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

Abstract

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: https://doi.org/10.33029/0206-4952-2022-43-3-320-332 (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.

References

1. Fire A., Xu S., Montgomery M., Kostas S., Driver S., Mello C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391: 806–11. DOI: https://doi.org/10.1038/35888

2. Milletti F. Cell-penetrating peptides: classes, origin, and current landscape. Drug Discovery Today. 2012; 17: 850–60. DOI: https://doi.org/10.1016/j.drudis.2012.03.002

3. Luo K., Li C., Wang G., Nie Y., He B., Wu Y., Gu Z. Peptide dendrimers as efficient and biocompatible gene delivery vectors: Synthesis and in vitro characterization. J Control Rel. 2011; 55 (1): 77–87. DOI: https://doi.org/10.1016/j.jconrel.2010.10.006

4. Takechi-Haraya Y., Saito H. Current understanding of physicochemical mechanisms for cell membrane penetration of arginine-rich cell penetrating peptides: role of glycosaminoglycan Interactions. Curr Prot Pept Sci. 2018; 19 (6): 623–30. DOI: https://doi.org/10.1016/j.jconrel.2010.10.006

5. Kumar V., Agrawal P., Kumar R., Bhalla S., Usmani S.S., Varshney G.C., Raghava G. P. Prediction of cell-penetrating potential of modified peptides containing natural and chemically modified residues. Front Мicrobiоl. 2018; 9: 725. DOI: https://doi.org/10.3389/fmicb.2018.00725

6. Hofland H.E., Shephard L., Sullivan S.M. Formation of stable cationic lipid/DNA complexes for gene transfer. Proc Natl Acad Sci. USA. 1996; 93: 7305–9.

7. Turetsky E.A., Koloskova O.O., Nosova A.S., Shilovsky I.P., Sebyakin Yu.L., Khaitov M.R. Physicochemical properties of lipopeptide-based liposomes and their complexes with siRNA. Biomedical Chemistry. 2017; 63 (5): 472–5. DOI: https://doi.org/10.18097/PBMC20176305472 (in Russian)

8. Regberg J., Srimanee A., Erlandsson M., Sillard R., Dobchev D.A., Karelson M., Langel U. Rational design of a series of novel amphipathic cell-penetrating peptides. Int J Pharm. 2014; 464 (1-2): 111–6. DOI: https://doi.org/10.1016/j.ijpharm.2014.01.018

9. Eggimann G.A., Blattes E., Buschor S., Biswas R., Kammer S.M., Darbre T., Reymond J.-L. Designed cell penetrating peptide dendrimers efficiently internalize cargo into cells. Chem Comm. 2014; 50: 7254. DOI: https://doi.org/10.1039/C4CC02780A

10. Shcharbin D., Shcharbina N., Bryszewska M. Recent patents in dendrimers for nanomedicine: Evolution 2014. Recent patents on nanomedicine. 2014; 4 (1): 25–31. DOI: https://doi.org/10.2174/1877912304666140609233256

11. Nikolskii A.A., Shilovskiy I.P., Yumashev K.V., Vishniakova L.I., Barvinskaia E.D., Kovchina V.I., Korneev A.V., Turenko V.N., Kaganova M.M., Brylina V.E., Nikonova A.A., Kozlov I.B., Kofiadi I.A., Sergeev I.V., Maerle A.V., Petukhova O.A., Kudlay D.A., Khaitov M.R. Effect of local suppression of Stat3 gene expression in a mouse model of pulmonary neutrophilic inflammation. Immunologiya. 2021; 42 (6): 600–14. DOI: https://doi.org/10.33029/0206-4952-2021-42-6-600-614 (in Russian)

12. Kozhikhova K.V., Andreev S.M., Shilovskiy I.P., Timofeeva A.V., Gaisina A.R., Shatilov A.A., Turetskiy E.A., Andreev I.M., Smirnov V.V., Dvornikov A.S., Khaitov M.R. A novel peptide dendrimer LTP efficiently facilitates transfection of mammalian cells. Org Biomol Chem. 2018; 16 (43): 8181–90. DOI: https://doi.org/10.1039/C8OB02039F

13. Allolio C., Magarkar A., Jurkiewicz P., Baxová K., Javanainen M., Mason P.E., Šachl R., Cebecauer M., Hof M., Horinek D., Heinz V., Rachel R., Ziegler C.M., Schröfel A., Jungwirth P. Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore. Proc Natl Acad Sci USA. 2018; 115: 11923–8. DOI: https://doi.org/10.1073/pnas.1811520115

14. Takechi-Haraya1 Y., Ohgita T., Kotani M., Kono H., Saito C., Tamagaki-Asahina H., Nishitsuji K., Uchimura K., Sato T., Kawano R., Sakai-Kato K., Izutsu K., Saito H. Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides. Sci Reports. 2022; 12: 4959. DOI: https://doi.org/10.1038/s41598-022-08876-9

15. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immun Methods. 1983; 65: 55–63. DOI: https://doi.org/10.1016/0022-1759(83)90303-4

