Importance of hydrophobic interactions in the single-chained cationic surfactant-DNA complexation

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Journal of Colloid and Interface Science on 1 July 2018, available at: https://doi.org/10.1016/j.jcis.2018.03.048 .” 1 Graphical abstract A decrease in the critical micelle concentration favors the surfactant-DNA complexation 2 IMPORTANCE OF HYDROPHOBIC INTERACTIONS IN THE SINGLE-CHAINED CATIONIC SURFACTANT-DNA COMPLEXATION Manuel López-López,1 Pilar López-Cornejo, Victoria Isabel Martín, Francisco José Ostos, Cintia Checa-Rodríguez,2 Rosario Prados-Carvajal,2 José Antonio Lebrón, Pablo Huertas,2 and María Luisa Moyá* Department of Physical Chemistry, University of Seville, C/ Profesor García González 1, 41012 Seville. Spain. Tlf. 34954557175 Fax: 34954557174 1Department 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 2Department of Genetics, University of Seville and Andalusian Center for Molecular Biology and Regenerative Medicine-CABIMER, University of Seville-CSIC-University Pablo de Olavide, 41092, Spain. Keywords: DNA, single-chained cationic surfactants, complexation, charge inversion, compaction, critical micelle concentration *Author to whom all correspondence should be directed 3 Abstract The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency. 1. Introduction Deoxyribonucleic acid, DNA, is a nucleic acid that contains the genetic instructions for the development and functioning of living organisms. The ability of DNA to bind polycation molecules plays an important role in the physical properties and biological functions of DNA [1]. It was found that DNA chains usually show an elongated coil state in aqueous solution. However, DNA molecules exist in a highly packed state in various organisms from viruses to eukaryotic cells in order to store, transport and preserve the genetic material. DNA chains undergo a coil–globule transition upon the increasing concentration of condensing agents in solution [2]. Compaction is caused by modifying electrostatic interactions between DNA segments, by changing DNA–solvent interactions, by excluding volume to the worm-like coil (and/or counterions), by provoking localized bending or distortion of the helical structure, or by some combination of these effects. The mechanism of 4 condensation of DNA is not only important for interpreting basic biological processes, but it is also relevant for the development of new techniques for DNA extraction, gene therapy and gene transfection [3]. In particular, for the design of non-viral gene therapy vectors it is necessary to transform the DNA macromolecule into the compact form. Multivalent metal cations, polyamines, cationic and non-ionic surfactants, catanionic liposomes, and polymers have been used as compacting agents [4-15]. Compaction may also be induced by changes in the relative permittivity of the medium [16]. The efficiency of several cationic surfactants as condensing agents has been investigated [8, 10-15, 17-19]. Initially, binding of cationic surfactants to the negatively charged DNA occurs via electrostatic interactions, the DNA cationic counterions being exchanged by the surfactant. At this stage practically no change in the global DNA complex charge occurs. The main driving force is the entropy gain resulting from the counterion release. As binding progresses, hydrophobic interactions between the incoming cationic surfactant and the layer of bound surfactant become more important, which explains the high degree of cooperativity observed in the binding isotherms. The relevance of the hydrophobic interactions in the association of cationic