Investigation of the Internalization of Fluorescently Labeled Lipophilic siRNA into Cultured Tumor Cells

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The attachment of lipophilic molecules of natural origin, which have natural means for cell internalization, to small interfering RNA (siRNA) is an effective way of delivering siRNA to cells for biomedical purposes in vitro and in vivo. Earlier, we showed that the attachment of cholesterol to the 5'-end of the sense strand of nuclease-resistant siRNA through the optimized linker allows it to penetrate the cells and suppress the expression of the target gene. However, the effectiveness of the conjugates is different for cells of different origin, and in hematopoietic cells, they are not active, despite effective accumulation. In this work, we investigated the accumulation of fluorescently labeled cholesterol conjugates of siRNA using endocytosis inhibitors and showed that fluorescently labeled 5'-cholesterol conjugate of siRNAs penetrate KB-3-1 and K562 cells in several ways whose contribution differs depending on cell type and the presence of serum. In a serum-free medium, it was found that macropinocytosis and clathrin-dependent endocytosis contribute to the accumulation of the conjugate in KB-3-1 cells, while clathrin-dependent endocytosis makes the main contribution in K562 cells, while inhibitors of different types of endocytosis do not reduce the biological activity of the conjugate without a fluorescent label.

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  1. 1

    Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C., Nature, 1998, vol. 391, pp. 806–811.

  2. 2

    Chernikov, I.V., Vlassov, V.V., and Chernolovskaya, E.L., Front. Pharmacol., 2019, vol. 10, p. 444.

  3. 3

    Kanasty, R.L., Whitehead, K.A., Vegas, A.J., and Anderson, D.G., Mol. Ther.: J. Am. Soc. Gene Ther., 2012, vol. 20, pp. 513–524.

  4. 4

    Zatsepin, T.S., Kotelevtsev, Y.V., and Koteliansky, V., Int. J. Nanomed., 2016, vol. 11, pp. 3077–3086.

  5. 5

    Lorenz, C., Hadwiger, P., John, M., Vornlocher, H.P., and Unverzagt, C., Bioorg. Med. Chem. Lett., 2004, vol. 14, pp. 4975–4977.

  6. 6

    Nishina, K., Unno, T., Uno, Y., Kubodera, T., Kanouchi, T., Mizusawa, H., and Yokota, T., Mol. Ther.: J. Am. Soc. Gene Ther., 2008, vol. 16, pp. 734–740.

  7. 7

    Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., John, M., Kesavan, V., Lavine, G., Pandey, R.K., Racie, T., et al., Nature, 2004, vol. 432, pp. 173–178.

  8. 8

    Wolfrum, C., Shi, S., Jayaprakash, K.N., Jayaraman, M., Wang, G., Pandey, R.K., Rajeev, K.G., Nakayama, T., Charrise, K., Ndungo, E.M., Zimmermann, T., Koteliansky, V., Manoharan, M., and Stoffel, M., Nat. Biotechnol., 2007, vol. 25, pp. 1149–1157.

  9. 9

    Raouane, M., Desmaele, D., Urbinati, G., Massaad-Massade, L., and Couvreur, P., Bioconjugate Chem., 2012, vol. 23, pp. 1091–1104.

  10. 10

    Dassie, J.P., Liu, X.Y., Thomas, G.S., Whitaker, R.M., Thiel, K.W., Stockdale, K.R., Meyerholz, D.K., McCaffrey, A.P., McNamara, J.O., 2nd, and Giangrande, P.H., Nat. Biotechnol., 2009, vol. 27, pp. 839–849.

  11. 11

    Song, E., Zhu, P., Lee, S.K., Chowdhury, D., Kussman, S., Dykxhoorn, D.M., Feng, Y., Palliser, D., Weiner, D.B., Shankar, P., Marasco, W.A., and Lieberman, J., Nat. Biotechnol., 2005, vol. 23, pp. 709–717.

