Influence of the degree of oligomerization of surfactants on the DNA/surfactant interaction

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: Colloids and Surfaces B: Biointerfaces on 2019, available at: https://doi.org/10.1016/j.colsurfb.2019.110399” 1 Influence of the degree of oligomerization of surfactants on the DNA/ surfactant interaction Alberto Lópeza, Pilar López-Cornejoa, Manuel López-Lópezb, José Antonio Lebróna, Francisco José Ostosa, David Pérez-Alfonsoa, Jaime Oviedoa, André Laschewskyc, María Luisa Moyáa,⁎ a Department of Physical Chemistry, University of Seville, C/ Profesor García González 1, 41012 Seville, Spain b Department of Chemical Engineering, Physical Chemistry and Materials Science, Faculty of Experimental Sciences, Campus de El Carmen, Avda. de las Fuerzas Armadas s/n, 21071 Huelva, Spain c Institut für Chemie, Universität Postdam, Karl-Liebknecht Str. 24–25, 14476 Postdam-Golm, Germany and Fraunhofer Institute of Applied Polymer Research, Geiselbergstr. 69, 14476 Postdam-Golm, Germany *Author to whom all correspondence should be directed 2 ABSTRACT The interaction between calf thymus DNA, ctDNA, and a series of oligomeric surfactants derived from N-benzyl- N,N-dimethyl-N-(1-dodecyl)ammonium chloride is investigated. The influence of the surfactants' degree of oligomerization (2, 3 and 4) on the ctDNA/surfactant interaction is studied, as well as the effect of the structure of the spacer group linking the individual surfactant fragments. In particular, the effect of the distance between the positive charges and the hydrophobic chains within the oligomers on these interactions was examined, by using the three positional isomers (i.e., ortho−, meta−, and para−) with the rigid xylidene moiety as spacer. Results show that the dimeric ("gemini") surfactants are much more efficient in the inversion of the nucleic acid charge than the single-chained (monomeric) surfactant. Whereas the ortho − isomer causes a partial condensation, the meta − and para − isomers can completely condense ctDNA. The meta − and para − isomers of the trimeric surfactants can also completely condense the polynucleotide. In contrast, the tetrameric surfactant investigated does not change the morphology of the nucleic acid from an elongated coil into a compacted form, in spite of effectively inverting the nucleic acid's charge in their complex. Accordingly, the capacity for ctDNA compaction of oligomeric surfactants is not simply correlated to their degree of oligomerization, but depends on a complex balance of the number and relative distance of cationic charges and/or hydrophobic tails in the surfactants for effectively interacting with the nucleic acid to form the appropriate complex. This information will help to design more effective cationic surfactants as non-viral vectors for gene therapy. 1.-Introduction The condensation of deoxyribonucleic acid, DNA, is an essential process for gene regulation in living systems. In this process, the DNA molecules are compacted from an elongated form to a globular one. The study of this packing process is relevant for many applications in medicine and biotechnology [1,2]. Among others, polycations, multivalent metal cations, and cationic surfactants have been used as compacting agents [3–12]. In particular, cationic surfactants are known to be efficient DNA compacting agents [13–19]. While the DNA/surfactant binding is mainly based on strong attractions between the anionic polynucleotide and the cationic surfactants, both electrostatic and hydrophobic interactions play significant roles in the DNA/surfactant complex formation. The importance of the hydrophobic interactions is made evident by the high degree of cooperativity observed in the DNA/ surfactant binding isotherms, as it was recently demonstrated for singlechained DNA/surfactant complexes [20]. The structure of the cationic surfactant strongly influences its interactions with DNA [10,11]. The most evident surfactant characteristic is its charge. Electrostatic repulsion disfavors the formation of DNA/ anionic surfactant complexes. In fact, negatively charged surfactants are poor DNA compacting agents, but can be used to decompact previously formed DNA/cationic surfactant complexes due to the formation of mixed micelles [21]. Nonionic surfactants can condense polynucleotides, monopole − dipole interactions presumably contributing. Yet by 3 virtue of charge complementarity, cationic surfactants are by far much more efficient to induce the DNA morphological transition from an elongated to a globular form. Since enhanced electrostatic attraction a priori favors the interaction of surfactants with DNA, an increase in the positive charge of the surfactant head as well as in the number of cationic head groups (e.g. in dicephalic surfactants) tends to improve the performance of surfactants as DNA condensing agents [22,23]. The counter − ion nature also affects the condensing capacity of surfactants. Recently, Zhang et al. found for a single-chained sugar-derived surfactant that counter − ions that favor self-aggregation of the surfactant in water also increase its efficiency to compact DNA [24]. In addition to electrostatic effects, hydrophobic interactions play a key role in DNA