surfactants to DNA is shown by the fact that binding isotherms shift to lower surfactant concentrations (i.e. the binding is stronger) when the hydrophobic chain length of the cationic surfactant increases [20]. For all surfactants in water, formation of micelles (or aggregates) is made possible by the negative hydrophobic Gibbs energy contribution to the Gibbs energy of self-aggregation. This term considers the Gibbs energy change accompanying the transfer of the surfactant hydrophobic tail (or tails) from the bulk aqueous phase into the aggregate. In terms of absolute magnitude, it is the largest Gibbs energy term and controls the surfactant concentration at which aggregation occurs [21]. Taking this into account, it would be reasonable to expect a close relation between the strength of the surfactant association to DNA, that is, their DNA compacting efficiency, and 5 its tendency to self-aggregate. As was mentioned above, in previous works the importance of the hydrophobic interactions in the surfactant-DNA complexation has been suggested, but a parameter directly linked to the self-association tendency of surfactants which could be used as a criterion to foresee the surfactant DNA compaction capacity has not been found. With this in mind, the authors focused their attention in the study of the binding of single-chained cationic surfactants to DNA with the goal of understanding the key factors determining the DNA compacting capacity of these surfactants. The results of this work will be of interest for researchers investigating the design and preparation of new non-viral vectors for gene therapy, especially for those focused on the use of cationic lipids. 2. Materials and methods 2.1. Chemicals and reagents Materials were purchased from Sigma-Aldrich and Fluka, of the highest purity available. Some of the cationic surfactants used in this work were commercial, such as dodecyl-, tetradecyl-, and hexadecyltrymethyalmmonium bromide (DTAB, TTAB, CTAB, the purity of the three surfactants was 99%), hexadecyl- and tetradecyltrimethylammonium chloride (CTAC and TTAC, the purity of both of them being >98%), hexadecyl- and dodecylpyridinium bromide and chloride (CPB and DPB, CPC and DPC, the purity of the four surfactants was 98%), dodecyldimethylbenzylammonium bromide (DDBAB, the purity was >99%) or 1-dodecyl-3-methylimidazolinium bromide (DMIB, the purity was 98%). The dodecyltriethylammonium bromide (DTEAB), (S)-5-acetamido-6-(dodecylamino)-N,N,N- trimethyl-6-oxohexan-1-ammonium chloride (ADTAC), N-benzyl-N,N-dimethyl-N-(1- dodecyl)ammonium bromide (BDDAB), and N-cyclohexyl-N,N-dimethyl-n-(1- dodecyl)ammonium bromide (CDDAB) surfactants were synthesized and thoroughly purified 6 according to refs. 22-24. The structure of the surfactants used in this work is shown in Scheme 1. N CH3 Br ( )15 CH3 H3C CH3 N CH3 Br ( )13 CH3 H3C CH3 N CH3 Br ( )11 CH3 H3C CH3 N CH2-CH3 Br ( )11 CH3 CH3-CH2 CH3-CH2 CTAB TTAB DTAB DTEAB N H3C CH3 CH3 Br ( )11 N H3C CH3 CH3 Br ( )11 N N CH3 CH2(CH2)10CH3 Br BDDAB CDDAB DMIB O N H Br ON(CH2)11CH3 H3C CH3 N CH3Br ( )11 CH3 H2C CH3 BEDDAB BDAB Br N CH2(CH2)14CH3 Br N CH2(CH2)10CH3 N CH3 Cl ( )15 CH3 H3C CH3 N CH3 Cl ( )13 CH3 H3C CH3 CPB DPB CTAC TTAC NMe3 H N N HO Cl O CH2(CH2)10CH3 Cl N CH2(CH2)14CH3 Cl N CH2(CH2)10CH3 ADTAC CPC DPC Scheme 1.-Structure of the surfactants used in this work. 7 Calf thymus DNA concentration, given by phosphate groups, was determined spectrophotometrically from molar absorptivity (6600 mol−1 dm3 cm−1 at 260 nm) [25]. An agarose gel electrophoresis test using ethidium bromide, EB, indicated that the average number of base pairs per DNA molecule is above 10000 bp [26]. Absence of proteins was suggested by ratios in absorbance at 260 and 280 nm of the solutions between 1.8 and 1.9 [27]. DNA solutions were all buffered by using 40 mM HEPES (Sigma-Aldrich, purity> 99.5%) at pH=7.4. Distilled water was deionized using a system Super Q Millipore (resistivity >18 M cm). 