Bis-quaternary ammonium gemini surfactants for gene therapy: Effects of the spacer hydrophobicity on the DNA complexation and biological activity

Journal Pre-proof Bis-quaternary ammonium gemini surfactants for gene therapy: Effects of the spacer hydrophobicity on the DNA complexation and biological activity Delvis R. Acosta-Mart´ınez (Formal analysis), Eustolia ´ Rodr´ıguez-Velazquez (Conceptualization) (Supervision) (Formal analysis), Fernanda Araiza-Verduzco (Formal analysis), Pablo Taboada (Formal analysis), Gerardo Prieto (Formal analysis), Ignacio A. Rivero (Formal analysis), Georgina Pina-Luis (Formal analysis), Manuel Alatorre-Meda (Conceptualization)supervision) (Formal analysis) (Funding acquisition)

PII:

S0927-7765(20)30047-3

DOI:

https://doi.org/10.1016/j.colsurfb.2020.110817

Reference:

COLSUB 110817

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

15 September 2019

Revised Date:

25 December 2019

Accepted Date:

20 January 2020

´ Please cite this article as: Acosta-Mart´ınez DR, Rodr´ıguez-Velazquez E, Araiza-Verduzco F, Taboada P, Prieto G, Rivero IA, Pina-Luis G, Alatorre-Meda M, Bis-quaternary ammonium gemini surfactants for gene therapy: Effects of the spacer hydrophobicity on the DNA complexation and biological activity, Colloids and Surfaces B: Biointerfaces (2020), doi: https://doi.org/10.1016/j.colsurfb.2020.110817

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Bis-quaternary ammonium gemini surfactants for gene therapy: Effects of the spacer hydrophobicity on the DNA complexation and biological activity

Delvis R. Acosta-Martínez,1,2 Eustolia Rodríguez-Velázquez,1,3,*

Fernanda Araiza-Verduzco,1 Pablo Taboada,2 Gerardo Prieto,4 Ignacio A. Rivero,5

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Georgina Pina-Luis,5 and Manuel Alatorre-Meda6,**

Tecnológico Nacional de México/I. T. Tijuana, Centro de Graduados e Investigación en

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Química-Grupo de Biomateriales y Nanomedicina, Blvd. Alberto Limón Padilla S/N 22510 Tijuana, B. C., México.

Colloids and Polymers Physics Group, Particle Physics Department, Universidade de

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Santiago de Compostela, 15782 Santiago de Compostela, Spain Facultad de Odontología, Universidad Autónoma de Baja California, Campus Tijuana,

Calzada de Universidad 14418, 22390 Tijuana, B. C., México. Biophysics and Interfaces Group, Applied Physics Department, Universidade de Santiago

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de Compostela, 15782, Santiago de Compostela, Spain. 5

Tecnológico Nacional de México/I. T. Tijuana, Centro de Graduados e Investigación en

Química, Blvd. Alberto Limón Padilla S/N 22510 Tijuana, B. C., México. Cátedras CONACyT-Tecnológico Nacional de México/I. T. Tijuana, Centro de Graduados

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e Investigación en Química-Grupo de Biomateriales y Nanomedicina, Blvd. Alberto Limón Padilla S/N 22510 Tijuana, B. C., México.

Authors to whom correspondence should be addressed: 1

* [email protected] ** [email protected]

Highlights

The GS spacer hydrophobicity modulates the DNA complexation and biological

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Graphical abstract

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The higher the spacer hydrophobicity the lower the GS CMC

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The higher the spacer hydrophobicity the higher the DNA-GS complexation ratio

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The higher the spacer hydrophobicity the stronger the DNA-GS binding

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The higher the spacer hydrophobicity the lower the GS cytocompatibility

ABSTRACT 2

Gemini surfactants (GS) have been highlighted as attractive gene carriers for a few years now; however, key aspects of the role of the GS chemical structure on the DNA-GS complexation and subsequent biological activity remain to be determined. Aiming to elucidate the effects of the GS spacer hydrophobicity, this work was focused on the biophysical characterization of the self-assembly, DNA complexation, cytocompatibility, and DNA transfection of a series of bis-quaternary ammonium GS with fixed side alkyl

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chains of 14 carbons and varying head-to-head alkyl chain spacers of 4, 6, and 14 carbons

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(referred to as GS4, GS6, and GS14, respectively). The characterization was carried out by a battery of experimental techniques including UV-vis, and fluorescence sprectroscopies, 

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potential, dynamic light scattering (DLS), isothermal titration calorimetry (ITC), and flow

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cytometry, among others. Overall, the spectroscopic results showed that the self-assembly of the GS was favored with the spacer hydrophobicity since lower values of critical micelle

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concentration (CMC) were observed for samples with longer spacer chains. On the other hand, the ITC results revealed that the DNA-GS complexation was driven by an initial

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electrostatic attraction between DNA and GS monomers/micelles followed by complementary hydrophobic interactions which strengthen the DNA-GS binding, the latter being more pronounced for GS with longer spacers. Finally, the biological tests demonstrated that while GS with moderate hydrophobicity (GS4 and GS6) yielded

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outstanding levels of cytocompatibility and DNA transfection over a range of concentrations, the most hydrophobic sample (GS14) proved to be cytotoxic upon administration to cultured HeLa cells (p < 0.05). In our opinion, the fundamental information here presented might be pivotal not only for understanding the DNA-GS

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complexation mechanism, but also for developing efficient GS-based carriers for gene therapy.