  12. 12

    Xia, C.F., Boado, R.J., and Pardridge, W.M., Mol. Pharm., 2009, vol. 6, pp. 747–751.

  13. 13

    Cuellar, T.L., Barnes, D., Nelson, C., Tanguay, J., Yu, S.F., Wen, X., Scales, S.J., Gesch, J., Davis, D., van Brabant, SmithA., Leake, D., Vandlen, R., and Siebel, C.W., Nucleic Acids Res., 2014, vol. 43, pp. 1189–1203.

  14. 14

    Hu, J., Xiao, F., Hao, X., Bai, S., and Hao, J., Mol. Ther. Nucleic Acids, 2014, vol. 3. e209.

  15. 15

    Thomas, M., Kularatne, S.A., Qi, L., Kleindl, P., Leamon, C.P., Hansen, M.J., and Low, P.S., Ann. N.Y. Acad. Sci., 2009, vol. 1175, pp. 32–39.

  16. 16

    Nair, J.K., Willoughby, J.L., Chan, A., Charisse, K., Alam, M.R., Wang, Q., Hoekstra, M., Kandasamy, P., Kel’in, A.V., Milstein, S., Taneja, N., O’Shea, J., Shaikh, S., Zhang, L., van der Sluis, R.J., et al., J. Am. Chem. Soc., 2014, vol. 136, pp. 16 958–16 961.

  17. 17

    Cesarone, G., Edupuganti, O.P., Chen, C.P., and Wickstrom, E., Bioconjugate Chem., 2007, vol. 18, pp. 1831–1840.

  18. 18

    Koehn, S., Schaefer, H.W., Ludwig, M., Haag, N., Schubert, U.S., Seyfarth, L., Imhof, D., Markert, U.R., and Poehlmann, T.G., J. RNAi Gene Silencing, 2010, vol. 6, pp. 422–430.

  19. 19

    Arthanari, Y., Pluen, A., Rajendran, R., Aojula, H., and Demonacos, C., J. Contr. Release, 2010, vol. 145, pp. 272–280.

  20. 20

    Brunzell, J.D., Davidson, M., Furberg, C.D., Goldberg, R.B., Howard, B.V., Stein, J.H., and Witztum, J.L., Diabetes Care, 2008, vol. 31, pp. 811–822.

  21. 21

    Brown, M.S. and Goldstein, J.L., Cell, 1997, vol. 89, pp. 331–340.

  22. 22

    Kruglova, I.S., Meshchaninova, M.I., Ven’iaminova, A.G., Zenkova, M.A., Vlasov, V.V., and Chernolovskaia, E.L., Mol. Biol. (Moscow), 2010, vol. 44, pp. 284–293.

  23. 23

    Petrova, N.S., Chernikov, I.V., Meschaninova, M.I., Dovydenko, I.S., Venyaminova, A.G., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., Nucleic Acids Res., 2012, vol. 40, pp. 2330–2344.

  24. 24

    Chernikov, I.V., Gladkikh, D.V., Meschaninova, M.I., Ven’yaminova, A.G., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., Mol. Ther. Nucleic Acids, 2017, vol. 6, pp. 209–220.

  25. 25

    Chernikov, I., Meschaninova, M., Venyaminova, A., Zenkova, M., Vlassov, V., and Chernolovskaya, E., J. Hematol. Oncol. Res., 2016, vol. 2, p. 13.

  26. 26

    Chernikov, I.V., Gladkikh, D.V., Meschaninova, M.I., Karelina, U.A., Ven’yaminova, A.G., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., Nucleic Acid Ther., 2019, vol. 29, pp. 33–43.

  27. 27

    Volkov, A.A., Kruglova, N.S., Meschaninova, M.I., Venyaminova, A.G., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., Oligonucleotides, 2009, vol. 19, pp. 191–202.