2.2.Preparation of [2-(2-benzoylammino-etoxy)ethyl]-N-dodecyl-N,N-dimethyl-ammonio bromide, BEDDAB The synthesis of [2-(2-benzoylammino-etoxy)ethyl]-N-dodecyl-N,N-dimethyl- ammonio bromide (BEDDAB), 3, was performed according to Scheme 2. Cl O DCM/H2O O N HOOH H2NOOH HSO4 NBu4 O N HOBr PPh3 CBr4 O N H Br Acetone ON(CH2)11CH3 H3C CH3 (CH3)2N(CH2)11CH3 12 3 Scheme 2.-Preparation of BEDDAB. A solution of benzoyl chloride (4 mL) is added dropwise to a solution of 2-(2-amino- ethoxy)ethanol (4 mL) in dichloromethane (3 mL), DCM, and NaOH 1.8 M (8 mL), in the presence of a catalytic amount of tetrabutylammonium hydrogensulfate. The mixture was stirred for 12 hours and intermediate 1 was obtained as a whittish oil. Subsequently, to a solution of 1 (3.52 g) and triphenylphosphine (6.2 g), Ph3P, in DCM, carbon tetrabromide (7.82 g), CBr4, was added under inert atmosphere. After 1 hour of stirring, 7 mL of water is 8 added and the agitation goes on for 5 more minutes. The solution is evaporated and compound 2 is obtained. In the next step N,N-dimethyldodecylammine (3.87 g) is added to an acetone solution of 2 (2.46 g), keeping the mixture under reflux for 1 day. The reaction mixture was then concentrated to dryness and the residue purified by crystallization from hexanol to give a dark orange oil (3) (4.05 g, 97 %). Procedures for the preparation of the new surfactant and intermediates are described in detail in Supplementary Material as well as the characterization of the new compounds. 2.3. Determination of the critical micelle concentration, cmc, of BEDDAB Conductivity was measured with a Crison GLP31 conductimeter. A dispenser Crison Burette 1S was programmed to add the adequate quantities of a concentrated surfactant solution in order to change the [surfactant] from concentrations well below the cmc, up to at least two to three times the cmc concentration. This method allows one to obtain a large number of experimental conductivity data, the estimation of the cmc being more accurate. The conductimeter was calibrated with KCl solutions of the appropriate concentration range. 2.4. UV-visible absorption spectroscopy The stability of the buffered surfactant-DNA complex solutions was investigated using a CARY 500 SCAN UV-vis-NIR spectrophotomer (Varian) connected to a water flow Lauda cryostat. Temperature was kept at 303.00.1 K. A standard quartz cell of 10 mm path length was used. The absorbance of the solutions at 260 nm was found to be stable for more than three hours for all the surfactants investigated. Besides, no turbidity was observed in the solutions for all the surfactant concentrations used. 2.5. Competitive binding experiments Fluorescence emission measurements were done by using a Hitachi F-2500 fluorescence spectrophotometer. The temperature was kept at 303.00.1 K by a water flow Lauda cryostat connected to the cell compartment. Ethidium bromide (EB) emission spectra 15 Morphology changes in DNA molecules can be followed by circular dichroism spectroscopy, CD [41]. Figure 4 shows the CD spectra of some surfactant-DNA complex solutions at 303 K. The black solid line in this figure corresponds to the pure DNA spectrum 220 240 260 280 300 -3 -2 -1 0 1 2 3 /nm obs/mdeg DNA BEDDAB-DNA Z+/-=15 BEDDAB-DNA Z+/-=10 BEDDAB-DNA Z+/-=5 BEDDAB-DNA Z+/-=20 Figure 4.