compaction. The increased condensing efficacy of surfactants upon increasing the length of the hydrophobic tail(s) has been demonstrated by different authors [25–28] as well as the effects of additional side chains in the hydrophobic tails [29]. In contrast, fluorinated surfactants were found less efficient to compact DNA than their hydrogenated analogues [30]. The interaction between DNA and magnetic surfactants has also been investigated [31]. In addition to the variation of the nature and size of the cationic groups and the hydrophobic chains in surfactants, several authors have investigated the interactions between nucleic acids and dimeric surfactants ("gemini surfactants") for condensing DNA [15,32–41]. This included studies on the influence of the spacer length [29,38,40–44], which is an additional structural variable specific for dimeric surfactants [45]. It was found that generally, for a given tail length, dimeric surfactants are more efficient DNA condensing agents than single-chained surfactants. This was explained by the more favorable hydrophobic interactions helping the formation of the DNA/surfactant complexes. In any case, the complexation of dimeric cations by DNA will be always favored over the complexation of simple cations for entropic reasons. Still, except for a recent study on the interactions between a star–shaped hexameric surfactant and DNA [46], an investigation about the effect of the surfactant's degree of oligomerization beyond dimers on the DNA/surfactant interaction has not been carried out yet to the authors’ knowledge. In fact, such complexes of oligomeric surfactants and DNA may be considered as intermediate structures between classical lipoplexes and polyplexes. Therefore, we have studied the interaction between calf thymus DNA, ctDNA, and a series of homologous oligomeric surfactants derived from N-benzyl-N,N-dimethyl-N-(1-dodecyl)ammonium chloride. The surfactants investigated are: i) the parent single-chained surfactant N-benzyl- N,N-dimethyl-N-(1-dodecyl)ammonium chloride (monomer X-1); ii) the dimeric surfactant isomers: N,N'-(1,2-phenylenebis(methylene))bis (N,N-dimethyl-N-(1- dodecyl)ammonium dichloride (o-X-2), N,N'-(1,3- phenylenebis(methylene))bis(N,N- dimethyl-N-(1-dodecyl)ammonium dichloride (m-X-2), and N,N'-(1,4- phenylenebis(methylene))bis(N,N- dimethyl-N-(1-dodecyl)ammonium dichloride (p-X-2); iii) the linear trimeric surfactants: bis(3-(N′,N′-dimethyl-N′-dodecylammoniomethy- lene)phenylenemethylene)-N-dodecyl-N- methylammonium trichloride (m-X-3) and bis(4- (N′,N′-dimethyl-N′-dodecylammoniomethylene)phenylenemethylene)-N-dodecyl-N- methylammonium trichloride (p-X-3); and iv) the linear tetrameric surfactant 1,3-bis(N-(3- (N′-dodecyl-N′,N′- dimethylammoniomethyl)phenylenemethylene)-N-dodecyl-N-methy- lammoniomethyl) benzene tetrachloride (m-X-4), as displayed in Scheme 1. Besides, by 4 comparing the behavior of dimeric surfactant isomers with rigid spacers (ortho−, meta−, and para − xylylene), this work will also give information about the influence of the distance and relative positions of the cationic charges and the hydrocarbon tails on their capacity as DNA compacting agents. Thus, our study will provide new physical insights in the DNA/surfactant interactions since they will help to discern which could be the more appropriate number of hydrophobic tails Scheme 1.- Structure of the surfactants used in this work. as well as their position in the head group in order to have an efficient surfactant for condensing nucleic acids from an elongated form to a compacted globular one. Moreover, it may also shed some light on the relationship between lipoplexes and polyplexes. This is important for understanding DNA compaction, and will help to design more effective cationic surfactants as non-viral vectors for gene therapy. 5 2.-Materials and methods 1.1. Chemicals and reagents Materials were from Sigma-Aldrich and Fluka, of the highest purity available. Calf thymus DNA concentration (given by phosphate groups) was estimated from its molar absorptivity at 260 nm (6600 mol −1 dm 3 cm −1 ) [47]. The average number of base pairs is above 10,000 bp, according to an agarose gel electrophoresis test utilizing ethidium bromide, EB [48]. The ratios in absorbance at 260 and 280 nm of the ctDNA solutions were between 1.8 and 1.9, indicating the absence of proteins [49]. ctDNA solutions were all buffered by using 10 mM HEPES at pH = 7.4. The syntheses of the surfactants were reported before [50,51]. Water was purified by a MilliQ water purification system (Millipore, resistivity > 18 MΩ cm −1 ). 1.2. UV–vis absorption spectroscopy A Hitachi UV–vis 3900 spectrophotometer, connected to a water flow Lauda cryostat, was used to check the stability of the buffered ctDNA/surfactant solutions. Temperature was maintained at 303.0 ± 0.1 K. The absorbance of the solutions at 260 nm was stable for more than 4 h for all systems investigated. All the solutions were clear, with no turbidity even at the highest surfactant concentrations used. 1.3. Fluorescence measurements Ethidium bromide, EB, emission fluorescence intensity was measured using a Hitachi F- 2500 with a cell compartment connected to a water flow Lauda cryostat. Temperature was kept at 303.0 ± 0.1 K. The excitation and emission wavelengths were 520 and 592 nm, respectively. EB emission spectra were recorded from 500 to 700 nm, with a scan rate of 60 nm/min, and excitation and emission slits equal to 5 nm both. The EB concentration was 4.9 × 10 −6 M and the ctDNA concentration was 3 × 10-5 M, both of them were kept constant. The precise surfactant concentrations depended on their nature. All the ctDNA/surfactant solutions studied were buffered (HEPES 10 mM, pH = 7.4). 