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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.

Petrova NS, Chernikov IV, Meschaninova MI, Dovydenko IS, Venyaminova AG, Zenkova MA, Vlassov VV, Chernolovskaya EL - Nucleic Acids Res. (2011)

Bottom Line: A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev ave., 8, Novosibirsk 630090, Russia.

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.

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Silencing of P-gp expression in KB-8-5 cells by non-modified or cholesterol-containing siRNAs. (A) Kinetics of P-gp level suppression following incubation with cholesterol-modified siRNAs or transfection of siRNA with Lipofectamine 2000. The data were obtained via western blotting carried out after 3–8 days of incubation followed by data quantitative analysis by GelPro 4.0. program (Media Cybernetics, USA). The human β-actin protein was used as an internal standard. Data were normalized to the ratio of the P-gp/β-actin levels in untreated cells (control). Mean values (±SD) from three independent experiments are shown. (B) Levels of P-gp in KB-8-5 cells at Day 5 post-transfection. An untreated sample of KB-8-5 cells was taken as a control.
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gkr1002-F5: Silencing of P-gp expression in KB-8-5 cells by non-modified or cholesterol-containing siRNAs. (A) Kinetics of P-gp level suppression following incubation with cholesterol-modified siRNAs or transfection of siRNA with Lipofectamine 2000. The data were obtained via western blotting carried out after 3–8 days of incubation followed by data quantitative analysis by GelPro 4.0. program (Media Cybernetics, USA). The human β-actin protein was used as an internal standard. Data were normalized to the ratio of the P-gp/β-actin levels in untreated cells (control). Mean values (±SD) from three independent experiments are shown. (B) Levels of P-gp in KB-8-5 cells at Day 5 post-transfection. An untreated sample of KB-8-5 cells was taken as a control.

Mentions: The silencing activity of the cholesterol-conjugated siMDR was investigated using drug resistant KB-8-5 cells. These cells are capable of growing in the presence of 300 nM vinblastine, due to the over-expression of the MDR1 gene. The level of the MDR1 gene product–P-gp was estimated by western blotting after 3–8 days of incubation of cells with 5 µM conjugates in the absence of cytostatic in order to avoid the negative selection of living cells. The start time point (3 days) was chosen taking into account the half-life time of P-gp: 48–72 h (50). The level of β-actin in the same samples was used as an internal standard. Scrambled siRNA (siScr) and its lipophilic analogs were used as controls of specificity. The levels of P-gp were normalized to the levels of β-actin for data presentation (Figure 5A and B). It was found that the efficiency of the MDR1 gene silencing increased when the linker length augmented from 0 to 6-10 carbon atoms and correlated with the efficacy of cellular accumulation of the lipophilic siRNAs. The further increase of linker length, to aminododecyl, resulted in a decrease of the silencing activity and retardation of P-gp level reduction (Figure 5A). The 40% P-gp suppression was observed at Day 4 after addition of Ch6-siMDR to the cells, the action of Ch8-, Ch10- and Ch12-siMDR was less pronounced (30, 25 and 18% reduction of P-gp level, respectively) and was detected at the same time point, the levels of the P-gp in the cells incubated with Ch0- and Ch3-siMDR did not differ from the controls. The maximum levels of P-gp silencing were detected in the cells incubated with cholesterol-containing siMDRs with linkers of 3–10 carbon atoms for 5 days (60% for Ch6-, Ch8- and Ch10-siMDRs and 50% for Ch3-siMDR) (Figure 5). It is noteworthy that there is no statistically relevant difference between the silencing activities of conjugates containing linkers with 3-10 carbon atoms; whereas Ch3-siMDR has a tendency to be less efficient than others. Ch12-siMDR was reliably less efficient than Ch6-, Ch8- and Ch10-siMDRs: this conjugate induced only 30% reduction of the P-gp level after 4 days of incubation (P < 0.02, Ch12-siMDR versus Ch6-, Ch8- or Ch10-siMDR). At Day 6 of incubation the silencing activities of Ch6-, Ch8- and Ch10-siMDRs were similar (Figure 5A). In contrast, the silencing activity of Ch12-siMDR increased to 57% and did not differ to a reliable degree from the activity of Ch3-, Ch6- and Ch10-siMDRs at Day 6, the activity of Ch8-siMDR was slightly higher (P < 0.05, Ch8-siMDR versus Ch12-siMDR). The silencing effect decreased at days 7–8 and did not exceed 15–20% for these conjugates (Figure 5A). Ch0-siMDR was inactive. Hence, the linker length between cholesterol residue and siRNA is critical for the biological activity of the conjugates.Figure 5.


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.