- Circular dichroism spectra in 40 mM HEPES at pH=7.4. T=303 K. of the B-DNA structure characterized for two bands: a positive peak close to 278 nm, caused by stacking interactions between the bases, and a negative peak at 247 nm, caused by the 220 240 260 280 300 -3 -2 -1 0 1 2 3 /nm obs/mdeg DNA DTEAB-DNA Z+/-=100 16 helical supra-structure of the nucleic acid that provide an asymmetric environment for the basis. For a DTEAB-DNA solution with Z+/-=100, no substantial changes in the DNA CD spectrum are observed. However, addition of increasing amounts of BEDDAB to the DNA solution results in important variations in the DNA spectrum. The intensities of both the negative and positive bands decrease upon increasing [BEDDAB] and the two peaks practically disappear at high Z+/- values. These changes indicate the DNA compaction and partial denaturation of the double strand which agree with a morphological change from an elongated structure to a globular one [42]. In order to visualize the morphological changes caused in the nucleic acid by the surfactant binding, atomic force microscopy, AFM, experiments were done. Figure 5 shows the AFM topographic images of the solutions adsorbed onto an APTES modified mica surface for DTEAB and BEDDAB and varying Z+/- values. These structures were stable in a time scale of hours. The first image in Figure 5 corresponds to a pure DNA solution, with its coil conformation. In the case of DTEAB, even at high Z+/- values, DNA in an elongated state is observed and only some globular structures were found. This is consistent with , fluorescence, electrophoresis and CD results which showed that DTEAB binding to DNA is not strong enough to neutralize and invert the nucleic acid charge, the surfactant association not resulting in the compaction of the nucleic acid. For BEDDAB at Z+/-=12.5 more than one structure coexist, but at Z+/-=35 no extended coils are observed and only globules were found. Images 5C and 5D indicate that BEDDAB is an efficient compacting agent. Figure S3 (Supplementary Material) shows the AFM images corresponding to the systems DTAB-DNA and CDDAB-DNA. One can see that the images are consistent with the  values observed for the different systems. Surfactants which cannot neutralize the DNA (and, consequently, do not invert its charge) do not compact the nucleic acid and elongated structures of the DNA are observed. When the binding of the surfactants to the DNA results in DNA neutralization 17 Figure 5.- AFM topographic images of some surfactant-DNA complex solutions, 40 mM HEPES at pH=7.4, adsorbed on APTES modified mica surface. Pure DNA; A)DTEAB-DNA Z+/-=50; B) DTEAB-DNA Z+/-=100; C) BEDDAB-DNA Z+/-=12.5; D)BEDDAB-DNA Z+/- =35. followed by a charge inversion, a total compaction of the nucleic acid occurs and only globular structures are observed by AFM measurements. The [surfactant] necessary to 1.93 nm 0.04 nm 180nm 2.01 nm -0.29 nm 180nm C D Pure DNA 18 compact the DNA depends on its cmc, being lower the higher the tendency to selfaggregation is. The experimental results show the relevance of the surfactant cmc for predicting its efficiency as a DNA compacting agent. With this in mind, it is interesting to make some comments about the different Gibbs energy contributions determining the cmc value and, consequently, the Gibbs energy of micellization, GoM [43, 44]. These contributions are: i)The surfactant tail transfer Gibbs energy, also known as the hydrophobic contribution, accounts for the Gibbs energy change accompanying the transfer of the surfactant tail from the solution into the micellar interior; ii)The aggregate-core solvent interfacial Gibbs energy takes into