www.acsabm.org Article Efficient DNA Condensation Induced by Chiral β‑Amino Acid-Based Cationic Surfactants Bernat Pi-Boleda, Sravani Ramisetty, Ona Illa, Vicenç Branchadell, Rita S. Dias,* and Rosa M. Ortuño* Cite This: ACS Appl. Bio Mater. 2021, 4, 7034−7043 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ABSTRACT: Four cationic chiral amino acid-based surfactants, cis- and trans-1 and cis- and trans-2, have been studied as DNA- condensing agents with enhanced properties and the absence of cell toxicity. The polar head of the surfactant is made of a cyclobutane β-amino acid in which the amino group is a hydrochloride salt and Downloaded via 83.39.143.78 on September 20, 2021 at 14:06:26 (UTC). the carboxyl group is involved in an amide bond, allowing the link with hydrophobic C12 (surfactant 1) or C16 (surfactant 2) chains. The ability of these surfactants to condense DNA was investigated using a dye exclusion assay, gel electrophoresis, and circular dichroism and compared with the well-studied dodecyltrimethy- lammonium bromide (DTAB) and cetyltrimethylammonium bro- mide (CTAB). The surfactant with the longest chain length and the trans stereochemistry (trans-2) was found to be the most efficient in condensing the DNA, including CTAB. Surfactant cis-2 was found to be less efficient, probably due to its poorer solubility. The β- amino acid surfactants with the shorter chain length behaved similarly, such that the cis/trans stereochemistry does not seem to play a role in this case. Interestingly, these were also found to induce DNA condensation for the same concentration as trans-2 and CTAB but showed a lower binding cooperativity. Therefore, a longer alkyl chain only slightly improved the effectiveness of these surfactants. Further, atomic force microscopy revealed that they compact DNA into small complexes of about 55−110 nm in diameter. KEYWORDS: DNA−surfactant complexes, surfactant headgroup, critical association concentration, dye exclusion, circular dichroism ■ INTRODUCTION Gene therapy has gained significant attention over the past two cationic polymers,15,16 metal cations,17 dendrimers,18,19 poly- peptides,20,21 and nanoparticles.22,23 decades as a potential method for treating genetic disorders Special attention has been dedicated to cationic amphiphilic such as Severe Combined Immunodeficiency,1 Hemophilia B,2 molecules due to their properties.24 The cationic polar heads cystic fibrosis,3 Leber’s Congenital Amaurosis,4 and Parkin- interact with negatively charged phosphate groups of DNA, son’s disease5 as well as an alternative method to traditional while the hydrophobic chains stabilize the formed aggregates. chemotherapy for treating cancer.6 It consists of introducing a The degree of DNA condensation, the size of the complexes, target gene, which is encoded in a DNA or RNA chain, into a and the neutralization of negative charges have been identified specific cell. Research efforts are currently focused on as crucial factors to transfect DNA into cells.25 Complexes with designing effective carrier vectors for gene therapy that a radius below 200 nm are likely to be transfected into cells efficiently compact and protect DNA,7 as DNA is rapidly through endocytosis and end up in endosomes that, in time, degraded by serum nucleases in the blood when injected will develop into lysosomes where the complexes are degraded intravenously.8 Moreover, they must transfect into cells and due to the presence of enzymes in these compartments.26 release the target DNA within. Not surprisingly, viral carriers Thus, it is most desirable that the vector facilitates the DNA such as retroviruses and adenoviruses are found to exhibit a release from the endosomes.27,28 high efficiency in delivering both DNA and RNA to numerous cell lines. However, fundamental problems associated with viral vector systems, including toxicity, immunogenicity, and Received: June 13, 2021 limitations with respect to scaled-up procedures, encouraged Accepted: August 11, 2021 the investigation of other potential vectors for introducing the Published: August 25, 2021 DNA into the targeted tissues.9,10 The role of chemists in the field of gene therapy is to design and prepare new nonviral vectors, based on cationic lipids,11−13 cationic surfactants,12,14 © 2021 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsabm.1c00683 7034 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article Chart 1. Structure of Chiral Cyclobutane β-Amino Acid-Based Cationic Amphiphiles45 Studied in This Work and of Commercial DTAB and CTAB The structure of the surfactant plays a very important role in the DNA condensation and complex formation, which can be ■ EXPERIMENTAL SECTION Materials. Oligonucleotide primers were received from Sigma- controlled by modifying the structure of the polar head and the Aldrich. For circular dichroism (CD) studies, which require high length of the hydrophobic chain.29−31 However, the cytotox- concentrations and volumes, DNA from salmon sperm (10 mg mL−1 icity of the most commonly studied cationic