## 3 Results
### Conjugates with Longer Lipidic Chains Accumulated in Cells with Higher Efficiency  
The ability of LONs to accumulate in eukaryotic cells in the absence of a transfection agent (gymnosis) was studied by flow cytometry using fluorescein-labeled LONs and murine macrophages RAW264.7 or THP-1-induced human macrophages. The efficiency  
Figure 3: Dose-response curves of 2\(\AA\) and 2n LONs for RAW264.7 (**A**,**B**) and human THP-1 induced macrophages (**C**,**D**). Cells were incubated with LONs in a complete medium for 24 h. The results are shown as a percentage of viable cells observed after cell incubation with LON relative to untreated cells (100%). All experimental points were run in triplicate for statistical analysis. Data are presented as MEAN \(\pm\) SD. The dotted curve shows extrapolation to 50% of viable cells. Figure 2: Hydrodynamic diameters of nanoparticles formed spontaneously in water by 2\(\AA\) and 2n (**B**) lipid-oligonucleotide conjugates. Dynamic light scattering (DLS) data. of intracellular accumulation of LONs was assessed by two parameters: the number of fluorescein-positive cells in the population and average level of the conjugate content in the cells (mean fluorescence intensity, MFI). Accumulation of LONs in RAW264.7 and THP-1-induced macrophages differed considerably. LONs accumulated in RAW264.7 cells with moderate efficiency: 20-50% of cells were transfected with a MFI of 6-11 \(\times\) 10\({}^{3}\) RFU (Figure 4A,B). As expected, the cells incubated and control oligonucleotide ODN-\(F\) displayed the lowest efficiency: 10% of fluorescent cells with MFI of 4 \(\times\) 10\({}^{3}\) RFU, which corresponded to the baseline level of oligonucleotide taken up by the macrophages and/or adhered to the cell surface. We observed that more hydrophobic LONs, that is, the ones containing either two hydrocarbon chains instead of only one, or those having a longer aliphatic group: hexadecyl instead of dodecylphenyl, were showing more efficient accumulation in RAW264.7 cells (Figure 4A,B: compare conjugates having two vs. one group, and \(\eta\)- vs. \(\delta\)-modified LONs) than their less hydrophobic counterparts and unmodified control ODN-\(F\). A similar but much more prominent accumulation of lipid-conjugated oligodeoxynucleotides was observed in human THP-1-induced macrophages. LONs accumulated in these cells with higher efficiency both in terms of the number of transfected cells and mean fluorescence intensity (Figure 4C,D). Conjugates 1\(\delta\)-\(F\) and 1\(\eta\)-\(F\) containing one lipophilic group displayed similar transfection efficiency regardless of the type of lipid chains, either dodecylphenyl (8) or hexadecyl (\(\eta\)): 43% cells were transfected with MFI of 8 \(\times\) 10\({}^{3}\) RFU, which was fourfold higher as compared to the control ODN-\(F\) (Figure 4C,D). Increasing the number of lipophilic groups from one to two resulted in a significant enhancement of transfection efficiency. 28 LON yielded 85% cells with fluorescence intensity 25 \(\times\) 10\({}^{3}\) RFU whereas 2\(\eta\) conjugate was taken up with the highest efficiency, accumulating in nearly 100% cells with fluorescence intensity 150 \(\times\) 10\({}^{3}\) RFU (Figure 4C,D), which is on par with the level achieved by liposome-mediated oligonucleotide delivery [34]. Lipid-Oligonucleotide Conjugates are Efficiently Taken up by Cells in the Absence of Transfection Agents and Localize Mostly in the Cytoplasm within Endosomes  
Confocal microscopic analysis was carried out to verify and characterize intracellular localization of LONs. RAW 264.7 and human THP-1-induced macrophages were incubated with 5.0 \(\mu\)M fluorescein-labeled LONs in complete medium for 24 h. After incubation, cells  
Figure 4: Accumulation of fluorescein-labeled LONs in murine macrophages RAW264.7 (**A,B**) and THP-1-induced human macrophages (**C,D**). The percentage of fluorescent cells (**A,C**) and mean fluorescence intensity (**B,D**) were measured by flow cytometry 24 h post transfection. Data are presented as MEAN \(\pm\) SD.