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

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Abstract

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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ACKNOWLEDGMENTS

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.

Author information

Correspondence to E. L. Chernolovskaya.

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COMPLIANCE WITH ETHICAL STANDARDS

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

Conflict of Interests

Authors declare no conflict of interest.

Additional information

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.

Corresponding author: phone: +7 (383) 363-51-61; e-mail: elena_ch@niboch.nsc.ru.

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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). https://doi.org/10.1134/S1068162019060128

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Keywords:

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