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Gene therapy has a great potential for treating many human diseases that are currently considered incurable. Viral vectors based on adenoviruses or ret.

ISSN 16076729, Doklady Biochemistry and Biophysics, 2012, Vol. 445, pp. 197–199. © Pleiades Publishing, Ltd., 2012. Original Russian Text © I.V. Grigoriev, V.A. Korobeinikov, S.V. Cheresiz, A.G. Pokrovsky, L.Ya. Zakharova, M.A. Voronin, S.S. Lukashenko, A.I. Konovalov, Yu.F. Zuev, 2012, published in Doklady Akademii Nauk, 2012, Vol. 445, No. 3, pp. 349–352.

BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY

Cationic Gemini Surfactants as New Agents for Plasmid DNA Delivery into Cells I. V. Grigorieva, V. A. Korobeinikova, S. V. Cheresiza, b, A. G. Pokrovskya, L. Ya. Zakharovac, M. A. Voroninc, S. S. Lukashenkoc, Academician A. I. Konovalovc, and Yu. F. Zueva, d Received April 10, 2012

DOI: 10.1134/S1607672912040047

Gene therapy has a great potential for treating many human diseases that are currently considered incurable. Viral vectors based on adenoviruses or ret roviruses are very effective in gene delivery. Neverthe less, in view of the problems associated with immuno genicity and biosafety of viral vectors, there remains a need to develop nonviral vectors. As an alternative to viral vectors, cationic polymers [1–3] and liposomes [4], which bind to DNA through electrostatic interac tions and form nanosized complexes, have been inves tigated [5, 6]. Cationic agents protect DNA from deg radation by nucleases and serve as mediators in the penetration into the cell and subsequent release from endosomes, which increases the efficiency of transfec tion. The use of cationic surfactants as nonviral vectors was described in several papers [6, 7]. It is shown that they are effective agents that provide the compaction of DNA and recharging of the complex. Despite the obvious advantages (simplicity of synthesis and for mulation, low concentration, availability, and high complexing ability), a significant drawback of syn thetic agents based on cationic surfactants is the low transfection efficiency. In view of this, the search for new cationic vector systems is a relevant problem. In this study, we investigated alkylammonium gem ine surfactants (AGSs) with the formula

a

Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia b Research Institute of Molecular Biology and Biophysics, Siberian Branch, Russian Academy of Medical Sciences, Novosibirsk, Russia c Arbuzov Institute of Organic and Physical Chemistry, Kazan Research Center, Russian Academy of Sciences, ul. Akademika Arbuzova 8, Kazan, 420088 Tatarstan, Russia d Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences, ul. Lobachevskogo 2/31, Kazan, 420111 Russia

Br両



R

R1

R1

N

N

⊕ Br



(CH2)m R CH3 CH3

,

where m = 4–12, R = СН3, С2Н4ОН, R1 = nCnН2n + 1, where n = 10, 12, 14, and 16. Aqueous solutions of these compounds ensure efficient delivery of DNA into cells. Gemine surfactants (alkanedylα,ω bis(methyldialkylammonium bromides)) were pre pared under laboratory conditions as described in [8]. The structure of the obtained compounds was con firmed by elemental analysis as well as by IR and NMR spectroscopy data. Cell line 293T (human embryonic kidney epithe lium cells), which was used as a producer of pseudo lentiviruses and as a model target cell line, was main tained by the standard procedure. We used the peGFPN1 plasmid (Clontech, United States), which is 5100 bp in length and expresses the green fluores cent protein, and the pCINEO plasmidbased vector (Promega, United States), which is 5000 bp in length and does not contain the green fluorescent protein gene. HEK293T cells were replated 40 thousand cells per well one day before transfection. To transfect HEK293T cells, 50–100 thousand cells per well of a 96well plate were used. One hour before transfection, the cells were placed in a fresh DMEM medium free of serum and antibiotics. The solution of transfectants in phosphatebuffered saline (PBS×1), pH 7.0, at con centrations of 10–3, 10–2, 0.1, and 1 mg/ml was added to 0.6 g of plasmid DNA; after incubation for 15 min, the lipoplexes in a total volume of 20 ml were added to the cells. The fluorescence intensity of cells was ana lyzed by flow cytometry 48 h after transfection. The optimal incubation time with the transfectants was 4 h, after which the medium in the cells was replaced with the complete DMEM medium with serum and antibiotics. The cytotoxicity of the compounds was determined using 3(4,5dimethylthiazol2yl)2,5 diphenyltetrazolium bromide (MTT reagent) accord ing to the standard protocol for lymphoid cells.