1.4. Zeta potential measurements Zeta potential measurements were done with a Zetasizer Nano ZS Malvern Instrument Ltd. (UK) in the General Services of the University of Seville. Temperature was maintained at 303.0 ± 0.1 K and a fixed ctDNA concentration of 3.0 × 10 −5 M was present in all the solutions, at pH = 7.4 (HEPES 10 mM). The reported values are the arithmetic average of at least 9 measurements. 6 1.5. Circular dichroism measurements A Biologic Mos-450 spectrometer with a cell compartment connected to a circulation thermostatic bath was used to register the circular dichroism, CD, spectra. Temperature was kept at 303.0 ± 0.1 K. A scan speed of 50 nm min −1 was used. It was checked that the HEPES buffer does not contribute to the absorption spectra. For each solution investigated, the reported spectra were the average of 10 runs, with a 5 min equilibration time before each scan. All measurements were done at a fixed ctDNA concentration of 3.0 × 10−5 M, while varying the surfactant concentration at pH = 7.4 (HEPES 10 mM). 1.1. Melting measurements A Biologic Mos-450 spectrometer equipped with a Peltier temperature controller was used for studying ctDNA thermal denaturation, applying a heating rate of 0.2 °C min −1 . The apparatus reads the differences in temperature and absorbance (at 260 nm) between the reference and the sample cuvettes. The ctDNA concentration was kept constant at 3.0 × 10 -5 M. A phosphate buffer of pH = 7.4 was used instead of 10 mM HEPES buffer, because the latter interferes with absorbance measurements at 260 nm. The DNA melting point, T m , was estimated as the temperature corresponding to the midpoint of the steep slope of the temperature dependent absorbance curve. At least three profiles were acquired for each ctDNA/surfactant solution studied and the Tm values were averaged. The standard deviation was estimated to be close to 0.5 °C. 1.2. Atomic force microcospy, AFM, measurements A Molecular Imaging PicoPlus 2500 AFM (Agilent Technologies) with silicon cantilevers (Model Pointprobe, Nanoworld) with a resonance frequency around 240 kHz and nominal force constant 42 N/m was used. AFM imaging was recorded in air in the tapping mode, with scan speeds of about 0.5 Hz and data collection at 256 × 256 pixels. It was necessary to use dilute solutions of ctDNA (0.30 μM) in order to molecules, due to their large size. AFM images of the ctDNA/surfactant buffered solutions were obtained by drying a 30 μL droplet of the solution deposited on a modified freshly cleaved mica surface. The mica surface was prepared as follows: 0.1% (v/v) APTES aqueous solution was dropped onto a freshly cleaved mica surface and incubated for 20 min. Subsequently, the surface was washed with ultrapure water, and air-dried. Then, a total of 30 μL of ctDNA (or ctDNA/surfactant) solution was dropped onto the mica surfaces. After 60 min of incubation, the surfaces were washed with pure water, and air dried before AFM imaging. The software WSxM 4.0 Beta 6.2, from Nanotec [52] was used for data treatment. 7 3.-Results and discussion Prior to the experiments, the stability of the ctDNA/surfactant solutions investigated was checked by UV–vis spectroscopy. No turbidity was observed even at the highest surfactant concentrations. In the initial experiments, the competitive binding of ethidium bromide, EB, and of the various oligomeric surfactants to ctDNA was studied. EB binds to double stranded DNA by intercalation, the molecules lying parallel to the base pairs due to their planar structure [53]. When the phenanthridine fluorophore of EB is intercalated between adjacent base pairs of the double helix, the radiationless deactivation modes are attenuated, resulting in fluorescence enhancement [54]. The fluorescence emission intensity decreases sharply when EB changes from this position to a more polar environment, such as the aqueous bulk phase. On this basis, information about the interaction of the surfactants under study and calf thymus DNA, ctDNA, can be obtained from the variations in the fluorescence emission intensity of solutions containing EB and ctDNA upon adding the surfactants. Fig. S1 (Supplementary Material) illustrates the dependence of I/Io on the charge ratio cationic surfactant:ctDNA, Z+/Z-, for the surfactants studied. Io and I are the fluorescence emission intensities of the dye in the absence and in the presence of surfactant, respectively. Z+/Z- va- lues were calculated by considering surfactant charges equal to +1, +2, +3, and +4 for the mono-, di-, tri-, and tetrameric surfactants, respectively. A charge of Z − =- 2 was attributed to ctDNA [55]. Fig. S1 shows a diminution in the emission intensity of EB upon increasing Z + / Z- for all the surfactants investigated, the I/Io value reaching a constant