Keywords: Gemini surfactants; Gene therapy; Surfactant spacer; Cytocompatibility; DNA

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complexation; and DNA transfection.

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INTRODUCTION

Non-viral gene therapy is considered as a much safer alternative to the well-

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established viral approach. So far, non-viral vectors show the promise of being administered repeatedly with minimal host immune response, target-ability, stability on

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storage, and ease of production. Moreover, when optimized, they are able to compact the

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DNA, reverse its negative electrostatic charge, protect it against enzymatic degradation, and facilitate its delivery to cells [1].

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Most commonly studied non-viral vectors (also referred to as gene carriers) include natural and synthetic cationic polymers (polycations) [2-7], lipid-based cationic liposomes [8, 9], and single-chained cationic surfactants [10]. These systems present pros and cons one with respect to each other. In general, polymeric carriers display a greater structural

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and chemical versatility for manipulating their physicochemical properties, a higher stability upon storage and reconstitution, and a larger capacity to compact the genetic material. However, they are prone to yield low levels of transfection efficiency due to the quite strong interactions they can establish with DNA (and also RNA) [5, 7], requiring the use of additional cosolutes and/or external stimuli to enhance or even promote the gene 4

delivery. On the other hand, liposomes are considered as highly optimal from a biological point of view given their outstanding performance in terms of biodegradability, low immunogenicity, and capability of transferring DNA of essentially unlimited size (like polycations), being perhaps the systems of choice of the vast majority of reported publications and clinical trials [11]. However, they present some pitfalls as gene carriers related to a quite unpredictable transfection efficiency, which dramatically depends on

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various factors such as composition, preparation conditions, size, surface charge, and the

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method of introduction of the liposome-DNA complexes to the cell cultures. Finally, single-chained cationic surfactants have demonstrated to compact the DNA in a

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straightforward way prompted by their spontaneous self-assembly into micelles followed

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by DNA complexation, leading to outstanding levels of transfection. Remarkably, they have even been used as additives to improve the transfection efficiency of polyamines [12,

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13] and lipopeptides [14-17]. However, due to their rather high CMC, they in general entail moderate to high levels of cytotoxicity.

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Circumventing to some extent the biological limitations of conventional singlechained surfactants, GS have been proposed for a few years now as attractive carriers for gene therapy and other biomedical applications such as drug delivery and cosmetics [1721]. GS are a novel class of surfactants which, different to single-chained surfactants, are

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composed of two polar heads and two long alkyl chains linked by a spacer at the level or close to the head group. In general, GS present a lower CMC, a higher stability against dilution, and a higher loading capacity of hydrophobic compounds inside their micelles, just to cite a few [22-24].

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GS of different nature have been employed in gene delivery, giving rise to appealing results in terms of physicochemical properties, cytocompatibility, and transfection efficiency. Of interest to this study, very recent publications have studied the proper conditions at which bis-quaternary ammonium GS yield high levels of DNA transfection in parallel with low cytotoxicity, paying particular attention to the optimization of the systems with the use of helper lipids [18, 19, 21]. These studies pave indeed the way

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for the design of systems with advanced characteristics; however, key aspects on the

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correlation of GS structure with the DNA-GS complexation mechanism and subsequent biological activity of the forming DNA-GS complexes remain still unclear.

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Aiming to elucidate the effects of the GS spacer hydrophobicity, this work was

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focused on the biophysical characterization of the self-assembly, DNA complexation process, cytocompatibility, and DNA transfection efficiency of a series of bis-quaternary

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ammonium GS with fixed side alkyl chains of 14 carbons and varying head-to-head alkyl chain spacers of 4, 6, and 14 carbons, herein referred to as GS4, GS6, and GS14,

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respectively. These surfactants were chosen to cover a representative range of GS molecules with spacer chains composed from a low (4C) to a high number of spacing carbons (14C), having as the uppermost limit the same number of carbons as for the side chains (14C). Very importantly, this study was done in the absence of additional cosolutes

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(such as helper lipids) in order to assess the kind and extent of DNA-GS interactions without interferences of any class. As described in the following sections, the characterization was carried out by a battery of experimental techniques including conductometry, UV-vis and fluorescence sprectroscopies,  potential, DLS, agarose gel

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electrophoresis, isothermal titration calorimetry (ITC), flow cytometry, and ELISA based tests.

EXPERIMENTAL SECTION Materials

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GS4 (1,4-Bis(tetradecyl dimethyl ammonium)butane dibromide, Mw = 698 Da),

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GS6 (1,6-Bis(tetradecyl dimethyl ammonium)hexane dibromide Mw = 726 Da), and GS14 (1,14-Bis(tetradecyl dimethyl ammonium)tetradecane dibromide, Mw = 838 Da) were

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synthesized and kindly donated by the ReactyCat Group (GI-1935) of Universidade de Santiago de Compostela, Spain. GS4 and GS6 were synthesized from the corresponding

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´-dibromide, either 1,4-dibromobutane or 1,6-dibromohexane (5 mmol), which were

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reacted with anhydrous N,N-dimethyltetradecylamine (10 mmol) in 50 mL of acetone upon boiling under reflux for 96 h. The products were obtained after removal of the solvent with