account that the formation of a micelle creates an interface allowing for contact between the hydrophobic core and the solution; iii)The head groups interaction Gibbs energy considers the electrostatic repulsions between the surfactant head groups at the micelle surface; iv)The deformation Gibbs energy takes into account that the surfactant tail inside the micelle has a conformation different from that in a pure hydrocarbon liquid because of the molecular packing requirements inside the micelle; and v)The steric Gibbs energy considers that the head groups at the micelle surface experienced steric repulsions. As was mentioned above, the hydrophobic contribution is considered the driving force for the self-association process of surfactants [45]. A simple approach to quantify the tendency of a molecule to bind the DNA is to estimate the apparent association equilibrium constant of the molecule to the nucleic acid, Kapp. This equilibrium constant can be obtained from the following equation [46]: 𝐾𝐸𝐵[𝐸𝐵]= 𝐾𝑎𝑝𝑝[𝑠𝑢𝑟𝑓𝑎𝑐𝑡𝑎𝑛𝑡50%] (1) where [EB] is the ethidium bromide concentration used in the competitive binding experiments, KEB is the equilibrium binding constant of EB to the nucleic acid [47], and 19 [surfactant50%] is the surfactant concentration corresponding to a 50% reduction in the fluorescence intensity of DNA bound EB. Figure 6 shows the plot of ln(Kapp) vs. ln(cmc) for the different bromide surfactants investigated. A reasonable good linear correlation was observed, including surfactants with different hydrophobic chain length. ln(Kapp/M-1) -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 9 10 11 12 13 14 15 BEDDAB CDDAB BDDAB DPB DMIB DTAB DTEAB BDAB TTAB CTAB CPB CTAC CPC TTAC ADTOAC DPC ln(cmc/M) Figure 6.- Plot of ln(Kapp) vs. ln(cmc) for the different surfactants investigated. Black circles: bromide surfactants; Red circles: chloride surfactants. T=303 K. In relation to the counterion nature, it is known that it influences the surfactant-DNA interactions, basically following the Hofmeister series [30]. But the cmc values, for a given surfactant, also correlate generally well with the Hofmeister series [48-51]. This experimental observation can be explained by considering the influence of the counterion nature on the reduction in the electrostatic repulsions between the ionic surfactant heads resulting from the adsorbed counterions [52, 53]. With this in mind, the authors also investigated the interactions between the DNA and several chloride single-chained cationic surfactants. As one can see in Figure 6 (red circles), the chloride surfactants fit in the same linear correlation found for the 20 bromide surfactants. This could be taken as indicative that the influence of the counterion nature on the surfactant-DNA interactions mirrors its influence on the cmc. The meaning of the correlation shown in Figure 6 has to be explained. The binding of the single-chained surfactants to DNA can be modelled considering that the cationic headgroups would be localized within several Å from DNA phosphates, while hydrophobic tails would lay down on the DNA surface, displacing all or most water molecules from the DNA hydration layer [38]. The exposed side of the bound surfactant molecules then binds to other surfactant molecules similarly layered on the adjacent DNA helix. That is, the surfactant- DNA interactions can be considered the sum of two processes: i)cationic headgroup electrostatic interaction with DNA phosphate by displacing sodium cations from counterion atmosphere, and ii) cooperative hydrophobic interaction between aliphatic tails that also brings multiple DNA molecules together. All the surfactants investigated have the same charge, but substantially different tendencies to self-aggregate, as is shown by the cmc values summarized in Table S1. Figure 6 indicates that the binding strength of the cationic singlechained surfactants to DNA is mainly controlled by their self-aggregation tendency and, consequently, when comparing their interactions with DNA, the cmc can be used as a criterion to predict the surfactant DNA compaction capacity. 