Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group

Abstract

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5′-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5′-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells

“…Likewise, the transport of ODNs first to and then out of the nucleus has already been reported [63]. These observations are also in agreement with the results achieved by other authors who reported that the efficiency of cellular accumulation of lipophilic siRNAs was dependent upon the type of cell line tested [29].…”

“…Cells were seeded at 1.25 × 10 5 cells/well in 24-well plates and incubated overnight in appropriate growth medium. Then cells were incubated in the presence of Cy5-or Alexa 488-labeled LOCs, ODN and ODN-lipoplexes at 37°C for 4 h. All these experiments were performed using OptiMEM because the transfection efficiency of several standard transfection reagents is reduced in the presence of serum [26][27][28][29][30]. After incubation, cells were completely dissociated in 0.5% Trypsin-EDTA (Invitrogen).…”

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (A). Mechanisms of interaction

“…Three different anticancer siRNAs and one scrambled siRNA were chosen from previously published results (Bellon, 2001;Braicu et al, 2013;Guoan et al, 2010;Jagani et al, 2011Jagani et al, , 2013Petrova et al, 2012). The sequences of the siRNAs are presented in the supporting information…”

Bioconjugates for targeted delivery of therapeutic oligonucleotides

“…Cholesterol-siRNA conjugates can form particles with lipoproteins in blood circulation, which increases circulation time and promotes uptake into the hepatocytes via lipoprotein receptors [39]. The lipid-siRNA conjugates have been further optimized by using new lipids such as α-tocopherol [40] and by controlling the length of the linker between siRNA and lipid [41]. …”