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a rotary evaporator and crystallization from ethanol-ether. The obtained crystals were recrystallized from methanol and dried in a vacuum desiccator at room temperature. GS14 was synthesized in a two-step process. First, 1,14-dibromotetradecane was prepared by Kolbe´s electrolysis of 8-bromooctanoic acid in methanol [25]. Next, the obtained ´-

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dibromide was reacted with anhydrous N,N-dimethyltetradecylamine in acetone upon boiling and reflux for 96 h. The product was obtained after removal of the solvent with a rotary evaporator and crystallization from ethanol-ether. The obtained crystals were dried in a vacuum desiccator at room temperature. The synthesized GS were characterized by 1HNMR spectroscopy to confirm their chemical structure and purity. GS4 and GS6 were 7

dissolved in D2O (3.3% w/v), while GS14 was dissolved in CDCl3 (3.3% w/v). The 1HNMR spectra were acquired with a 9.4 T Bruker Avance III spectrometer operating at a 400 MHz proton frequency and processed with the MestreNova software (Mestrelab Research Inc., Spain). Chemical shifts are expressed in ppm (Figures S1-S3, Supplementary Material). The proton count from the integration confirmed the molecular formulas of the three samples and the absence of noticeable impurities (purity of the three GS ≥ 99%). GS4

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(400 MHz, D2O, 25 °C): δ = 3.60-3.40 (8H), δ = 3.30-3.10 (12H), δ = 2.00-1.75 (8H), δ =

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1.55−1.15 (44H), and δ = 0.95-0.85 (6H). GS6 (400 MHz, D2O, 25 °C): δ = 3.45-3.35 (8H), δ = 3.20-3.10 (12H), δ = 1.85-1.70 (8H), δ = 1.55−1.25 (48H), and δ = 0.95-0.85 (6H).

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GS14 (400 MHz, CDCl3, 25 °C): δ = 3.70-3.40 (8H), δ = 3.40-3.30 (12H), δ = 1.80-1.60

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(8H), δ = 1.50−1.10 (64H), and δ = 0.95-0.85 (6H). Calf thymus DNA sodium salt with a reported molecular weight of 10-15 million Da and plasmid DNA (pCMV-GFP suspended

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in water for injection, 3.5 kbp, 1 mg mL−1) were bought from Sigma-Aldrich and PlasmidFactory, respectively. TRIZMA and EDTA (both with a purity grade of 99%) were

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purchased from Sigma Aldrich. Dulbecco’s modified Eagle medium (DMEM), heatinactivated fetal bovine serum (FBS), MEM non-essential amino acids (NEAA; 100×), sodium pyruvate (100 mM), L-glutamine (L-Glu; 200 mM), penicillin-streptomycin (penstrep; 100 mg/mL), and trypsin-EDTA (0.25%, 0.913 mM EDTA) solutions were bought

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from Invitrogen. Cell Counting Kit-8 (CCK-8) was bought from Dojindo Molecular Technologies. Lipofectamine® transfection reagent was obtained from Invitrogen. HeLa (CCL-2) cells were purchased from the American Type Culture Collection. Finally, tissue culture plates (TCPs), cell culture flasks, and disposable sterile filters (pore size of 0.22 μm) were bought from Corning Costar. Sterile filtered Milli-Q water was used throughout. 8

All other chemicals were bought from Sigma Aldrich and were of reagent grade. Unless otherwise stated, all experiments were carried out under sterile conditions at least in triplicate.

CMC determination The CMC of all studied GS was characterized by conductometry and spectroscopies

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of UV-vis and fluorescence inside an air-conditioned room at a constant temperature of 25

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°C. Conductance data were collected with a PC650 conductivity meter from OAKTON. To perform the study, 25 mL of a GS aqueous solution (1 mM for GS4 and GS6, and 0.4 mM

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for GS14) were placed in a conductivity cell that was continuously stirred (80 rpm); then,

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constant volumes of water were added (5 mL) up to completion of 180 mL. On the other hand, the UV-vis characterization was carried out employing methyl orange (MO) as a

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sensing probe. Aqueous solutions of each GS were prepared at different concentrations in the presence of MO (0.05 mM). The UV-vis spectra of each sample were recorded between

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250 and 700 nm employing a SHIMADZU UV-2700 UV-vis spectrophotometer. All characterized samples were prepared 5 min in advance with respect to each measurement. Finally, the fluorescence characterization was carried out employing rhodamine B (RhoB) as a sensing probe. Aqueous solutions of each GS were prepared at different concentrations

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in the presence of RhoB (1×10-7 M). The fluorescence measurements for each sample were run employing a Horiba NanoLog fluorescence spectrophotometer, equipped with a Xe lamp as the excitation source, and working at excitation and emission wavelengths of 555 and 580 nm, respectively. The samples in this case were prepared immediately before each measurement. 9

DNA-GS complex preparation The DNA-GS complexes were prepared at different nitrogen-to-phosphate charge ratios (N/P = 0.5, 1, 1.5, 2, 3, 5, and 10), employing each GS under study. Briefly, 500 μL of the GS solutions at the desired concentrations were added to 500 μL of a DNA solution prepared at a fixed concentration of 0.02 mg/mL. The resulting mixtures were vortexed for

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10 s and allowed to stand for at least 10 min for their characterization. Unless otherwise

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stated, the GS solutions were prepared by dissolving each sample into water; meanwhile, the DNA solution was prepared using a 10 mM TRIZMA/EDTA/HCl buffer solution (pH

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7.4). The N/P ratios were calculated from the molecular weight of each GS and the average

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molecular weight of DNA nucleotides [26], as summarized in Table 1.

