Surfactants as a means of delivering a reporter genetic construct based on binase suicide gene to tumor cells

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Resumo

Among modern gene therapy methods for combating oncology, suicidal gene therapy based on the delivery of a cytotoxic agent to target cells is of particular importance and promise. As one of such genes, the gene for ribonuclease of Bacillus pumilus 7P, binase, can be considered; the enzyme has a high antitumor potential and low immunogenicity. In addition to the choice of a transgene, another factor influencing the effectiveness of gene therapy is the method of delivering the nucleic acid to target cells. Surfactants have high functional activity and are promising means of delivering therapeutic nucleic acids. The aim of this work was to evaluate the possibility of using geminal surfactants as a means of delivering a genetic construct based on the cytotoxic binase gene into tumor cells. To optimize the transfection conditions, a reporter genetic construct carrying the binase gene fused to the gene for the green fluorescent protein TurboGFP was created, which made it possible to evaluate the delivery efficiency by the fluorescence intensity. To eliminate the toxic effect of binase on recipient cells, the RNase inhibitor gene, barstar, was introduced into the genetic construct. A high complexing ability of geminal surfactants in relation to the reporter system was shown by methods of dynamic light scattering and fluorescence spectroscopy. For surfactant 16-6-16OH, the highest transfecting activity together with a low level of cytotoxicity was found. Thus, the study proved the possibility of using geminal surfactants for the delivery of therapeutic nucleic acids to target cells.

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Sobre autores

E. Dudkina

Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University

Email: ulyanova_vera@mail.ru
Rússia, ul. Kremlevskaya 18, Kazan 420008

E. Vasilieva

Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences

Email: ulyanova_vera@mail.ru
Rússia, ul. Akad. Arbuzova 8, Kazan, 420088

V. Ulyanova

Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University

Autor responsável pela correspondência
Email: ulyanova_vera@mail.ru
Rússia, ul. Kremlevskaya 18, Kazan 420008

L. Zakharova

Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences

Email: ulyanova_vera@mail.ru
Rússia, ul. Akad. Arbuzova 8, Kazan, 420088

O. Ilinskaya

Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University

Email: ulyanova_vera@mail.ru
Rússia, ul. Kremlevskaya 18, Kazan 420008

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2. Fig. 1. Electropherogram of the products of cloning the “binase-barstar” genetic cassette into the pTurboGFP-C vector: M – 1 kb DNA length marker (Sibenzyme, Russia); 1 – PCR product “binase-barstar”; 2 – the resulting genetic construct pTurboGFP-Bi-Brst, restricted at the XhoI and BamHI sites.

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3. Fig. 2. Distribution of aggregates by size, averaged by the number of particles: 16-6-16OH/DNA (a), 16-6-16/DNA (b) and CTAB/DNA (c), at different surfactant/DNA ratios. SDNA = 10 µM; 25°C.

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4. Fig. 3. Changes in the electrokinetic potential of surfactant–DNA complexes at different ratios of components. SDNA = 10 µM; 25°C.

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5. Fig. 4. Fluorescence spectra of ethidium bromide/DNA complexes in the presence of 16-6-16OH (a), 16-6-16 (b) and CTAB (c). SDNA = 10 µM; SEB = 0.5 µM; 25°C.

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6. Fig. 5. Dependence of the degree of binding of surfactants 16-6-16OH, 16-6-16 and CTAB to DNA on the ratio of components at 25°C.

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7. Fig. 6. Evaluation of the efficiency of transfection of Lipofectamine 3000 A549 cells by fluorescence microscopy with a magnification of 100×.

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8. Fig. 7. Structural formulas of the studied cationic surfactants.

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