## 3 Results
### Conjugates with Longer Lipidic Chains Accumulated in Cells with Higher Efficiency
were stained with Phalloidin-iFluor532 or Phalloidin-TRITC (actin microfilaments) and NucBlue (nuclei) (Figures 5-7). Firstly, to prove that LONs do not expose on the cell membranes but do penetrate into the cells, confocal microscopic analysis was performed and Z-stack images were built. It was demonstrated that LON 2n, the most efficient one according to flow cytometry data, was indeed localized in the cytosol of RAW264.7 cells and THP-1-induced macrophages (Figure 5). In addition, LON 1n was shown to efficiently accumulate in KB-8-5 cells (see Supplementary Materials, Figure S4). Figure 5: Intracellular localization of 2n LON in murine macrophages RAW264.7 (**A**) and THP-1-induced human macrophages (**B**). Analysis was performed by confocal fluorescent microscopy (\(\alpha\)Plan-Apochromat 100x/1.46 Oil DIC M27 objective) 24 h post transfection. Data are presented as orthogonal projections. Blue signal is DAPI (nuclear staining), green signal is fluorescein-labeled 2n LON, red signal is Phalloidin-iFluor532 (**A**) or Phalloidin-TRITC (**B**). Scale bar 10 μm. Figure 6: Intracellular accumulation of LONs in RAW264.7. Analysis of samples was performed 24 h after addition of fluorescein-labeled conjugates (5 μm) to the cells. Analysis was performed using \(\alpha\)Plan-Apochromat 100x/1.46 Oil DIC M27 objective. Three-channel (**BGR**) pictures were obtained using staining by DAPI (nuclei staining) (**B**); fluorescein-labeled LONs (**G**); Phalloidin-iFluor532 (cytoskeleton staining) (**R**). Scale bars 20 μm. ODN-F—control unmodified oligonucleotide labeled with fluorescein. It can be seen that the efficiency of intracellular accumulation of LONs according to confocal microscopy (Figures 6 and 7) correlate well with the obtained flow cytometry data (Figure 4). Oligonucleotides containing two lipophilic groups accumulated in cells with much higher efficiency in comparison with LONs containing only one group, LON 2\(\eta\) with two hexadecyl groups being the best penetrator. LONs 18-\(F\) and 1\(\eta\)-\(F\) containing only one lipophilic modification accumulated in cells with efficiency not significantly higher than that of the control unmodified oligonucleotide ODN-\(F\). However, these LONs penetrated into the cells whereas ODN-\(F\) mainly adhered to the cell membrane (Figure S5, Supplementary Materials). In detail, LONs were evenly distributed in the cytosol, localizing mainly in endosomes; this was conjectured from the presence of numerous bright green fluorescent dots containing LONs in the cytosol of both cell lines tested (Figures 6 and 7). Lipid-Conjugated RGMa siRNAs Demonstrated Gene-Silencing Ability Similar to Unmodified RGMa siRNA in PK-59 Human Pancreatic Carcinoma Cell Culture  
The gene silencing ability of lipid-conjugated siRNAs in comparison with unconjugated siRNA was elucidated in human pancreatic carcinoma PK-59 cells expressing repulsive guidance molecule a (RGMa). RGMa is a glycoprotein, which is a potent inhibitor of axonal re-myelination and, thus, is regarded as a potential therapeutic target for multiple sclerosis (MS) treatment [35]. Inhibition of RGMa by human monoclonal antibodies promotes neuronal regeneration and repair [36]. RGMa siRNA sequence was obtained from literature [29]. Lipid-conjugated RGMa siRNAs were modified at the 3'-end by either two (passenger strand only) or four (both passenger and antisense strands) dodecylphenyl (\(\Delta\)) or hexadecyl (H) groups. To compare silencing activity under similar conditions, transfection was carried out at concentrations of siRNA 1, 10 and 100 nM in the presence of Lipofectamine 2000. The levels of RGMa expression as assessed by RT-PCR are shown in Figure 8.  
Figure 7: Intracellular accumulation of LONs in human THP-1-induced macrophages. Analysis of samples was performed 24 h after addition of fluorescein-labeled conjugates (5 \(\mu\)M) to the cells. Analysis was performed using \(\alpha\)Plan-Apochromat 100x/1.46 Oil DIC M27 objective.