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Characterization of the compaction ratio and size of the DNA-GS complexes The N/P compaction ratio, (N/P)c, and size of the DNA-GS complexes were

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assessed by  potential, agarose gel electrophoresis, and dynamic light scattering (DLS). For the  potential and DLS characterizations, the complexes were prepared as above described at the desired N/P ratios and characterized with a Zetasizer NanoSeries spectrophotometer (Malvern). All measurements were carried out at 25 ° C using folded

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capillary cells for  potential measurements and polystyrene disposable cells for DLS ones. The results are the average of five measurements. For the gel electrophoresis characterization, the complexes were prepared in the presence of ethidium bromide (50 μM) at the desired N/P ratios in a buffer consisting of 10 mM TRIZMA/HCl and 1 mM EDTA (pH 7.4). The complexes and pure DNA (N/P = 0) were characterized using an 10

agarose gel (1.0%) at 110 V for 45 min. DNA was visualized under UV illumination at room temperature.

Characterization of the DNA-GS complexation thermodynamics The complexation thermodynamics was characterized by ITC employing a VP-ITC titration microcalorimeter from MicroCal Inc., Northampton, MA, with a cell volume of

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1.355 mL at 25 °C. Samples were degassed in a ThermoVac system (MicroCal) prior to

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use. The sample cell was filled with the DNA solution and the reference cell with buffer solution only. GS solutions were introduced into the thermostated cell by means of a

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syringe and stirred at 250 rpm, which ensured rapid mixing but did not cause foaming on

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solutions. Each titration consisted of an initial 2 μL injection (neglected in the analysis) followed by subsequent 27 injections of 10 L programmed to occur at 1500 s intervals,

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sufficient for the heat signal to return to the baseline. We present the results of the ITC experiments in terms of the enthalpy change per injection, ΔHi, as a function of N/P. Heat

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contributions of dilution/micellization from titrations of GS solutions into buffer solution only (without DNA) were subtracted from the heat resulting from their titration into the DNA solution in order to obtain the net binding heats. Raw ITC data of GS binding to DNA were processed as described previously [3, 5, 7]. The isotherms were fitted by using

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the single-site binding model supplied by MicroCal (Origin v. 5.0). This model employs the following fitting equations, where Q is the heat per injection, M is the macromolecule concentration, V is the volume of the cell, n and ΔH are the stoichiometry and enthalpy of complexation, respectively, and Θ is the fraction of ligand bound to the macromolecule:

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𝑄 = 𝑀𝑉𝑛ΘΔ𝐻

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Θ

𝐾 = (1−Θ)[𝑋] and [𝑋] = 𝑋 − 𝑀𝑛Θ

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One can solve for Θ using the equilibrium equation for the binding constant K, being X the concentration of ligand and [X] the concentration of free ligand. To achieve an

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accurate fit of all three floating parameters to our data, multiple attempts were performed

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starting from different initial parameters. The same set of values was reached at the

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minimum χ2, regardless of the values of initialization.

Cell culture

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HeLa cells were cultured in DMEM supplemented with 10% FBS, 0.1 mM NEAA, 2 mM L-Glu, 1 mM sodium pyruvate, and 1% pen-strep and incubated at 37 °C in a

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humidified atmosphere with 5% CO2. Cells were passaged when they reached an 80−90% optical confluence. Confluent monolayers were treated with the trypsin-EDTA solution and incubated for 4 min at culture conditions for detachment. Cells were then pelleted,

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resuspended in culture medium, and passaged or seeded onto TCPs for experimentation as required.

Cytocompatibility

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HeLa cells were seeded into 96-well TCPs (100 L, 1×104 cells/well) and grown for 24 h at culture conditions. Afterwards, the GS solutions, prepared in 10 mM phosphate buffered saline (PBS), were pipetted into the cell-containing wells (100 L, at the concentrations needed to prepare DNA-GS complexes with N/P = 1.5, 2, 3, and 5; see Table 1 for details on how N, P, and N/P were calculated) and incubated for 24 h. Then, the culture medium was discarded, and the cells were added with fresh culture medium (100

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L) containing the CCK-8 reagent (10 L), followed by gentle shaking for 1 min and

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incubation for 60 min at culture conditions. The optical density (OD) of the formazan was measured afterwards at 450 nm using an ELISA microplate reader (BIO-RAD model 680,

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Hercules, CA, USA). The metabolic activity of the cells after exposure to the GS solutions,

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represented as the percentage of cell viability, was calculated by normalizing the formazan OD reading from the cells exposed to the GS solutions with respect to control, non-exposed

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DNA transfection

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cells (100% viability). The results are the average of three independent experiments.