4. Conclusions The efficiency of several cationic surfactants as DNA condensing agents has been investigated by several authors [8, 10, 15, 12, 17, 23, 25, 31, 33, 35, 46, 54], the association of the surfactants to the nucleic acid being explained by electrostatic as well as hydrophobic interactions. The importance of the hydrophobic interactions in the surfactant-DNA complexation has been suggested [21], but no conclusive results have been reached. With the goal of investigating this issue further, in this work the interactions between several bromide 21 and chloride single-chained cationic surfactants and calf thymus DNA have been studied. It was observed that the surfactants with the lower cmc´s were the most efficient in compacting the DNA from an elongated structure to a globular form; that is, a higher tendency of the surfactant to self-aggregate makes the surfactant-DNA complexation more favorable. The plot of ln(Kapp) vs. ln(cmc) was found to be a reasonably good linear correlation for all the surfactants studied, where Kapp, is estimated from the experimental results obtained in the EB competitive binding experiments. Therefore the cmc can be used as a criterion to predict the cationic single-chained surfactant DNA compaction efficiency. Since the cmc is mainly controlled by the hydrophobic Gibbs energy contribution to the Gibbs energy of micellization, this result shows the importance of hydrohobic interactions in the single-chained cationic surfactant-DNA complexation. To the authors´ knowledge this is the first time that only one parameter, the cmc, can be used as a criterion to predict the surfactant DNA compaction capacity. The results of this work will provide a further understanding of the main factors controlling the surfactant-DNA complexation, which is relevant for researchers investigating the design and preparation of non-viral vectors based on cationic lipids for gene transfection. The study of the binding of surfactants to DNA, and the resulting DNA compaction, is worth further investigation. Future work will involve the study of interactions between dimeric surfactants and DNA. This investigation is of particular interest since many dimeric surfactants has been found to be efficient non-viral vectors in gene transfection [8, 12, 15, 18, 19, 35, 55]. Acknowledgements This work was financed by the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía (P12-FQM-1105), FQM-274 and FQM-206, University of Seville 22 (2017/1004), R+D+i grant from the Spanish Ministry of Economy and Competitivity (SAF2013-43255 and SAF2016-74855) and FEDER funds. 23 References [1] Z. Guo, Y. Wang, A. 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Dias, DNA surfactants interactions. in Encyclopedia of Surface ad Colloid Science, Ed. P. Somasundaran, 3rd. ed. (2016) 1810-1824. [18] 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. 