## 4 Discussion  
A previously developed methodology of Staudinger reaction-mediated in-line oligonucleotide modification [26; 27; 28] was employed herein to obtain lipophilic conjugates of oligodeoxynucleotides and siRNAs carrying one or more extended hydrocarbon chains, 4-dodecylphenyl or hexadecyl, attached to the internucleotic phosphate groups via the (sulfonyl)phosphoridate linkage, which is hydrolytically stable and carries negative charge under physiological conditions (Figure 1A,B). The scheme allows one to modify any internucleotic or terminal phosphate group within a DNA or 2'-OMe RNA chain from the 5'- to 3'-end by a facile chemical reaction with a stable sulfonyl azide precursor during automated DNA/RNA assembly without recourse to a special phosphormidite monomer, which is usually obtained by a multi-step synthesis [18; 20; 21]. The obtained lipid-oligonucleotide conjugates (LONs) can be conveniently isolated by the usual C18 reverse-phased HPLC, due to significant difference in retention times with unmodified oligonucleotides (see Supplementary Materials) for HPLC profiles and ESI-MS spectra). Our preliminary data have shown that such lipophilic groups are fully compatible with other phosphate group modifications such as phosphorothioate, mesyl phosphoramidate [27; 37; 38] and phosphoryl guanidine [39]. Notably, we have observed spontaneous nanoparticle formation in aqueous solution by LONs containing two adjacent hydrophobic chains, with nanoparticle size dependent on the type of the side-chain (Figure 2). Nanoparticle formation by oligonucleotides is thought to be essential for good cellular uptake [40], structured RNA studies [41] and design of stimuli-responsive drug delivery systems [42]. Low cytotoxicity is another beneficial characteristic of such LONs, which allows one to vary the concentration of an oligonucleotide over a wide range to optimize its therapeutic dosage for further in vitro and in vivo studies. We have confirmed that LONs  
Figure 8: Inhibition of RGMa expression in PK-59 human pancreatic carcinoma cell culture by lipid conjugates of RGMa siRNAs vs. unmodified siRNA (RGMa). The conjugates were modified near the 3’-end by two (passenger chain only) or four (both chains) lipophilic groups (\(\Delta\)—dodecylphenyl, H—hexadecyl). used in this study evinced low cytotoxicity. Calculated IC\({}_{50}\) of studied LONs fell within the 75 to 120 \(\upmu\)M range, which was significantly higher than concentrations of antisense oligonucleotides targeting therapeutically relevant RNAs commonly used in vitro and in vivo: nanomolar concentrations [43; 44; 45; 46] or <5 \(\upmu\)M [47] or therapeutic doses of siRNAs and antisense oligonucleotides approved by FDA and EMA for clinical practice: for siRNAs 21 nmol/kg for Patisiran [48] and 153 nmol/kg for Givosiran [49]; for antisense oligonucleotides <5 \(\upmu\)moles/kg for Giodorisen [50], Volanesorsen [51] and Indersen [52]. It is worth noting that the increase in hydrophobicity of the conjugates have no influence on their cytotoxicity (Figure 3). This result prompts us to expect that the increase of the number of side-chains or introduction of more hydrophobic groups, for example, hydrocarbon chains longer than 16 carbon atoms into LONs will not increase significantly the overall cytotoxicity of conjugates. We demonstrated that LONs accumulated in the cells with intracellular distribution analogous to that of oligonucleotides delivered by liposomal formulations (Figures 4 and 5) [53; 54; 55]. It appears that LONs penetrated through the outer membrane and localized in cytosol predominantly in endosomes 24 h after addition (Figures 6 and 7). It is known that endosomal escape of therapeutic oligonucleotides is one of the rate-limiting steps of their delivery and the key to successful intracellular performance of nucleic acid therapeutics [56].