HeLa cells were seeded into 12-well TCPs (2 mL, 2×105 cells/well) and grown for 24 h at culture conditions. Afterwards, the DNA-GS complexes, prepared in 10 mM phosphate buffered saline (PBS), were pipetted into the cell-containing wells (200 L, N/P

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= 1.5 and 2; see Table 1 for details on how N, P, and N/P were calculated) and incubated for 24 h. Then, the culture medium was discarded, and the cells were harvested upon addition of fresh culture medium (1 mL) containing trypsin-EDTA (750 L) followed by incubation at culture conditions for 5 min and centrifugation (1200 rpm, 4 min). The cell pellets were resuspended in 2 mL of PBS for washing and retrieved by centrifugation (1200 13

rpm, 4 min). The washed pellets were resuspended again in 2 mL of fresh PBS and 200 L of each cell suspension were taken for characterization. The DNA transfection was determined by measuring the fluorescence of the GFP protein encoded in the plasmid DNA. The fluorescence data were collected by flow cytometry (BD FACSAria IIu, BD, Franklin Lakes, NJ). In all analyses, 106 events were acquired, scored using a FACSAria II analyzer

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(BD, Franklin Lakes, NJ), and processed by employing the Flowjo software program (v10.6.1). Highly dispersed edge-data were excluded from the region of interest (ROI) by

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gating from 102 to 2×105 events in the fluorescence axis. The plasmid concentration employed for these experiments was 0.05 mg/mL. Lipofectamine 2000® was employed as a

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transfection blank following the recommendations of the manufacturer (1:1 in volume with

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respect to the plasmid solution). The results are the average of three independent

Statistical analysis

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experiments.

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Statistical analysis was performed by the one-way analysis of variance (ANOVA) for repeated measurements. The post hoc Tukey test was used to perform multiple comparisons and differences were considered significant at a level of p < 0.05.

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RESULTS AND DISCUSSION Determination of CMC

The DNA complexation mediated by cationic surfactants is a process that occurs

upon the cooperative binding of surfactant monomers/micelles to the surface of the negatively charged DNA molecule by ion correlation effects, up until the latter is saturated 14

and thus compacted [27]. Having this in mind, the first step in our investigation was to characterize the GS in terms of their CMC, the concentration at which micellization takes place. The CMC of each sample was determined by conductometry and spectroscopies of UV-vis and fluorescence. The obtained results are presented in Figures 1, S4-S8, and Table 2. Figure 1A shows the dependence of the specific conductance, κ, on the concentration of GS4. The plot depicts that the conductance of the bulk solution grows linearly, although

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with a clear change in slope at the GS4 concentration of 0.177 mM, which corresponds to

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the CMC of the sample [28]. The conductance plots of GS6 and GS14 show a similar behavior (Figures S4 and S5, Supplementary Material). On the other hand, Figure 1B

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shows the evolution of the maximum absorption wavelength of methyl orange, MO max,

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as a function of GS4 concentration. A sharp increase in the maximum wavelength can be observed at concentration values between 0.1 and 0.2 mM. Meanwhile, the absorption

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maxima fluctuate slightly around 410-420 nm at higher concentrations, close to the absorption peak characteristic of MO in pure water (ca. 465 nm). As elsewhere described,

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the abrupt change in max of a selected probe denotes a change in the hydration state of the surfactant molecules in the bulk solution, which is ascribed to the formation of micelles [29]. Therefore, the zone wherein this change is observed can be designated as the surfactant CMC. The CMC in the portrayed graph was determined as the concentration at

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the maximum of the first derivative plot; it was found to correspond to 0.159 mM. The UVvis plots of GS6 and GS14 are shown in Figures S4 and S5 (Supplementary Material). Finally, Figure 1C shows the evolution of the maximum fluorescence emission of RhoB (normalized scale) as a function of GS4 concentration. A sharp decrease in the maximum emission can be observed at GS4 concentration values between 0.10 and 0.13 mM, 15

followed by stabilization at higher concentrations. As described for other fluorescence probes, the observed drop in the maximum emission reveals an abrupt change in polarity related to the formation of surfactant micelles [30, 31]. Therefore, as concluded for the UVvis characterization, the zone at which this change is observed can be designated as the GS4 CMC (= 0.122 mM, denoted by the minimum of the first derivative of the plot). Table 2 shows a summary of the CMC values of the studied GS as determined by the implemented

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techniques. As observed, the obtained CMC’s prove to decrease with the spacer length,

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adopting values of ca. 0.12-0.18, 0.09-0.16, and 0.02-0.05 mM for GS4, GS6, and GS14, respectively. Interestingly, the plotting of the average of these values against the number of

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carbons of the spacer chains reveals a linearly decreasing trend of the CMC with the spacer

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length (i.e. with the spacer hydrophobicity; see Figure S8 in Supplementary Material). Obviously, this linear trend could be further adjusted in future publications by including

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more GS samples with intermediate spacer lengths (e.g. GS8, GS10, and GS12). Very importantly, however, the obtained CMC values are consistent with those published by

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other authors [32], and one order of magnitude lower than that of their equivalent singlechained tetradecyl trimethyl ammonium bromide (TTAB) [33].

Determination of the compaction ratio and size of the DNA-GS complexes

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The (N/P)c ratio and size of the DNA-GS complexes were determined by

electrophoretic mobility experiments and DLS; the obtained results are presented in Figures 2 and 3.

