Supercationic peptide dendrimers as vectors for nucleic acids delivery to mammalian cells


Introduction. The usage of nucleic acids (NA) for modulation of gene expression or introduction new genes is a cardinal trend in modern biomedical science. However, such manipulations require the use of efficient delivery vehicles that could provide transmembrane transfer, nucleotide chain stability, and delivery vehicles themselves. Promising delivery vehicles for NA described in this work are supercationic dendrimeric peptides (СDPs), which have a branched structure due to the presence of ε-amide bonds and are relatively stable under biological conditions.

The aim of the study – design and synthesis of non-toxic CDPs which, as we expect, will be capable to penetrate efficiently into mammalian cells and stimulate transfection of eukaryotic cultures with model DNA plasmids. Other aim was to access the relationship between the structure and activity of the CDP and also an assessment of nucleolin and nucleophosmin chaperone molecule’s role in the transmembrane transfer of the CDPs.

Material and methods. All peptides for this study were synthesized using solid-phase synthesis method with Fmoc-protection of α-groups, purified by solid-phase HPLC and analyzed by mass spectrometry. Their cytotoxicity was assessed by the MTT assay using cell cultures HeLa and FEH. We studied the relationship between the structure of CDPs and their transfection activity, which was characterized in cell cultures Hela and A549 by the expression of reporter genes for luciferase and green fluorescent protein. Transport activity and analysis of intracellular localization of the CDPs were studied by confocal fluorescence microscopy using specific fluorescently labeled antibodies and fluorescently labeled CDP.

Results. The synthesized CDPs had 3 functional regions (modules): the N-terminal supercationic module, represented by arginine residues, the central module, represented by a hydrophobic core of lysine residues with short hydrophobic inserts and the C-terminal hydrophobic module including cysteine residue intended for attaching a reporter group. The study made it possible to access the relationship between the transfection activity of the peptide with its charge and the hydrophobicity of the C-terminal fragment. It has been shown that chaperone molecules, nucleolin and nucleophosmin, are involved in process of transmembrane transfer of CDP into the cell nucleus.

Conclusion. New dendrimeric peptides promising for effective delivery of NA molecules into mammalian cells have been obtained. The role of the chaperone proteins nucleolin and nucleophosmin in the transmembrane transport of the CDPs has been established. In the cells of most malignant tumors, the level of expression of these chaperone proteins is increased; therefore, these peptides can also be considered as potential antitumor agents.

Keywords:dendrimers; cationic peptides; solid-phase synthesis; non-viral vectors; gene therapy; nucleic acids; RNA interference; transfection; nucleolin

For citation: Kozhikhova K.V., Andreev S.M., Uspenskaya D.V., Shatilova A.V., Turetsky E.A., Shatilov A.A., Lushnikova A.A., Vishnyakova L.I., Shilovskiy I.P., Smirnov V.V., Kudlay D.A., Khaitov M.R. Supercationic peptide dendrimers as vectors for nucleic acids delivery to mammalian cells. Immunologiya; 2022; 43 (3): 320–32. DOI: (in Russian)

Funding. The study was supported by RSF within the competition for Grants 2020 «Initiative research conducting by young scientists» of Presidential research funding program, being implemented by lead scientists, including young scientists, at project number 20-73-00368.

Conflict of interests. Authors declare no conflict of interests.

Author’s contribution. Concept development and design of the study – Kozhikhova K.V., Andreev S.M.; conducting experiments on toxicity and transfection – Uspenskaya D.V., Turetsky E.A., Vishnyakova L.I.; peptide synthesis and purification – Shatilova A.V., Shatilov A.A.; statistical processing and calculations – Shilovskiy I.P., Smirnov V.V.; design of experiments with labeled peptides – Lushnikova A.A.; discussion of the concept, editing and approval of the final version of the article – Khaitov M.R.

Acknowledgments. The authors thank the graduate students who took part in this work: Kumasheva R.A. [I.M. Sechenov First MSMU (Sechenov University), MOH of Russia] – participated in implementation of transfection, Babikhina M.O. and Khokhlova A.M. (RTU MIREA of the MSHE of Russia) – participated in peptide synthesis.


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Musa R. Khaitov

Corresponding member of Russian Academy of Sciences, MD, Professor, Director of the NRC Institute of Immunology FMBA of Russia