197

198

GRIGORIEV et al. N/Pt 64

N/Pt 16

4

1

0.25

64

16Bz16(OH)

N/Pt 16

4

1

0.25

64

10610

N/Pt 16

4

1

16616(OH)

0.25

4

1

0.25

16416(OH)

N/Pt 64

16

N/Pt 64

16

4

16616

1

0.25

64

16

4

1

0.25

161216(OH)

Fig. 1. Changes in the electrophoretic mobility of the plasmid in the agarose gel upon the formation of complexes of cationic sur factants with DNA at different N/P ratios.

The degree of cell transfection was determined using flow cytometry by calculating the percentage of cells (FACS Diva software, Becton Dickinson, United States) incubated in the presence of cationic surfac tants that cause the eGFP expression. Metafectene was used as a reference drug. To determine the electrophoretic mobility of pEGFP– transfectants complexes, eGFP (0.8 µg) was added to transfectant solutions at concentrations of 0.003, 0.01, 0.03, and 0.1 mg/mL, and the resulting mixtures were incubated at room temperature for 15 min. The reaction products were separated by hor izontal electrophoresis in 1% agarose gel at an ethid ium bromide concentration of 0.5 µg/mL using a DNA marker (SibEnzyme, Russia). Electrophoresis was performed in TAE buffer (×1) at an electric field intensity of 10–15 V/cm for 30–45 min. Visualization of DNA in the gel was performed using a Vilber Lour mat ECX15.M transilluminator. The formation of DNA–surfactant complexes was tested using a Shi madzu RF5301 PC spectrofluorophotometer by the decrease in the ethidium bromide fluorescence inten sity. Figure 1 presents data on the complexation of cat ionic agents with the plasmid DNA, obtained by agar ose gel electrophoresis. For all compounds tested, the proportion of free plasmid DNA decreased as the con centration of potential transfectants increased until an N/P charge ratio of ~1, which was evidence of the for mation of complexes. It is noteworthy that, at N/P > 1,

the fluorescence intensity of 161216(OH) decreased to an almost complete disappearance of the signal, indicating a higher efficiency of complex formation. For other potential transfectants, the formation of intermediates was observed, which could be seen in the lanes of the gel and had a lower mobility relative to DNA without lipoplexes. It can be assumed that the differences observed for the DNA/161216 (OH) lipoplex were due to the specific aggregation of this surfactant, because the presence of a flexible spacer can facilitate morphological rearrangements in the system (in particular, micelle–vesicle structural tran sitions). The compounds without DNA were more toxic (Fig. 2). The lack of the possibility to form complexes with plasmid DNA in the medium had an adverse effect on the viability of cells because of interaction with their surface. The transfection efficiency values obtained for dif ferent compounds are summarized in the table. At high concentrations of the compounds, marked cell death in the cell population as well as a wide variation in the transfection efficiency were observed. The per centage of the fluorescent population relative to the proportion of living cells is shown. Transfection efficiency and cytotoxicity are two most important parameters of genetic material deliv ery systems. The efficiency of transfection with gem ine surfactants containing the alkylammonium head group is maximal in the case of 161216(OH), 164

DOKLADY BIOCHEMISTRY AND BIOPHYSICS

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CATIONIC GEMINI SURFACTANTS AS NEW AGENTS CD50, mg/mL 0.12

DNA+ DNA−

0.08 0.04

16 1 2 16 (O H )

10 6 1 0

16 6 1 6

16 4 1

6( O H

) 16 B z 16 (O H ) 16 6 1 6( O H )

0

Fig. 2. Cytotoxicity of potential transfectants determined in the MTT test. The black columns show the DNA + transfectants samples, and the crosshatched columns show the samples with potential transfectants alone.