31 SUPPLEMENTARY MATERIAL IMPORTANCE OF HYDROPHOBIC INTERACTIONS IN THE SINGLE-CHAINED CATIONIC SURFACTANT-DNA COMPLEXATION Manuel López-López,1 Pilar López-Cornejo, Victoria Isabel Martín, Francisco José Ostos, Cintia Checa-Rodríguez,2 Rosario Prados-Carvajal,2 José Antonio Lebrón, Pablo Huertas,2 and María Luisa Moyá* Department of Physical Chemistry, University of Seville, C/ Profesor García González 1, 41012 Seville. Spain. Tlf. 34954557175 Fax: 34954557174 1Department 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 2Department of Genetics, University of Seville and Andalusian Center for Molecular Biology and Regenerative Medicine-CABIMER, University of Seville-CSIC-University Pablo de Olavide, 41092, Spain. *Author to whom all correspondence should be directed 32 Experimental procedure for [2-(2-benzoylammino-etoxy)ethyl]-N-dodecyl-N,N- dimethyl-ammonio bromide, BEDDAB. General techniques. The characterization of the new compounds was performed by their spectral data. 1H and 13CNMR spectra were obtained for solutions in CDCl3 and CD3OD in a Bruker AMX 300 and Bruker advance AV-300 spectrometers; J values are given in Hz and  in ppm. The FABMS spectra were measured with a KRATOS MS-80 RFA instrument. The HRFABMS spectra were measured with a Micromass AutoSpeQ instrument. The NMR and MS spectra for all compounds were performed in CITIUS (Research General Service for the University of Seville). The completion of the reactions were monitored by TLC (silica gel HF254 (Merck) CH2Cl2/MeOH = 4:1 and AcOEt: Hexane = 2:3) with detection by UV light and charring with H2SO4 and ethanolic solution of ninhydrine. Elemental analysis of the new surfactant was also carried out. BEDDAB was prepared according reaction indicated in Scheme 1. N-[2-(2-Hidroxy-ethoxy)ethyl]-benzamide (1) A solution of 2-(2-amino-ethoxy)ethanol (4 mL, 39.87 mmol, 1 eq) in dichloromethane (3 mL), DCM, was added dropwise to a DCM (3 mL) benzoyl chloride (4.63 mL, 39.87 mmol, 1 eq) and NaOH (4.63 mL, 39.87 mmol, 1 eq) solution in the presence of a catalytic amount of tetrabutyammonium hidrogensulfate. The mixture was stirred for 12 hours. The progress of the reaction was controlled by TLC (DCM: MeOH= 20:1). The reaction mixture then was concentrated to dryness. The residue was purified in DCM (80 mL), washed with NaHCO3 (3 x 10 mL) and the water was eliminated with Na2SO4. The product is obtained as a transparent oil (7.1 g, 85%). 1H-RMN (300 MHz, CDCl3): δ(ppm)=7.83-7.73 (m, 2H, Ph), 7.53-7.33 (m, 3H, Ph), 6.87 (sa, 1H, NH), 3.74 (t, 2H, O-CH2-CH2-OH), 3.68-3.62 (m, 4H, NH-CH2-CH2-O), 3.59 (t, 2H, O- CH2-CH2-OH), 2.46 (sa, 1H, OH). 13C-NMR (175.4 MHz, CDCl3): δ(ppm)=167.93 (CO), 134.56, 131.59, 128.63, 127.15 (5C, Ar), 72.39, (O-CH2-CH2-OH), 70.05, 39.94 (2C, NH-CH2-CH2-O), 61.83 (O-CH2-CH2-OH). CIMS m/z 210 [28%, (M+H)+]. HRCIMS m/z obs. 210.0000, calcd. para C11H16NO3 [M]+ 210.1130. N-[2-(2-Bromo-ethoxy)ethyl]-benzamide (2) To a solution of N-[2-(2-hidroxy-ethoxy)ethyl]-benzamide (3.52 gr, 16.84 mmol) and triphenylphosphine (6.20 gr, 23.58 mmol), Ph3P, in DCM (3.45 mL), carbon tetrabrimide (7.82 gr, 23.58 mmol), CBr4, was added under an inert atmosphere. The mixture was stirred for 1 hour. Then, 7 mL of H2O were added and the mixture was shaked for 5 min. The solution was concentrated to dryness and the obtained residue was purified using column chromatography with AcOEt:Hexane=1:3 as eluent. The product was obtained as a yellowish oil (3.24 g, 71%). 33 1H-RMN (300 MHz, CDCl3): δ(ppm)=7.83-7.78 (m, 2H, Ph), 7.53-7.39 ( m, 3H, Ph), ), 6.67 (sa, 1H, NH), 3.82 (t, 2H, O-CH2-CH2-Br), 3.69 (m, 4H, NH-CH2-CH2-O), 3.50 (t, 2H, O- CH2-CH2-Br). 13C-NMR (175.4 MHz, CDCl3): δ (ppm) = 167.60 (C=O), 133.60, 130.61, 127.69, 126.13 (5C, Ar), 69.80 (O-CH2-CH2-Br), 68.77, 38.77 (2C, NH-CH2-CH2-O), 30.05 (1C, -O-CH2- CH2-Br). CIMS m/z 272 [10%, (M+H)+]. HRCIMS m/z obs. 272.0000, calcd. para C11H15NO279Br [M]+ 272.0286 y m/z obs. 274.0000, calcd. para C11H15NO281Br [M]+ 274.0266 . Preparation of [2-(2-benzoylammino-etoxy)ethyl]-N-dodecyl-N,N-dimethyl-ammonio bromide, BEDDAB (3).