New cationic carbohydrate-containing amphiphiles and liposomes based on them for effective delivery of short nucleic acids into eukaryotic cells

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Abstract

New cationic amphiphiles containing lactose or D-mannose residues were synthesized and cationic liposomes with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) were obtained. The cytotoxicity and transfection activity of new carbohydrate-containing amphiphiles and cationic liposomes against HEK 293, BHK and BHK IR-780 cells were studied. It has been shown that cationic amphiphiles effectively deliver only short fluorescein-labeled oligodeoxyribonucleotide into eukaryotic cells, while cationic liposomes formed by lactose containing amphiphile and DOPE effectively mediate the transport of short oligonucleotide and small interfering RNA and were non-toxic to cells. The resulting cationic amphiphiles can be used for intracellular delivering of nucleic acids both individually and part of cationic liposomes.

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About the authors

E. V. Shmendel

MIREA – Russian Technological University

Author for correspondence.
Email: elena_shmendel@mail.ru

Lomonosov Institute of Fine Chemical Technologies

Russian Federation, prosp. Vernadskogo 86, Moscow, 119571

A. O. Buyanova

MIREA – Russian Technological University

Email: elena_shmendel@mail.ru

Lomonosov Institute of Fine Chemical Technologies

Russian Federation, prosp. Vernadskogo 86, Moscow, 119571

O. V. Markov

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences

Email: elena_shmendel@mail.ru
Russian Federation, prosp. ak. Lavrent’eva 8, Novosibirsk, 630090

N. G. Morozova

MIREA – Russian Technological University

Email: elena_shmendel@mail.ru

Lomonosov Institute of Fine Chemical Technologies

Russian Federation, prosp. Vernadskogo 86, Moscow, 119571

M. A. Zenkova

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences

Email: elena_shmendel@mail.ru
Russian Federation, prosp. ak. Lavrent’eva 8, Novosibirsk, 630090

M. A. Maslov

MIREA – Russian Technological University

Email: elena_shmendel@mail.ru

Lomonosov Institute of Fine Chemical Technologies

Russian Federation, prosp. Vernadskogo 86, Moscow, 119571

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2. Fig. 1. Carbohydrate-containing cationic amphiphiles D1–D3.

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3. Fig. 2. D2-DOPE liposome cytotoxicity assay. HEK 293 cell viability was assessed in real time using the xCELLigence instrument. HEK 293 cells were seeded in 16-well plates at a density of 5 × 103 cells/well. After 24 h, cationic D2-DOPE liposomes were added to the cells at a concentration of 4 to 64 μM, and after 4 h of incubation with cationic liposomes, FBS was added to the medium to a concentration of 10%.

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4. Fig. 3. Accumulation of FITC-ODN complexes with cationic amphiphiles D1–D3 in HEK 293 cells in the presence of 10% FBS. FITC-ODN/cationic amphiphiles D1–D3 complexes were formed at N/P = 1/1.5, 1/1, and 1.25/1 ratios. The percentage of FITC-positive cells (a) and the level of average fluorescence intensity of cells in the population (b) were determined by flow cytometry after 4 h of cell incubation with the complexes. The standard deviation does not exceed 8%.

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5. Fig. 4. Accumulation of FITC-ODN complexes with cationic liposomes D1-DOPE, D2-DOPE, and D3-DOPE in HEK 293 cells. FITC-ODN/cationic liposomes D1-DOPE, D2-DOPE, and D3-DOPE complexes were formed at N/P = 1/2, 1/1, and 2/1 ratios. Transfection was performed in the absence (a, b) or presence of 10% FBS (c, d) in the cell medium. The percentage of transfected cells (a, c) and the level of average fluorescence intensity of cells in the population (b, d) were measured by flow cytometry after 4 h of cell incubation with the complexes. The standard deviation does not exceed 9%.

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6. Fig. 5. Delivery of pEGFP-C2 plasmid DNA using cationic amphiphiles D1–D3 into HEK 293 cells in the absence (a, b) or presence of 10% FBS (c, d) in the cell medium. The percentage of EGFP-positive cells (a, c) and the level of average fluorescence intensity (EGFP expression) of cells in the population (b, d) were measured by flow cytometry 48 h after incubation of cells with the complexes. The standard deviation does not exceed 8%.

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7. Fig. 6. Delivery of pEGFP-C2 plasmid DNA complexed with cationic liposomes D1-DOPE, D2-DOPE, and D3-DOPE into HEK 293 cells in the absence (a, b) or presence of 10% FBS (c, d) in the cell medium. The percentage of EGFP-expressing cells (a, c) and the level of average fluorescence intensity (EGFP expression) of cells in the population (b, d) were measured by flow cytometry 48 h after incubation of the cells with the complexes. The standard deviation does not exceed 7%.

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8. Fig. 7. Inhibition of EGFP protein expression in transgenic BHK IR-780 cells after siRNA delivery using cationic amphiphiles D1–D3 (a, b) or cationic liposomes D1-DOPE and D2-DOPE (c, d) in the absence (a, c) or in the presence of 10% FBS (b, d) in the cell medium.

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9. Scheme 1. Synthesis of new cationic amphiphiles containing lactose or D-mannose residues. a – 7AcLacBr/4AcManBr, CdCO3; b – Pd/C, NH4+CHOO–; c – succinic anhydride, DMAP, Et3N; d – N1,N4,N9-tri-tert-butoxycarbonyl-1,12-diamino-4,9-diazadodecane, HBTU, N,N-diisopropylethylamine; e – 1) TFA, 2) 0.04 N MeONa/MeOH, 3) 4 N HCl in dioxane.

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