16. Frommel C. The apolar surface area of amino acids and its empirical correlation with hydrophobic free energy. J Theor Biol. 1984; 111: 247–260. DOI: https://doi.org/10.1016/S0022-5193(84)80209-X

17. Ponkratova D. A., Lushnikova A.A. Structural features and expression of NPM and NCL in cutaneous melanoma. Molecular Biology. 2019; 53 (4): 663–73. DOI: https://doi.org/10.1134/S0026898419040098 (in Russian)

18. Kozhikhova K.V., Shilovskiy I.P., Shatilov A.A., Timofeeva F.V., Turetskiy E.A., Vishniakova L.I., Nikolskii A.A., Barvinskaya E.D., Karthikeyan S., Smirnov V.V., Kudlay D.A., Andreev S.A., Khaitov M.R. Linear and dendrimeric antiviral peptides: Design, chemical synthesis and activity against human respiratory syncytial virus. J Mater Chem. B. 2020; 8: 2607–17. DOI: https://doi.org/10.1039/C9TB02485A

19. Feng Z., Xu L., Xie Z. Receptors for respiratory syncytial virus infection and host factors regulating the life cycle of respiratory syncytial virus. Front Cell Infect. Microbiol. 2022; 12: 858629. DOI: https://doi.org/10.3389/fcimb.2022.858629

20. Vazdar M., Heyda J., Mason P.E., Tesei J., Allolio C., Lund M., Jungwirth P. Arginine «magic»: Guanidinium like-charge ion pairing from aqueous salts to cell penetrating peptides. Acc. Chem. Res. 2018; 51: 1455−64. DOI: https://doi.org/10.1021/acs.accounts.8b00098

21. Chua B.Y., Zeng W., Jackson D.C. Simple branched arginine-based structures can enhance the cellular uptake of peptide cargos. Int J Pep Therap. 2007; 13 (3): 431–7. DOI: https://doi.org/10.1007/s10989-006-9063-y

22. Das U., Hariprasad G., Ethayathulla A.S., Manral P., Das T.K., Pasha S., Mann A., Ganguli M., Verma A.K., Bhat R., Chandrayan S.K., Ahmed S., Sharma S., Kaur P., Singh T.P., Srinivasan A. Inhibition of protein aggregation: Supramolecular assemblies of arginine hold the key. PLoS ONE. 2007; 2 (11): e1176. DOI: https://doi.org/10.1371/journal.pone.0001176

23. Huang J., Wang J., Li Y., Wang Z., Chu M., Wang Y. Tuftsin: A natural molecule against SARS-CoV-2 infection. Front Mol Biosci. 2022; 9: 859162. DOI: https://doi.org/10.3389/fmolb.2022.859162

24. Simeoni F., Morris M.C., Heitz F., Divita G. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. NAR. 2003; 31 (11): 2717–24.

25. Fu Y., Chen Y., Luo X., Liang Y., Shi H., Gao L., Zhan S., Zhou D., Luo Y. The heparin binding motif of endostatin mediates its interaction with receptor nucleolin. Biochemistry. 2009; 48 (49): 11655–63. DOI: https://doi.org/10.1021/bi901265z

26. Lushnikova A.A., Morozova L.F., Pankratova D.A., Balbutsky A.V., Andreev S.M., Mikhailov A.E., Khaitov M.R. Application of cationic peptides for induction of human skin melanoma cell death. Patent. RU26 20170 C1

27. Khaitov M., Nikonova А., Shilovskiy I., Kozhikhova K., Kofiadi I., Vishnyakova L., Nikolsky A., Gattinger P., Kovchina V., Barvinskaya E., Yumashev K., Smirnov V., Maerle A., Kozlov I., Shatilov A., Timofeeva A., Andreev S., Koloskova O., Kuznetsova N., Vasina D., Nikiforova M., Rybalkin S., Sergeev I., Trofimov D., Martynov A., Berzin I., Gushchin I., Kovalchuk A., Borisevich S., Valenta R., Khaitov R., Skvortsova V. Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation. Allergy. 2021; 76 (9): 2840–54. DOI: https://doi.org/10.1111/all.14850

EDITOR-IN-CHIEF
EDITOR-IN-CHIEF
Musa R. Khaitov

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

Вскрытие
Medicine today

II Всероссийская конференция с международным участием "Воспаление глаза" 12 ноября 2022 года, Москва Воспалительные заболевания глаза - широко распространенная и многогранная проблема, с которой может столкнуться в своей практике любой специалист. Найти оптимальные алгоритмы...

Масштабное событие в области дерматовенерологии и косметологии - II конференция InteDeCo 2022 "Интегративная дерматовенерология и косметология. Новые стандарты взаимодействия" - состоится 16-17 декабря 2022 года. Программа мероприятия пройдет на современной...

VIII Московский Городской Съезд педиатров с межрегиональным и международным участием "Трудный диагноз в педиатрии" Приглашаем педиатров, детских эндокринологов, реаниматологов, гинекологов, неонатологов, кардиологов, хирургов, урологов, психологов, специалистов по лучевой...


JOURNALS of «GEOTAR-Media»