value, independent of Z+/Z-. Accordingly, the EB molecules are displaced from the complex with ctDNA to the bulk solution. This could be explained by a competition of the surfactants for the ctDNA sites between the base pairs, and/or by a conformational change of the nucleic acid caused by the ctDNA/surfactant interactions, resulting in insufficient available space for the accommodation of the EB molecules. Later on in the text, circular dichroism experiments will provide more information about this issue. Fig. S1 demonstrates that the change from a single-chained to a dimeric structure results in a large increase in the ctDNA/surfactant interaction since the displacement of EB molecules occurs at much lower Z+/Z- values for the monomeric than for the dimeric surfactants. When the degree of oligomerization, i.e. the number of surfactant units per molecule, increases further from 2 to 3, and finally from 3 to 4, the differences in the Z+/Z- values at which the EB displacement takes place are much smaller. Furthermore, Fig. S1c shows that for a given degree of oligomerization, the isomeric surfactants (containing ortho−, meta−, and para − xylylidene spacers) behave similarly, within experimental errors. The study of the zeta potential variations of the nucleic acid upon changing surfactant concentration provides further information about the ctDNA/surfactant interaction [20,21,40,56]. Fig. 1 shows the dependence of the relative zeta potential, ζ/ζo, where ζ and ζo are the zeta potential of the solutions in the presence and in the absence of surfactant, respectively, on the charge ratio Z+/Z- for the various surfactants. The ratio Z+/Z- could not 8 be increased higher than ∼ 22 in the case of X1, because solubility problems precluded the experiments. The data in Fig. 1 indicate that, in all cases, a continuously increasing surfactant concentration results not only in the neutralization of ctDNA, but eventually in a charge inversion of the ctDNA/surfactant complex. This charge inversion is caused by the binding of the surfactant molecules to the nucleic acid. It is driven by the electrostatic attraction between the negatively charged phosphate groups and the positively charged ammonium head groups of the surfactants. The sodium counterions of ctDNA originally present are increasingly displaced by the cationic surfactants from the negatively charged sites on the nucleic acid. This process is favored by an increase in the entropy of the system due to the release of the Na+ ions [57] as well as due to the charge fractionalization mechanism proposed by Shklovskii [58]. In analogy to Fig. S1, the Z+/Z- values at which the charge inversion occurs, is significantly higher for the monomeric surfactant than for its dimeric homologues. In fact, the value of Z+/Z- at which the ζ/ζo is zero, the isoneutrality value (where ξ-potential of the ctDNA/surfactant complex is zero), is close to 16.3 for X1 and 2.5, 2.1 and 2.4 for o-X-2, m- X-2 and p-X-2, respectively. A higher Z+/Z- is necessary for the ctDNA charge inversion by the dimeric surfactants than by the trimeric surfactants (ζ/ζo =0 at Z+/Z- = 0.93 and 0.58 for m-X-3 and p-X-3, respectively), although the difference is much smaller. Finally, similar Z+/Z- values of m-X-3 and m-X-4 induce the charge inversion for ctDNA ((ζ/ζo = 0 at Z+/Z- = 0.90 for m-X-4). As in the case of the EB competitive assays, compared to the pronounced differences observed between the behaviors of the monomeric and the dimeric surfactants, the differences between the dimeric and the trimeric series were much smaller, and the differences between the trimers and the tetramers became even marginal. Besides, the degree of oligomerization also affects the magnitude of the charge inversion observed. This is the smallest for the monomeric surfactant and the largest for the tetrameric surfactant. In regard to the effect of the spacer group on the magnitude of charge inversion, differences between the isomers are found, in contrast to their virtually identical performance in the competitive EB assays. The zeta potential data show that the dimeric as well as trimeric isomers incorporating the meta − and para − substituted xylylidene spacers behave similarly, but for the dimeric o-X-2, containing the ortho − substituted spacer, the maximum positive charge reached when Z + /Z- increases is substantially smaller than for the para − and meta − isomers. That is, the ortho − substituted spacer is significantly less effective for charging positively the nucleic acid than m-X-2 and p-X-2 (see Fig. 1c). Additional studies will be necessary in order to explain these experimental findings. Circular dichroism, CD, measurements inform about conformational changes in chiral molecules, e.g. caused by their interaction with other species. The right-handed B form of double stranded ctDNA has a characteristic CD spectrum in the far UV region (220–320 nm) at pH = 7.4. A positive band at about 280 nm, due to the stacking interactions between bases, and a negative band around 247 nm, resulting from the right-handed helicity of the nucleic acid that provides an asymmetric environment for the bases, are observed [59]. Changes in the DNA's CD spectrum upon the addition of different species provide information about their interaction with the nucleic acid [60–64]. 