Figure 2 shows the evolution of the  potential values of the DNA-GS complexes as a function of N/P. Consistent with the notion that the DNA complexation process is driven 16

by ion correlation effects (electrostatic interactions), this figure reveals a gradual decrease in the -potential of DNA with N/P, presenting a shift of negative to positive values at N/P ≈ 1.5 for the DNA-GS4 system and at N/P ≈ 2.0 for the DNA-GS6 and DNA-G14 complexes. These N/P ratios at which the DNA charge is reversed represent (N/P)c for each system. (N/P)c marks the concentration of GS required to compact DNA into stables

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complexes. Interestingly, regardless of the length of the spacer, the  potential of the three DNA-GS complexes reaches a plateau around 3-5 mV at N/P > (N/P)c, values that are

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equivalent to those exhibited by other systems including DNA-poly(amino acid) [34] and DNA-polycation complexes [3, 5, 35]. As shown in the inset, the formed complexes present

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sizes of ca. 250 nm at (N/P)c, which demonstrated to gradually increase up to ca. 500 nm at

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higher N/P ratios of 3 and 5 (in particular, the DNA-GS14 system).

The (N/P)c values obtained by -potential experiments were confirmed by agarose

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gel electrophoresis. This technique allows a visual characterization of charged species in solution [36, 37]. Figure 3 shows the gel electrophoresis photographs of the studied DNA-

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GS complexes. The figure depicts the migration in the electric field of free (N/P = 0) and not fully compacted DNA (N/P < (N/P)c) to the cathode, as well as the full retardation of complexes in which DNA is fully compacted (N/P ≥ (N/P)c). Thus, it corroborates that the GS cationic monomers/micelles indeed reverse the negative charge of DNA, resulting in

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the formation of stable complexes from (N/P)c on. Worth mentioning, by comparing these results with those presented in Figure 2, it is possible to observe that the -potential and gel electrophoresis findings coincide in the very same (N/P)c ratios of 1.5, 2.0, and 2.0 for the DNA-GS4, DNA-GS6, and DNA-GS14 complexes, respectively.

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Thermodynamic characterization of the DNA-GS complexation process Recent literature has highlighted the influence of binding energetics on the complexation of biomolecules in solution and on the subsequent biological performance of the resulting complexes [3, 5, 7, 38, 39]. Thus, it was also of our interest to characterize by

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ITC the DNA-GS binding affinity and the interactions involved in their complexation

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process. Figure 4 shows the enthalpy of injection, Hi, as a function of N/P resulting from the titration of the GS solutions into the DNA one after the subtraction of

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dilution/micellization effects. Solid lines represent the numerical fits of the single-site

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binding model to the experimental data. Table 3 summarizes the DNA-GS binding stoichiometry (n), binding equilibrium constant (K), and changes in enthalpy (H) and

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entropy (S) derived from the numerical fittings.

As can be seen from Figure 4, the isotherms of all experiments display a monotonic

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curve ending in a plateau, although with some variations. The DNA-GS binding in the three cases proceeded as an exothermic process at early stages of complexation (wherein DNA is in excess), which leveled off upon subsequent titration of each GS. Being typical of the complexation of charged species in biological milieu, a similar outcome was previously

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found by our group and others for the complexation process of distinct polyelectrolyteligand systems [5, 40]. In general, negative values of enthalpy resulting from the association of polylectrolytes in solution denote the existence of long-range electrostatic interactions of the polyelectrolyte in question with its complexing counterions and/or buffer ionic species (DNA with complexing GS monomers/micelles in our case) [3]. In this 18

context, the observed gradual decrease in the heat release can be interpreted as an indicative of the progressive neutralization of negatively charged phosphate groups of DNA by positively charged GS monomers/micelles, constituting one of the driving forces behind the DNA-GS complexation, as further discussed below. Therefore, the zone of the isotherm at which the plateau is reached can be attributed to the complete DNA compaction [3, 5]. Interestingly, the enthalpogram of the DNA-GS14 system exhibited an exothermic-to-

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endothermic shift along the complexation process, revealing a prominent role of additional

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interactions over the DNA-GS binding [3, 41]. In this sense, it is well-accepted that the complexation of DNA with interacting ligands can occur by a synergy between

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electrostatics and complementary attractive forces, which are reliant on both the nature of

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the ligand and the binding stereochemistry; these forces might include hydrogen bonding, protonation effects, and hydrophobicity [3, 41-44]. From a mechanistic point of view, the

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long-range Coulombic attractions are assumed to initiate the approximation of interacting species, while the complementary force (or forces) are believed to strengthen the DNA-

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ligand association. Considering the chemical structure of the studied GS, in particular the quaternized state of the nitrogen atoms, interactions such as possible protonation effects and hydrogen bonding are expected to be negligible; hence, the non-electrostatic component of the DNA-GS14 binding enthalpy (the positive portion of the isotherm) can

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be ascribed to emergence of hydrophobic contributions preferably (not to say totally), which are more evident when working with GS with longer spacer chains. According to previous publications, the established hydrophobic effect not only favors the formation of surfactant micelles but also their intercalation within the DNA bases [41, 44]. Remarkably, as demonstrated by the increasing values of K for the DNA-GS6 and DNA-GS14 19

complexes with respect to DNA-GS4 (Table 3), it can be observed that the occurring hydrophobic interactions reinforce indeed the prime electrostatic attraction outlined above; that is, the DNA complexation and subsequent compaction is demonstrated to occur upon combination of these two driving forces. Additional support to this notion is given by the similar increasing trend in the values of n, which indicates that the longer the GS spacer (i.e. the higher the hydrophobicity of the system) the higher the concentration of GS that

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interacts with DNA (i.e. the bigger the population of GS monomers/micelles complexing

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Cytocompatibility and transfection efficiency

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the DNA) (see Table 3).