Petrova NS, Chernikov IV, Meschaninova MI, Dovydenko IS, Venyaminova AG, Zenkova MA, Vlassov VV, Chernolovskaya EL - Nucleic Acids Res. (2011)

Silencing of P-gp expression in KB-8-5 cells by non-modified or cholesterol-containing siRNAs. (A) Kinetics of P-gp level suppression following incubation with cholesterol-modified siRNAs or transfection of siRNA with Lipofectamine 2000. The data were obtained via western blotting carried out after 3–8 days of incubation followed by data quantitative analysis by GelPro 4.0. program (Media Cybernetics, USA). The human β-actin protein was used as an internal standard. Data were normalized to the ratio of the P-gp/β-actin levels in untreated cells (control). Mean values (±SD) from three independent experiments are shown. (B) Levels of P-gp in KB-8-5 cells at Day 5 post-transfection. An untreated sample of KB-8-5 cells was taken as a control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3299988&req=5

gkr1002-F5: Silencing of P-gp expression in KB-8-5 cells by non-modified or cholesterol-containing siRNAs. (A) Kinetics of P-gp level suppression following incubation with cholesterol-modified siRNAs or transfection of siRNA with Lipofectamine 2000. The data were obtained via western blotting carried out after 3–8 days of incubation followed by data quantitative analysis by GelPro 4.0. program (Media Cybernetics, USA). The human β-actin protein was used as an internal standard. Data were normalized to the ratio of the P-gp/β-actin levels in untreated cells (control). Mean values (±SD) from three independent experiments are shown. (B) Levels of P-gp in KB-8-5 cells at Day 5 post-transfection. An untreated sample of KB-8-5 cells was taken as a control.
Mentions: The silencing activity of the cholesterol-conjugated siMDR was investigated using drug resistant KB-8-5 cells. These cells are capable of growing in the presence of 300 nM vinblastine, due to the over-expression of the MDR1 gene. The level of the MDR1 gene product–P-gp was estimated by western blotting after 3–8 days of incubation of cells with 5 µM conjugates in the absence of cytostatic in order to avoid the negative selection of living cells. The start time point (3 days) was chosen taking into account the half-life time of P-gp: 48–72 h (50). The level of β-actin in the same samples was used as an internal standard. Scrambled siRNA (siScr) and its lipophilic analogs were used as controls of specificity. The levels of P-gp were normalized to the levels of β-actin for data presentation (Figure 5A and B). It was found that the efficiency of the MDR1 gene silencing increased when the linker length augmented from 0 to 6-10 carbon atoms and correlated with the efficacy of cellular accumulation of the lipophilic siRNAs. The further increase of linker length, to aminododecyl, resulted in a decrease of the silencing activity and retardation of P-gp level reduction (Figure 5A). The 40% P-gp suppression was observed at Day 4 after addition of Ch6-siMDR to the cells, the action of Ch8-, Ch10- and Ch12-siMDR was less pronounced (30, 25 and 18% reduction of P-gp level, respectively) and was detected at the same time point, the levels of the P-gp in the cells incubated with Ch0- and Ch3-siMDR did not differ from the controls. The maximum levels of P-gp silencing were detected in the cells incubated with cholesterol-containing siMDRs with linkers of 3–10 carbon atoms for 5 days (60% for Ch6-, Ch8- and Ch10-siMDRs and 50% for Ch3-siMDR) (Figure 5). It is noteworthy that there is no statistically relevant difference between the silencing activities of conjugates containing linkers with 3-10 carbon atoms; whereas Ch3-siMDR has a tendency to be less efficient than others. Ch12-siMDR was reliably less efficient than Ch6-, Ch8- and Ch10-siMDRs: this conjugate induced only 30% reduction of the P-gp level after 4 days of incubation (P < 0.02, Ch12-siMDR versus Ch6-, Ch8- or Ch10-siMDR). At Day 6 of incubation the silencing activities of Ch6-, Ch8- and Ch10-siMDRs were similar (Figure 5A). In contrast, the silencing activity of Ch12-siMDR increased to 57% and did not differ to a reliable degree from the activity of Ch3-, Ch6- and Ch10-siMDRs at Day 6, the activity of Ch8-siMDR was slightly higher (P < 0.05, Ch8-siMDR versus Ch12-siMDR). The silencing effect decreased at days 7–8 and did not exceed 15–20% for these conjugates (Figure 5A). Ch0-siMDR was inactive. Hence, the linker length between cholesterol residue and siRNA is critical for the biological activity of the conjugates.Figure 5.

Bottom Line: A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev ave., 8, Novosibirsk 630090, Russia.

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.

Show MeSH
Related in: MedlinePlus