16(OH), and 16Bz16(OH) transfectants (table). They belong to the same family and structurally differ only in length and type of linkers. The treatment of cells with other potential transfectants did not yield a sig nificant percentage of transfection. The closest monoca tionic analogues of the proposed compounds, which were chosen as reference compounds, are cetyltrimethy lammonium bromide (CTAB) and cetyl(2hydroxy ethyl)dimethylammonium bromide (CHAB) [7, 9– 11]. The disadvantages of the use of monocationic sur factants as a tool for delivering DNA into cells are (1) significantly higher values of critical micelle concen trations (CMC) (CMC of CTAB and CHAB were 0.87 and 0.75 mM, respectively); (2) a lower surface poten tial, which requires higher concentrations of surfac tants to reach comparable effects of DNA compaction and recharging surfactant/DNA complexes; and (3) reduced DNA transfection. The authors of [8, 12, 13] investigated the effect of ethidium bromide exclusion during complexation with oligo and polynucleotides by amphiphilic cat ionic agents. It is shown that the binding of DNA to cationic surfactants and calixarenes leads to a substan Transfection efficiency for different gemine surfactants Compound 161216(OH)

16416(OH)

16Bz16(OH)

Concentration, µmol/L 2.3 5.5 11 22 3 25 250 2500 232 116 58 29

GFP(+) transfec tion efficiency, % 0.8 ± 0.3 43.7 ± 6.5 51.3 ± 10.0 6.6 ± 1.5 31.7 ± 4.7 13.3 ± 4.2 5.0 ± 1.3 7.8 ± 2.5 14.1 ± 4.5 10.1 ± 4.3 3.9 ± 0.8 1.9 ± 0.4

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tial decrease in the fluorescence yield with coming to a plateau at N/P ≥ 1. These data are in good agreement with the data on the efficiency of interaction of amphiphilic agents with DNA, obtained by gel elec trophoresis and measuring the zeta potential of lipoplexes. Compounds with a long alkyl linker and greater hydrophobicity showed a significant drop in the fluorescence intensity, suggesting a more efficient complexation. In our case, a similar dependence was observed; the greatest efficiency was obtained when using the compound 161216(OH) with a side chain of C16, whereas compounds such as 10x10 showed much lower transfection efficiency. This finding sug gests that, along with the stoichiometric electrostatic interactions, the cooperative charge of aggregated sur factants also contributes to the formation of lipoplexes. Thus, the synthesized gemine surfactants with the alkylammonium head group may make a good alter native to the existing compounds to deliver genetic material into cells. The optimal conditions for trans fection were established. ACKNOWLEDGMENTS This study was supported by the SpecialPurpose Federal Program “Scientific and ScientificPedagogi cal Personnel of Innovative Russia in 2009–2013” (state contract no. 14.740.11.0384) and the Russian Foundation for Basic Research (project no. 1203 01085a). REFERENCES 1. Bagnacani, V., Sansone, F., Donofrio, G., et al., Org. Lett., 2008, vol. 10, no. 18, p. 3953. 2. Baldini, L., Casnati, A., Sansone, F., and Ungaro, R., Chem. Soc. Rev., 2007, vol. 36, no. 2, p. 254. 3. Sun, X. and Zhang, N., Mini Rev. Med. Chem., 2010, vol. 10, no. 2, p. 108. 4. Geromel, V., Cao, A., Briane, D., et al., Antisense Nucl. Acid Drug Dev., 2001, vol. 11, no. 3, p. 175. 5. Won, Y.W., Lim, K.S., and Kim, Y.H., J. Control Release, 2011, vol. 152, no. 1, p. 99. 6. Mintzer, M.A. and Simanek, E.E., Chem. Rev., 2009, vol. 109, no. 2, p. 259. 7. Mel’nikov, S.M., Sergeyev, V.G., and Yoshikawa, K., J. Am. Chem. Soc., 1995, vol. 117, no. 9, p. 2401. 8. Zakharova, L., Voronin, M., Gabdrakhmanov, D., et al., ChemPhysChem., 2012, vol. 13, no. 3, p. 788. 9. Mel’nikov, S.M., Sergeyev, V.G., and Yoshikawa, K., J. Am. Chem. Soc., 1995, vol. 117, no. 40, p. 9951. 10. Clamme, J.P., Bernacchi, S., Vuilleumier, C., et al., Biochim. Biophys. Acta, 2000, vol. 1467, no. 2, p. 347. 11. Pinnaduwage, P., Schmitt, L., and Huang, L., Biochim. Biophys. Acta, 1989, vol. 985, no. 1, p. 33. 12. Dasgupta, A., Das P.K., Dias R.S., et al., J. Phys. Chem. B., 2007, vol. 111, no. 29, p. 8502. 13. Rodik, R.V., Klymchenko, A.S., Jain, N., et al., Chem. Eur. J., 2011, vol. 17, no. 20, p. 5526. 2012

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