- N,N-dimethyldodecylammine (3.87 gr, 18.15 mmol) is added to an acetone solution (8.2 mL) of N-[2-(2-Bromo-ethoxy)ethyl]-benzamide (2) (2.46 gr, 9.08 mmol) and the mixture was kept under reflux for 1 day. ). The reaction mixture was then concentrated to dryness and the residue purified by crystallization from hexanol to give a dark orange oil (4.05 g, 97 %). 1H-RMN (300 MHz, CDCl3): δ(ppm)=8.07-8.03 (m, 2H, Ph), 7.97 (ta, 1H, NH), 7.48-7.38 (m, 3H, Ph), 4.02 (m, 2H, O-CH2-CH2-(CH3)2+N-CH2), 3.85 (m, 4H, CH2-O-CH2), 3.68 (m, 2H, Bz-NH-CH2-CH2), 3.45 (m, 2H, (CH3)2+N-CH2), 3.30 (s, 6H, (CH3)2+N-CH2), 1.64 (m, 2H, (CH3)2+N-CH2-CH2), 1.24 (m, 18H, (CH2)9), 0.88 (t, 3H, 3J = 6.9 Hz, (CH3). 13C-NMR (75,4 MHz, CDCl3) δ (ppm) = 167.81 (CO), 134.1, 131.52, 128.54, 127.78, (5C, Ar), 70.18, 64.19 (2C, CH2-O-CH2), 66.26, ((CH3)2-N+-CH2-), 64.42 (O-CH2-CH2-N+(CH3)2), 52.01 (2C, -N+(CH3)2), 39.60 (Bz-NH-CH2-CH2), 32.02, 29.69, 29.57, 29.32, 26.33, 23.02, 22.78 (10C, (CH2)10), 14.22 (CH3). CIMS m/z 485 [100%, (M+H)+]. HRCIMS m/z obs. 405.3492, calcd. para C25H45N2O2 [M]+ 405.3481. 34 Table S1.- Critical micelle concentration values, cmc, in aqueous solutions estimated by conductivity measurements, corresponding to the different surfactants used in this work. T=303 K. References are provided, when possible, for the readers to compare the data summarized in this table with those in the literature. Surfactant cmc/M 1CTAB 0.9810-3 1TTAB 3.710-3 1DTAB 1510-3 2DTEAB 1410-3 3BDDAB 5.810-3 3CDDAB 4.710-3 4DMIB 1010-3 4BEDDAB 3.310-3 5BDAB 5.710-3 6CPB 0.7010-3 7DPB 1210-3 7CTAC 1.610-3 8TTAC 5.610-3 9ADTOAC 5.310-3 7CPC 0.9910-3 7DPC 1610-3 CTAB:Cetyltrimethylammonium bromide; TTAB:Tetradecyltrimethylammonium bromide; DTAB:Dodecyltrimethylammonium bromide; DTEAB:Dodecyltrirthylammonium bromide; BDDAB: N-benzyl- N,N-dimethyl-N-(1-dodecyl)ammonium bromide; CDDAB: N-cyclohexyl-N,N-dimethyl-n-(1- dodecyl)ammonium bromide; DMIB: 1-dodecyl-3-methylimidazolinium bromide; BEDDAB: [2-(2- benzoylammino-etoxy)ethyl]-N-dodecyl-N,N-dimethyl-ammonio bromide; BDAB: benzyldimethylammonium bromide; CPB:Hexadecylpyridinium bromide; DPB:Dodecylpyridinium bromide; Cetyltrimethylammonium chloride, CTAC; Tetradecyltrimethylammonium chloride, TTAC; ADTOAC, (S)-5-acetamido-6- (dodecylamino)-N,N, N-trimethyl-6-oxohexan-1-ammonium chloride; CPC, Cetylpyridinium chloride; DPD, Dodecylpyridinium chloride. 1R. 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Thermochimica Acta 428 (2005) 147-155. 8N. Fatma, M. Panda, W. H. Ansari, Kabir-ud-din, Environment-friendly ester bonded gemini surfactant: Mixed micellization of 14-E2-14 with ionic and non ionic conventional surfactants J. Mol. Liq. 211 (2015) 247-255. 9V. 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. 35 0 2 4 6 8 0 100 200 300 400 cmc=3.3x10-3 M (micellar ionization degree)=0.45 103x[BEDDAB]/M /− m-1 Figure S1.- Dependence of the specific conductivity, , on surfactant concentration in BEDDAB aqueous solutions. Solid lines correspond to the Carpena fitting. T=303 K. Figure S2.- Electrophoretic mobility shift assay on an agarose gel (1%) for DTAB-DNA and BDDAB-DNA complexes. Experiments were run in TAE buffer (40 mM tris-acetate, 1 mM EDTA) and stained with Red Safe. Electrophoresis was performed at 90 V until completion. 36 Figure S3.- AFM topographic images of some surfactant-DNA complex solutions, 40 mM HEPES at pH=7.4, adsorbed on APTES modified mica surface. A)DTAB-DNA Z+/-=50; B) DTEAB-DNA Z+/-=87; C) CDDAB-DNA Z+/-=20; D) CDDAB-DNA Z+/-=50.