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The final step of the present work was the characterization of the biological activity of the GS under study. Figure 5 shows the cytocompatibility profiles of the three samples in

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the absence of DNA, as characterized over the range of GS concentrations (expressed in M of N) necessary to prepare DNA-GS complexes at N/P = 1.5, 2.0, 3.0, and 5.0. It can be

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observed from this figure that cytocompatibility depends on the spacer chain length and concentration of all studied GS, showing a clear decreasing trend with both parameters. Interestingly, GS4 and GS6 showed a higher cytocompatibility as compared to GS14 at all studied concentrations, with statistically significant difference (p < 0.05). However, the

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concentrations of 225 and 300 M of N for GS4 and GS6 (necessary to prepare DNA-GS complexes at N/P = 1.5 and 2.0, respectively) were the only ones displaying percentage values higher than or around 70%, resulting the only ones providing satisfactory levels of cytocompatibility as to be considered for transfection experiments. These results are consistent with the findings reported by Almeida et. al. [45] who found that GS with long20

chain spacers (12–10–12) had a marked cytotoxicity, while GS with short spacers (12–2–12 and 14–2–14) did not present cytotoxicity at all. The authors ascribed the cytotoxicity of the 12-10-12 GS to the fact that it provoked a leakage of aqueous contents from lipid models mimicking the composition of a cell membrane, while the less toxic 12–2–12 and 14–2–14 GS showed only a residual percentage of membrane destabilization [45]. Finally, the transfection efficiency of the DNA-GS complexes at the N/P ratios of

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1.5 and 2.0 was characterized by flow cytometry (Figures 6 and S12). In good agreement

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with the cytocompatibility results, GS4 and GS6 showed an adequate level of transfection efficiency, comparable to that of the Lipofectamine® transfection blank (around 60%, p >

-p

0.05). This result can be ascribed to the moderate binding affinity displayed by all DNA-GS

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complexes (in the order of 104-105 M-1), which is lower than those exhibited DNA-polymer complexes (in the order of 105-106 M-1; see Table 3) [3, 5, 7]. In general, low values of

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binding affinity give rise to high levels of transfection efficiency [5, 7, 46]. Conversely, the DNA-GS14 complex displayed a null transfection efficiency, which is very likely related to

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the low cytocompatibility of GS14.

CONCLUSIONS

The self-assembly, DNA interactions, and biological activity of a series of bis-

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quaternary ammonium GS with head-to-head spacers of varying lengths were characterized. The spacer hydrophobicity was found to govern the CMC, the DNA binding process, and the biological performance. With respect to the CMC, it was found that longer chain lengths gave rise to lower values of CMC, as revealed by conductometry and spectroscopies of UV-vis and fluorescence. On the other hand, concerning the DNA-GS 21

binding mechanism, it was found by ITC that the whole process is driven by an initial electrostatic attraction between DNA and GS monomers/micelles followed by complementary hydrophobic interactions which strengthen the DNA-GS binding, the latter being more pronounced for systems with longer spacer chain length. Finally, regarding the biological activity, GS with moderate hydrophobicity (GS4 and GS6) yielded the highest levels of both cytocompatibility and DNA transfection efficiency, the latter property being

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statistically equivalent to that displayed by the Lipofectamine® transfection reagent

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employed as a blank (p > 0.05). By contrast, the most hydrophobic sample (GS14) was found to be cytotoxic in the whole range of studied concentrations, with statistically

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significant difference with respect to the other samples (p < 0.05).

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Altogether, our results highlight the importance of elucidating the effect of GS hydrophobicity on the processes of DNA complexation, cytocompatibility, and DNA

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transfection. In our opinion, this fundamental information is pivotal not only for understanding the DNA-GS binding mechanism, but also for developing efficient GS-based

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carriers for gene therapy.

Declaration of interests

The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

Credit Author Statement Delvis R. Acosta-Martínez:

Experimental work and formal analysis

22

Eustolia Rodríguez-Velázquez:

Conceptualization, supervisión, formal analysis, and writing Formal analysis

Pablo Taboada:

Formal analysis

Gerardo Prieto:

Formal analysis

Ignacio A. Rivero:

Formal analysis

Georgina Pina-Luis:

Formal analysis

Manuel Alatorre-Meda:

Conceptualization, supervisión, writing, formal

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ro

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Fernanda Araiza-Verduzco:

ACKNOWLEDGMENTS

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analysis, and funding acquisition

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Authors thank the ReactyCat Group (GI-1935) of Universidade de Santiago de Compostela, for donating the GS herein studied. D. A.-M. thanks CONACyT (Mexico) for financial support through PhD Grant No. 597757 and PhD Research Stay Grant 291212. F. A.-V. thanks CONACyT (Mexico) for financial support through PhD Grant No. 306590. M.A.-

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M. thanks funding from CONACyT (Mexico) through Research Projects INFR-2015251863 and PDCPN-2015-89.