15 favoring the displacement of the single positively charged EB from the nucleic acid. Such enhanced interactions could also explain the facilitated ctDNA charge inversion observed in the zeta potential measurements, since for each 2- site in the nucleic acid, at least 4+ charges (corresponding to one surfactant molecule) are bound. In regard to the possibility of self-aggregation of the m-X-4 surfactant at the nucleic acid surface, note that the micellar aggregates of m-X-4, as well as of another tetrameric surfactants self-aggregation of the m-X- 4 surfactant at the nucleic acid surface, note that the micellar aggregates of m-X-4, as well as of another tetrameric surfactants in aqueous solution, are formed by very few surfactant molecules only (aggregation numbers ∼ 3) [50,69]. Therefore, if m-X-4 aggregates assembled at the polynucleotide surface, they would probably also be formed by few surfactant molecules. This would render the compaction of the ctDNA less favorable since it is the formation of highly positively charged surfactant aggregates at the ctDNA surface that favors nucleic acid compaction [40]. In line with this reasoning, one may speculate about the observed stagnation of the compacting efficiency when comparing the dimeric with the trimeric surfactant homologues. On the one hand, the molecular electrostatic interaction between the negative sites of ctDNA and the trications in the case of the trimeric surfactants are stronger than for the dications of the dimeric homologues. On the other hand, the notably smaller aggregates formed by m-X-3 and p-X-3 compared to the dimeric m-X-2 and p-X-2 [69] reduce the ability to compact nucleic acids. While for the dimeric surfactants compared to the single-chained, the first effect clearly outweighs the second one, the effects become more balanced for the trimeric surfactants, and finally, the second effect seems to overcome the first one in the case of the tetramer. 4.-Conclusions Several researchers have studied the efficiency of cationic surfactants as DNA compacting agents [10,11,13–19]. Results show that the structure of the surfactant strongly influences its interactions with DNA [10,11]. The effect of an increase in the positive charge of the surfactant head as well as in the number of cationic head groups on the formation of DNA/surfactant complexes has been examined [22,23]. The influence of changing the counterion nature [24], of increasing the length of the hydrophobic tail(s) [25–28] as well as the effects of additional side chains in the hydrophobic tails [29] on the DNA/surfactant interaction have also been investigated. In regard to the effect of the surfactant's degree of oligomerization on the DNA compaction capacity of surfactants, only dimeric surfactants have been studied [15,32–41]. The experimental observations show that, generally, for a given tail length, dimeric surfactants are more efficient DNA condensing agents than single-chained surfactants. However, with the exception of the study of Fan et al. about the interactions between a star–- shaped hexameric surfactant and DNA [46], the effect of the surfactant's degree of oligomerization, beyond dimers, on the DNA/ surfactant interaction has not been investigated yet to the authors’ knowledge. With this in mind, the capability to compact nucleic acids was investigated via a series of dimeric, trimeric and tetrameric surfactant homologues derived from N-benzyl-N,N-dimethyl-N-(1-dodecyl) ammonium chloride. Moreover, isomers within this series were compared, in which the distance between the individual surfactant units is controlled by different rigid spacer groups. In order to evaluate the behavior of the various surfactants, the results of several measurements were combined and compared, namely of the competitive displacement of DNA-bound ethidium bromide, of zeta potential measurements 16 of the DNA after surfactant addition, of circular dichroism due to changes of the DNA conformation induced by the surfactants, and of the visualization of the DNA exposed to the surfactants. The superimposed results demonstrate that the degree of oligomerization as well as the structure of the spacer groups linking the individual surfactant fragments within the oligomers are important parameters for their efficiency to compact DNA by changing the conformation of the nucleic acid from an elongated coil to a globular form. Still, the results also reveal that the effects are complex. There is no simple relationship between the degree of oligomerization of the surfactants and their ctDNA compaction capacity. That is, even if an increase in the number of hydrophobic chains is accompanied by a substantial diminution in the critical micelle concentration in aqueous solutions (as it is the case for the surfactants studied [50,69]), this does not imply that the surfactant efficacy to compact DNA increases. In addition to the electrostatic attraction between the cationic surfactants and the ctDNA, another important factor seems to be the tendency of the surfactant to self-aggregate at the nucleic acid surface. Moreover, it seems that efficient DNA compaction requires the correct placement of the cationic