  28. 28

    Kotula, J.W., Pratico, E.D., Ming, X., Nakagawa, O., Juliano, R.L., and Sullenger, B.A., Nucleic Acid Ther., 2012, vol. 22, pp. 187–195.

  29. 29

    Ivanov, A.I., Methods Mol. Biol., 2008, vol. 440, pp. 15–33.

  30. 30

    Ly, S., Navaroli, D.M., Didiot, M.C., Cardia, J., Pandarinathan, L., Alterman, J.F., Fogarty, K., Standley, C., Lifshitz, L.M., Bellve, K.D., Prot, M., Echeverria, D., Corvera, S., and Khvorova, A., Nucleic Acids Res., 2017, vol. 45, pp. 15–25.

  31. 31

    Alam, M.R., Ming, X., Dixit, V., Fisher, M., Chen, X., and Juliano, R.L., Oligonucleotides, 2010, vol. 20, pp. 103–109.

  32. 32

    Osborn, M.F., Alterman, J.F., Nikan, M., Cao, H., Didiot, M.C., Hassler, M.R., Coles, A.H., and Khvorova, A., Nucleic Acids Res., 2015, vol. 43, pp. 8664–8672.

  33. 33

    Gilleron, J., Paramasivam, P., Zeigerer, A., Querbes, W., Marsico, G., Andree, C., Seifert, S., Amaya, P., Stoter, M., Koteliansky, V., Waldmann, H., Fitzgerald, K., Kalaidzidis, Y., Akinc, A., Maier, M.A., et al., Nucleic Acids Res., 2015, vol. 43, pp. 7984–8001.

  34. 34

    Crooke, S.T., Wang, S., Vickers, T.A., Shen, W., and Liang, X.H., Nat. Biotechnol., 2017, vol. 35, pp. 230–237.

  35. 35

    Wang, S., Allen, N., Vickers, T.A., Revenko, A.S., Sun, H., Liang, X.H., and Crooke, S.T., Nucleic Acids Res., 2018, vol. 46, pp. 3579–3594.

  36. 36

    Chen, Y.H., Lin, W.W., Liu, C.S., Hsu, L.S., Lin, Y.M., and Su, S.L., PLoS One, 2014, vol. 9. e71862.

  37. 37

    Shukla, R.S., Jain, A., Zhao, Z., and Cheng, K., Nanomedicine, 2016, vol. 12, pp. 1323–1334.

  38. 38

    Bartlett, D.W. and Davis, M.E., Nucleic Acids Res., 2006, vol. 34, pp. 322–333.

  39. 39

    Bellon, L., Curr. Protoc. Nucleic Acid Chem., 2001.

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The authors would like to thank A.V. Vladimirova for cell maintenance.

This work was supported by the Russian Foundation for Basic Research grant no. 17-04-01100 and the Russian State-Funded Budget Project 2013–2020 AAAA-A17-117020210024-8.

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Correspondence to E. L. Chernolovskaya.

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This article does not contain any research involving humans and animals as research objects.

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Authors declare no conflict of interest.

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Translated by P. Vikhreva

Abbreviations: siRNA, small-interfering RNA; MDC, monodansylcadaverine; EIPA, 5-(N-Ethyl-N-isopropyl)amiloride; CDE, clathrin-dependent endocytosis; LR, lipid rafts; MPC, macropinocytosis; Nm, 2'-O-methyl nucleotide analog; PBS, phosphate buffered saline; FBS, Fetal bovine serum; RISC, RNA-induced silencing complex; RFU, relative fluorescence units; ASO, antisense oligonucleotide, PMT, photomultiplier tube.

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Chernikov, I.V., Karelina, U.A., Meschaninova, M.I. et al. Investigation of the Internalization of Fluorescently Labeled Lipophilic siRNA into Cultured Tumor Cells. Russ J Bioorg Chem 45, 766–773 (2019).

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  • siRNA
  • chemical modifications
  • cholesterol
  • conjugate
  • cell delivery mechanism
  • inhibitors of endocytosis