23

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FIGURE LEGENDS Figure 1. CMC of GS4 as obtained by conductometry (A) and spectroscopies of UV-vis (B) and fluorescence (C). The red lines in (A) represent linear fittings to the experimental data; the CMC value was determined as the GS4 concentration at the intersection of both lines. The red and black lines in (B) and (C) represent a sigmoidal fitting to the experimental data and its corresponding first derivative with respect to concentration,

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respectively. The CMC value was determined as the GS4 concentration at the maximum

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and minimum of the first derivative plots for (B) and (C), respectively. UV-vis absorption and fluorescence emission spectra of the employed probes are presented in Figures S6 and

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S7 (Supplementary Material).

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Figure 2.  potential of DNA-GS complexes as a function of N/P. The inset shows the hydrodynamic radius of the complexes, RH, at N/P ≥ (N/P)c. Table 1 illustrates how N, P,

lP

and N/P were calculated.

Figure 3. Agarose gel electrophoresis pictures of DNA-GS complexes as a function of N/P

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N/P = 0 corresponds to pure DNA in solution. Table 1 illustrates how N, P, and N/P were calculated.

Figure 4. Integrated heats of interaction of the titration of the GS into DNA as a function of N/P. Solid lines represent the numerical fittings to the experimental data. The heat

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contributions from GS dilution/micellization in each case were subtracted. The inset shows a zoom-in of the DNA-GS6 and DNA-GS14 thermograms. Table 1 illustrates how N, P, and N/P were calculated. Raw ITC data are presented in Figures S9-S11 (Supplementary Material).

30

Figure 5. Cytocompatibility of the studied GS as a function of N. The selected concentrations correspond to those employed for the preparation of the DNA-GS complexes for transfection experiments (N/P = 1.5, 2.0, 3.0, and 5.0 from the lowest to the highest, respectively; see Table 1 for details on how N, P, and N/P were calculated). Data represent the average of three independent experiments and the uncertainty bars stand for the standard deviation. Statistically significant difference between data (p < 0.05) is

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denoted with *.

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Figure 6. Transfection efficiency of DNA-GS complexes as a function of N/P. Data represent the average of three independent experiments and the uncertainty bars stand for

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the standard deviation. No statistically significant difference was observed either between

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analyzed data or with respect to the blank. (n.d. stands for non-detected). Table 1 illustrates how N, P, and N/P were calculated. Raw cytometry data are presented in Figure S12

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ur na

lP

(Supplementary Material).

31

Table 1. Summary of the DNA and GS concentrations employed for the preparation of the DNA-GS complexes. The P concentrations were calculated taking into account the average molecular weight of DNA nucleotides (330 g of DNA per 1 mol of P) [26]. On the other hand, the N concentrations were calculated considering the stoichiometric ratio of 1 mol of each GS per 2 mol of N. Numbers written in red correspond to the concentrations employed for the biological characterizations of cytocompatibility and transfection efficiency at the

0.02

0.061

1

0.02

0.061 0.05

0.02 0.05

2

3

0.05

0.02

5

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0.02

0.228

0.304 0.122

0.228 0.092

0.152

0.456 0.183

0.380 0.153

0.152 0.061

0.061

0.092

0.061

0.152

0.061

0.05 0.02

0.152

0.061

0.05

10

0.152

0.046

0.061

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0.02

0.114

0.152 0.061

mM of N 0.031

0.031

lP

1.5

0.016

-p

0.5

GS concentration mM

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DNA concentration mg/mL mM of P

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N/P

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selected N/P ratios.

0.760 0.305

0.760 0.305

1.520 0.610

32

Table 2. CMC values of the GS as determined by conductometry and spectroscopies of UV-vis and fluorescence. Data in brackets correspond to the R2 values of the linear fittings at the premicellar and postmicellar regions, respectively (conductometry); and to the standard error (SE) of the CMC values determined by the sigmoidal fittings (UV-vis and fluorescence). Differences in the attained CMC values can be ascribed to the higher

CMC Conductometry

(mM) CMC Fluorescence

GS4

0.177 (R2 = 0.997 and 0.999)

0.159 (SE = 1.09 × 10-3)

0.122 (SE = 2.76 × 10-4)

GS6

0.161 (R2 = 0.998 and 0.996)

0.086 (SE = 4.13 × 10-4)

0.102 (SE = 8.67 × 10-4)

GS14

0.047 (R2 = 0.989 and 0.996)

0.022 (SE = 1.53 × 10-4)

0.013 (SE = 9.31 × 10-4)

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Surfactant

(mM)

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ur na

lP

re

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(mM) CMC UV-vis

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sensitivity of the Uv-vis and fluorescence spectroscopies with respect to conductometry.

33

Table 3. Thermodynamic parameters of the DNA–GS complexation process. H (kcal/mol)

S (kcal/mol∙K)

0.19

-2.44

±

0.54

0.013

±

0.06

-0.23

±

0.01

0.024

±

0.25

-0.10

±

0.01

0.025

 n

K × 10-5 (M-1)

DNA-GS4

0.39

±

0.07

1.05

±

DNA-GS6

2.20

±

0.56

2.88

DNA-GS14

3.17

±

2.04

5.32

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re

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Complex

34

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Figure 1. Acosta-Martínez et. al.

35

of ro -p re

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lP

Figure 2. Acosta-Martínez et. al.

36

of ro -p re lP ur na

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Figure 3. Acosta-Martínez et. al.

37

of ro -p re

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lP

Figure 4. Acosta-Martínez et. al.

38

of ro -p re

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ur na

lP

Figure 5. Acosta-Martínez et. al.

39

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lP

Figure 6. Acosta-Martínez et. al.

40