charges and the hydrophobic tails within the oligomeric surfactants, thus asking for the proper design of the spacer group. In consequence, the trimeric surfactants studied and the analogous dimeric surfactants turned out to be the most effective for DNA compaction, whereas the tetrameric homologue showed low efficacy, as did the monomeric parent surfactant N-benzyl-N,N- dimethyl-N-(1-do- decyl)ammonium chloride. Since the star-shaped hexameric surfactant studied by Fan et al. was found to be an efficient DNA compacting agent [46], the study of the influence of the surfactant´s degree of oligomerization on the DNA/ surfactant interaction is worth further investigation. Future work will involve the study of interactions of different oligomeric star-like surfactants with rather extended flexible spacers and ctDNA. By comparison of the results obtained for the star-like surfactants with those found in this work for linear oligomers with rigid spacers, interesting similarities as well as differences could be found. This investigation will be of particular interest for researchers working on the design of more effective cationic surfactants as nonviral vectors for gene therapy. Acknowledgements This work was financed by the Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía (P12- FQM-1105, FQM-274 and FQM-206), Spain and University of Seville (2018/500), Spain. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version. 17 References [1] V.B. Teif, K. Bohinc, Condensed DNA: condensing the concepts, Prog. Biophys. Mol. Bio. 105 (2011) 208–222. [2] A.M. Maguire, F. Simonelli, E.A. Pierce, E.N. Pugh, F. Mingozzi, J. Bennicelli, S. Banfi, K.A. Marshall, F. Testa, E.M. Surace, S. Rossi, A. Lyubarsky, V.R. Arruda, B. Konkle, E. Stone, J. Sun, J. Jacobs, L. Dell’Osso, R. Hertle, J.-X. Ma, T.M. Redmond, X. Zhu, B. Hauck, O. Zelenaia, K.S. Shindler, M.G. Maguire, J.F. Wright, N.J. Volpe, J.W. McDonnell, A. Auricchio, K.A. High, J. Bennett, Safety and efficacy of gene transfer for Leber’s congenital amaurosis, N. Engl. J. Med. 358 (2008) 2240–2248. [3] J.C. Lindomar, K.A. Albuquerque, M.P. Milazzotto, B. Mattei, K.A. Riske, E. Jäger, J. Pánek, P. Štěpánek, P. Kapusta, P.I.R. Muraro, A.G.O. De Freitas, V. Schmidt, C. Giacomelli, J.J. Bonvent, F.C. Giacomelli, Efficient condensation of DNA into environmentally responsive polyplexes produced from block catiomers carrying amine or diamine groups, Langmuir 32 (2016) 577–586. [4] S.Y. Wong, J.M. Pelet, D. Putnam, Polymer systems for gene delivery–past, present, and future, Prog. Polym. Sci. 32 (2007) 799–837. [5] N.A. Kasyanenko, D.A. Mukhin, I.Yu. Perevyazko, Conformational changes of a DNA molecule induced by metal complexes formed in solution, Polym. Sci. Ser. C 52 (2010) 122–133. [6] J.A. Lebrón, F.J. Ostos, M. López-López, M.L. Moyá, O. Kardell, A. Sánchez, C.J. Carrasco, M. García-Calderón, C.B. García-Calderón, I.V. Rosado, P. López- Cornejo, Preparation and characterization of metallomicelles of Ru(II): cytotoxic activity and use as vectors, Colloid Surf. B 175 (2019) 116–125. [7] M.A. Mintzer, E.E. Simanek, Nonviral vectors for gene delivery, Chem. Rev. 109 (2008) 259–302. [8] U. Lächelt, E. Wagner, Nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond), Chem. Rev. 115 (2015) 11043–11078. [9] K. Kim, W.C.W. Chen, Y. Heo, Y. Wang, Polycations and their biomedical applications, Progr. Polym. Sci. 60 (2016) 18–50. [10] D. Zhi, Y. Bai, J. Yang, S. Cui, Y. Zhao, H. Chen, S. Zhang, A review on cationic lipids with different linkers for gene delivery, Adv. Colloid Interface Sci. 253 (2018) 117– 140. [11] D. Zhi, S. Zhang, S. Cui, Y. Zhao, Y. Wang, D. Zhao, The head group evolution of cationic lipids for gene delivery, Bioconj. Chem. 24 (2013) 487–519. [12] P. Dubruel, E. Schacht, Vinyl polymers as non-viral gene delivery carriers: current status and prospects, Macromol. Biosci 6 (2006) 789–810. [13] I. Zuhorn, J.B.F.N. Engberts, D. Hoekstra, Gene delivery by cationic lipid vectors: overcoming cellular barriers, Eur. Biophys. J. 36 (2007) 349–362. [14] V.I. Martín, B. Sarrión, M. López-López, P. López-Cornejo, I. Robina, M.L. Moyá, Reversibility of the interactions between a novel surfactant derived from lysine and biomolecules, Colloids Surf. B 135 (2015) 346–356. [15] A.J. Kirby, P. Camilleri, J.B.F.N. Engberts, M.C. Feiters, R.J.M. Nolte, O. Söderman, M. Bergsma, P.C. Bell, M.L. Fielden, C.L. García Rodríguez, P. Guédat, A. Kremer, C. McGregor, C. Perrin, G. Ronsin, M.C.P. van Eijk, Gemini surfactants: New synthetic vectors for gene transfection, Angew. Chem., Int. Ed. 42 (2003) 1448–1457. [16] A. Zinchenko, O. Pyshkina, A. Lezov, V. Sergeyev, K. Yoshikawa, R.S. Dias, B. Lindman (Eds.), DNA Interactions With Polymers and Surfactants, Wiley, Hoboken, 18 NJ, 2008, pp. 59–88. [17] N. Kasyanenko, I. Unksov, V. Bakulev, S. Santer, DNA interaction with head-to- tail associates of cationic surfactants prevents formation of compact particles, Molecules 23 (2018) 1576. [18] P.S. Gehlot, H. Gupta, A. Kumar, Paramagnetic surface active ionic liquids: interaction with DNA and MRI application, J. Colloid Interface Sci. Comm. 26 (2018) 14–23. [19] R.B. Gurpreet, G. Kaur, S.K. Mehta, Exploring interactions of copper hybrid surfactants with calf thymus-DNA, J. Mol. Liq. 241 (2017) 715–721. [20] M. López-López, P. López-Cornejo, V.I. Martín, F.J. Ostos, C. Checa-Rodríguez, R. Prados-Carvajal, J.A. Lebrón, P. Huertas, M.L. Moyá, Importance of the hydrophobic interactions in the single-chained cationic surfactant-DNA complexation, J. Colloid Interface Sci. 521 (2018) 197–205. [21] T. Chaudhuri, A. Pan, S. Das, S.P. Moulik, Ratimetric interactions of anionic surfactants with calf thymus DNA bound to cationic surfactants: study II, J. Surfact. Deterg. 