median value of their fluorescence intensity exceeded that of cells' auto-fluorescence 11 ± 1 times (Figure 7b). High efficacy of transfection was also reached for cells transfected with duplex D-17PG/FAM-17 in DMEM (Figure 7a). Surprisingly, the median value of cells' fluorescence was almost two times lower than for cells treated with a single stranded dodecyl-containing oligonucleotide (Figure 7b).

We attributed the difference in cells' fluorescence intensity to the different numbers of fluorescent groups in the aggregates. The formation of micellar structures of similar size with oligonucleotides exposed on surface and dodecyl groups forming a hydrophobic core requires a comparable number of oligonucleotide molecules. This comes from the fact that the size of oligonucleotide or duplex determines the curvature of the particle's surface and, therefore, its diameter. However, in the duplex, only one strand bears a fluorescent group, so the aggregate formed by a duplex with a diameter of 85.4 ± 17.0 nm (DLS data in PBS) contain twice less FAM groups than the aggregate formed by single strand conjugate with a diameter of 96.9 ± 47.8 nm.

The presence of BSA or BSA-containing FBS in the transfection media decreases the transfection efficacy (Figure 7a) and the accumulation of the oligonucleotide cargo in the cell (Figure 7b). We explain this result by the fact that aggregates formed by both oligonucleotides conjugated with three dodecyl groups and their duplexes appear in the BSA/DOCs associates (Figure 5, Lanes 7,11). The obtained data suggest that binding to albumin inhibits DOC's cell penetration since accumulation rates of oligonucleotide-albumin complexes are lower than those for aggregates or linear nucleic acids. Surprisingly, the efficacy of FAM-D-17PG accumulation in cells in the presence of FBS is higher than in the presence of BSA only (Figure 7a). This difference can be explained by the presence of other lipids in FBS, including fatty acids, which can compete with conjugate's dodecyl groups for interaction with

albumin [34,44,52]. In this case, the fraction of unbound FAM-D-17PG released from protein/DOCs associates can penetrate cells. At the same time, D-17PG/FAM-17 duplex in the presence of FBS demonstrated results comparable to those for BSA containing transfection. The reason for the difference between single stranded molecules of FAM-D-17PG and D-17PG/FAM-17 duplexes in this context is a larger negative net charge of the duplex [33].

The most intensive fluorescence was registered for the cells treated with DMEM-diluted FAM-D-17PG or D-17PG/FAM-17- (Figure 8a,d). Fluorescent signals of the medium intensity were found in cells transfected with FAM-D-17PG dissolved in DMEM supplemented with 10% FBS (Figure 8b); for other samples, FAM signals were too low (Figure 8c,e,f). In FAM-positive cells, a fluorescent signal was evenly distributed throughout the cytoplasm, and no co-localization with nuclei was revealed.

**Figure 8.** Qualitative (confocal fluorescent microscopy imaging) and quantitative (flow cytometry data) characterizations of HepG2 cells treated with 5 μM fluorescently labeled either FAM-D-17PG (**a**–**c**) DOC or D-17PG/FAM-17- (**d**–**f**) duplex in DMEM medium (**a**,**d**); in DMEM supplemented with 10% FBS (**b**,**e**); in DMEM supplemented with 30 μM BSA (**c**,**f**). The red bar in confocal fluorescent images corresponds to 20 μm. Our experiments are consistent with a previous study reporting micelle formation for other DNA-based amphiphilic conjugates depended on the presence of Mg2<sup>+</sup> [15,16]. In another study, the authors also suggested that Mg2<sup>+</sup> stabilizes all the lipid-oligonucleotides micellar assemblies, probably due to the Mg2<sup>+</sup>-mediated neutralization of the oligonucleotide negative charges [53].

These results indicate that oligonucleotides conjugated with three dodecyl groups can penetrate cultured human cells both as disassembled molecules and in aggregated form.