21 (2018) 127–137. [22] A. Piecuch, Ł. Lamch, E. Paluch, E. Obłazk, K.A. Wilk, Biofilm prevention by dicephalic cationic surfactants and their interactions with DNA, J. Appl. Microbiol. 121 (2016) 682–692. [23] A. Piecuch, Ł. Lamch, E. Paluch, E. Obłazk, K.A. Wilk, Biofilm prevention by dicephalic cationic surfactants and their interactions with DNA, J. Appl. Microbiol. 121 (2016) 682–692. [24] J.A. Lebrón, F.J. Ostos, M. López-López, M.L. Moyá, O. Kardell, A. Sánchez, C.J. Carrasco, M. García-Calderón, C.B. García-Calderón, I.V. Rosado, P. López- Cornejo, Preparation and characterization of metallomicelles of Ru(II): cytotoxic activity and use as vectors, Colloid Surf. B 175 (2019) 116–125. [25] L. Zhang, Y. Dong, X. Zhang, X. Guo, Micellization of lactosylammonium surfactants with different counter ions and their interaction with DNA, J. Chem. Eng. Data 61 (2016) 2969–2978. [26] L. Guo, Z. Zhang, H. Qiao, M. Liu, M. Shen, T. Yuan, J. Chen, D.D. Dionysiou, Spectroscopic study on interaction between three cationic surfactants with different alkyl chain lengths and DNA, Spectrochim. Acta A 151 (2015) 237–246. [27] S. Husale, W. Grange, M. Karle, S. Buergi, M. Hegner, Interaction of cationic surfactants with DNA: a single-molecule study, Nucleic Acids Res. 36 (2008) 1443–1449. [28] J. Akbar, N. Tavakoli, D.G. Marangoni, S.D. Wettig, Mixed aggregate formation in gemini surfactant/1,2-dialkyl-sn-glycero-3-phosphoethanolamine systems, J. Colloid Interface Sci. 377 (2012) 237–243. [29] V.A. Izumrudov, M.V. Zhiryakova, A.A. Goulko, Ethidium bromide as a promising probe for studying DNA interaction with cationic amphiphiles and stability of the resulting complexes, Langmuir 18 (2002) 10348–10356. [30] Z. Pietralik, J.R. Kumita, C.M. Dobson, M. Kozak, The influence of novel gemini surfactants containing cycloalkylside-chains on the structural phases of DNA in solution, Colloid Surf. B 131 (2015) 83–92. [31] E. Fisicaro, C. Compari, F. Bacciottini, L. Contardi, E. Pongiluppi, N. Barbero, G. Viscardi, P. Quagliotto, G. Donofrio, M.P. Krafft, Nonviral gene-delivery by highly fluorinated gemini bispyridinium surfactant-based DNA nanoparticles, J. Colloid Interface Sci. 487 (2017) 182–191. [32] L. Wang, S. Dong, J. Hao, Recent progress of magnetic surfactants: self-assembly, properties and functions, Curr. Opin. Colloid Interface 35 (2018) 81–90. [33] T. Ahmed, A.O. Kamel, S.D. Wettig, Interactions between DNA and gemini 19 surfactant: impact on gene therapy: parts I and II, Nanomedicine 11 (2016) 289-306 and 403-420. [34] E. Aicart, E. Junquera, Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors, Adv. Colloid Interface Sci. 233 (2016) 161–175. [35] K. Kumar, A.L. Barrán-Berdón, S. Datta, M. Múñoz-Úbeda, C. Aicart-Ramos, P. Kondaiah, E. Junquera, E. Aicart, A delocalizable cationic head group together with an oligo-oxyethylene spacer in gemini cationic lipids improves their biological activity as vectors of plasmid DNA, J. Mater. Chem. 2 (2015) 1495–1506. [36] X. Zhao, Y. Shang, J. Hu, H. Liu, Y. Hu, Biophysical characterization of complexation of DNA with oppositely charged gemini surfactant 12-3-12, Biophys. Chem. 138 (2008) 144–149. [37] E. Grueso, E. Roldan, P. Perez-Tejeda, E. Kuliszewska, B. Molero, L. Brecker, R.M. Giraldez-Perez, Reversible DNA compaction induced by partial intercalation of 16- Ph-16 gemini surfactants: evidence of triple helix formation, PCCP 20 (2018) 24902– 24914. [38] N. Barbero, C. Magistris, P. Quagliotto, L. Bonandini, C. Barolo, R. Buscaino, C. Compari, L. Contardi, E. Fisicaro, G. Viscardi, Guido, synthesis, physicochemical characterization, and interaction with DNA of long-alkyl-chain gemini pyridinium surfactants, ChemPlusChem 80 (2015) 952–962. [39] B. Sarrión, E. Bernal, V.I. Martín, M. López-López, P. López-Cornejo, M. García- Calderón, M.L. Moyá, Binding of 12-s-12 dimeric surfactants to calf thymus DNA: evaluation of the spacer length influence, Colloid Surf. B 144 (2016) 311–318. [40] V. Kumar, A. Chaterjee, N. Kumar, A. Ganguly, I. Chakraborty, M. Banerjee, d- Glucose derived novel gemini surfactants: synthesis and study of their surface properties interaction with DNA, and cytotoxicity, Carbohydr. Res. 397 (2014) 37–45. [41] L. Karlsson, M.C.P. van Eijk, O. Söderman, Compaction of DNA by gemini surfactants: effects of surfactant architecture, J. Colloid Interface Sci. 252 (2002) 290–296. [42] S.D. Wettig, R.E. Verrall, M. Foldvari, Gemini surfactants: a new family of building blocks for non-viral gene delivery systems, Curr. Gene Ther. 8 (2008) 9–23. [43] S.G. Silva, I.S. Oliveira, M.L.C. do Vale, E.F. Marques, Serine-based gemini surfactants with different spacer linkages: from self-assembly to DNA compaction, Soft Matter 46 (2014) 9352–9361. [44] C. Bombelli, S. Borocci, M. Diociaiuti, L. Galantini, P. Luciani, G. Mancini, M.G. Sacco, Role of the spacer of cationic gemini amphiphiles in the condensation of DNA, Langmuir 21 (2005) 10271–10274. [45] L. Hubčík, D. Galliková, P. Pullmannová, L. Lacinová, Z. Sulová, M. Hanulová, S.S. Funari, F. Devínsky, D. Uhríková, DNA-DOPE-gemini surfactants complexes at low surface charge density: from structure to transfection efficiency, Gen. Physiol. Biophys. 37 (2018) 57–69. [46] F.M. Menger, J.N. Keiper, Gemini surfactants, Angew. Chem. Int. Ed. 39 (2000) 1906–1920. [47] Y. Fan, H. Wang, C. He, F. Qiao, S. Wang, Y. Wang, DNA condensation induced by a star-shaped hexameric cationic surfactant, ACS Appl. Mater. Interfaces 9 (2017) 23333–23341. [48] Y.J. Liu, H. Chao, L.F. Tan, Y.X. Yuan, L.N. Ji, Ruthenium(II) mixed-ligand complexes containing 2-(6-methyl-3-chromonyl)-imidazol[4,5-f][1,10]-phenanthroline: synthesis, DNA-binding and photoclavage studies, Inorg. Chim. Acta 358 (2005) 1904– 1910. [49] F. Secco, M. Venturini, T. Biver, F. Sanchez, R. Prado-Gotor, E. Grueso, Solvent 20 effects on the kinetics of the interaction of 1-pyrenecarboxaldehyde with calf thymus DNA, J. Phys. Chem. B 114 (2010) 4686–4691. [50] W. Saenger, Principles of Nucleic Structure, Springer-Verlag, New York, 1984. [51] A. Laschewsky, L. Wattebled, M. Arotcarena, J.L. Habib-Jiwan, R.H. Rakotoaly, Synthesis and properties of cationic oligomeric surfactants, Langmuir 21 (2005) 7170–7179. [52] A. Laschewsky, K. Lunkenheimer, R.H. Rakotoaly, L. Wattebled, Spacer effects in dimeric cationic surfactants, Colloid Polym. Sci. 283 (2005) 469–479. [53] I. Horcas, R. Fernández, J.M. Gómez-Rodríguez, J. Colchero, J. Gómez-Herreo, A.M. Baro, WSxM: a software for scanning probe microscopy and a tool for nanotechnology, Rev. Sci. Instrum. 78 (2007) 013705. [54] V. Gabelica, E. De Pauw, F. Rosu, Interaction between antitumor drugs and a double-stranded oligonucleotide studied by electrospray ionization mass spectrometry, J. Mass. Spectrom. 34 (1999) 1328–1337. [55] G. Cosa, K.S. Focsaneau, J.R.N. Mc Lean, J.P. Mac Namee, J.C. Scaiano, Photophysical properties of fluorescent DNA-dyes bound to single- and doublestranded DNA in aqueous buffered solutions, Photochem. Photobiol. 73 (2001) 585–599. [56] M. Múñoz-Úbeda, S.K. Misra, A.L. Barrán-Berdón, C. Aicart-Ramos, M.B. Sierra, J. Biswas, P. Kondaiah, E. Junquera, S. Bhattacharya, E. Aicart, Why is less cationic lipid required to prepare lipoplexes from plasmid DNA than linear DNA in gene therapy? J. Am, Chem. Soc. 133 (2011) 18014–18017. [57] X. Zhao, Y. Shang, H. Liu, Y. Huang, Interaction of DNA with cationic gemini surfactants trimethylene-1,3-bis-(dodecyldimethylammonium bromide) and anionic surfactant SDS mixed system, J. Colloid Interface Sci. 314 (2007) 478–483. [58] D. Matulis, I. Rouzina, V. Bloomfield, Thermodynamics of cationic lipid binding to DNA and DNA condensation: roles of electrostatics and hydrophobicity, J. Am. Chem. Soc. 124 (2002) 7331–7342. [59] B.I. Shklovskii, Screening of a macroion by multivalent ions: correlation-induced inversion of charge, Phys. Rev. E: Stat. Phys. Plasmas Fluids Relat. Interdiscip. Top. 60 (1990) 5802–5811. [60] S. Neidle, Nucleic Acid Structure and Recognition, Oxford University Press, NewYork, 2002, pp. 89–138. [61] N. Berova, K. Nakanishi, R.W. Woody, Circular Dichroism: Principles and Applications, 2nd ed., Wiley-VCH, New York, 2000. [62] L. Wang, Y. Wang, X. Sun, G. Zhang, S. Dong, J. Hao, Versatile self-assembly and biosensing applications of DNA and carbon quantum dots coordinated cerium ions, Chem. Eur. J. 23 (2017) 10413–10422. [63] L. Wang, Y. Wang, J. Hao, S. Dong, Magnetic fullerene-DNA/hyaluronic acid nanovehicles with magnetism/reduction dual-responsive triggered release, Biomacromolecules 18 (2017) 1029–1038. [64] L. Wang, G. Wang, Y. Wang, H. Liu, S. Dong, J. Hao, Fluorescent hybrid nanospheres induced by single-stranded DNA and magnetic carbon quantum dots, New J. Chem. 43 (2019) 4965–4974. [65] P. Lincoln, A. Broo, B. Norden, Diastereomeric DNA-binding geometriesof intercalated ruthenium(ii) trischelates probed by linear dichroism: [Ru(phen) 2 DPPZ] 2+ and [Ru(phen) 2 BDPPZ] 2+ , J. Am. Chem. Soc. 118 (1996) 2644–2653. [66] E. Grueso, E. Kuliszewska, E. Roldán, P. Pérez-Tejeda, R. Prado-Gotor, L. Brecker, DNA conformational changes induced by cationic gemini surfactant: the key to switching DNA compact structures to elongated forms, RSC Adv. 5 (2015) 29433–29446. [67] M. Monnot, O. Mauffret, E. Lescot, S. Fermandjian, Probing intercalation and 21 conformational effects of the anticancer drug 2-methyl-9-hydroxyellipticinium acetate in DNA fragments with circular dichroism, Eur. J. Biochem. 204 (1992) 1035–1039. [68] B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, J.D. Watson, Molecular Biology of the Cell, 4th edn., Galland, New York, 1994. [69] R. Di Profio, L. Germani, R. Goracci, G. Grilli, M. Savelli, Tiecco, Interaction between DNA and cationic amphiphiles: a multi-technique study, Langmuir 26 (2010) 7885– 7892. [70] L. Wattebled, A. Laschewsky, A. Moussa, J.L. Habib-Jiwan, Aggregation numbers of cationic oligomeric surfactants: a time-resolved fluorescence quenching study, Langmuir 22 (2006) 2551–2557. 22