surfactants solution, average size of ≤2 kbp) was obtained from Invitrogen represents a shortcoming in their application. Amino acid- (Fisher Scientific). Surfactants cis- and trans-1 and -2 were synthesized and purified following the procedures described in ref based surfactants are gaining relevance due to their good levels 45, and CTAB and DTAB were obtained from Sigma-Aldrich. of biodegradability and biocompatibility.32−36 The combina- Tris(hydroxymethyl)aminomethane and hydrogen chloride for Tris- tion of amino acids or peptides with hydrocarbon chains of HCl buffer preparation were purchased from Sigma-Aldrich. All stock, variable length has given rise to a variety of compounds with intermediate, and final solutions were made, unless stated otherwise, good amphiphilic properties and the possibility of tuning the with 10 mM Tris-HCl, pH 7.4, prepared using Milli Q deionized amino acid composition to include, for example, pH-sensitive water (18.2 Ω/cm resistivity at 25 °C). DNA Preparation and Purification. For experiments other than moieties, believed to facilitate the endosomal escape.37 CD, a linear double stranded DNA (4145 bp) harboring the gene- Moreover, intermolecular hydrogen bonding promoted by encoding green fluorescent protein (GFP mut3) under the control of the amino groups can enhance the self-assembly of surfactants a T7 promoter was generated by polymerase chain reaction (PCR) in these systems.38 Furthermore, interactions of biodegradable using the plasmid pSB-E1 g46 as the DNA template and forward 5′- amino acid-based surfactants with anionic polyelectrolytes have GCTGGCCGATAAGCTCTAAG-3′ and reverse 5′-GGTGC- also been studied.39 Indeed, the role of polyelectrolytes and ATTGCAAACGCTAGG-3′ primers. After PCR, the product was purified using DNA clean and a concentrator kit from Zymogen. their interactions with surfactants have been highlighted.40,41 Methods. Dye Exclusion Assay (DEA). The accessibility of the Among amino acids, chiral cyclobutane-containing β-amino DNA molecules to the fluorescent dye GelStar was determined by acid-based surfactants have shown interesting abilities. They exciting the DNA−GelStar samples, possessing varying concentra- are tuned by the relative cis/trans stereochemistry and by the tions of surfactant, with light at a wavelength of 493 nm stereochemical constraints imposed by the cyclobutane ring as (corresponding to the excitation maximum of GelStar) and measuring well as the positive42 or negative43 charge or their nonionic the respective emitted fluorescence intensity at 500−550 nm. The character.44 All these features confer great versatility to these emitted fluorescence is linearly dependent on the “free” DNA concentration (in the absence of compacting agent).47 A reference derivatives as they strongly influence their aggregation sample with DNA and GelStar (without surfactant) was prepared properties. Recently, a new family of cationic surfactants, cis- simultaneously and used to normalize the data. Furthermore, to check and trans-1 and -2 (Chart 1), has been reported.45 Their that the surfactant aggregates did not interfere with the fluorescence physicochemical properties are strongly dependent on the pH intensity of the dye, control experiments were performed using the of the medium, such as the critical micellar concentration protocol described below but replacing DNA with the buffer solution, (CMC) and the surface tension. Further, the mode of that is, by measuring samples with buffer, GelStar, and the highest surfactant concentration studied, which was above the CMC. No aggregation is determined by the cis/trans stereochemistry significant differences were found in the fluorescence intensity of the with the cis-isomers forming micelles or vesicles while the dye in the presence or absence of surfactant micelles. trans-isomers predominantly form fibers. In addition, they were Samples were prepared in triplicate using the following procedure: shown to be nontoxic for HeLa cells. First, 5 μL of 20 μg mL−1 DNA was mixed with 5 μL of 100× GelStar With these qualities in mind, we have, in this work, studied (prepared from 10 000× GelStar stock solution obtained from Lonza) the condensation of DNA by chiral cyclobutane β-amino acid- and left to equilibrate for 15 min. Afterward, 5 μL of surfactant solutions with varying concentrations was added to the DNA−GelStar based cationic surfactants using a range of complementary mixtures. Finally, 35 μL of 10 mM Tris-HCl was added to the techniques and paying particular attention to the role of cis/ samples, which were left to incubate for 1 h. After incubation, the trans stereochemistry and the alkyl chain length. For fluorescence emission of the samples (500−700 nm) was measured comparison purposes, some of the experiments were using an Infinite M200 Pro Tecan spectrophotometer. The conducted using the commercial surfactants cetyltrimethylam- experimental parameters are shown in Table S1. As an example, the monium bromide (CTAB) and dodecyltrimethylammonium emission spectra of the DNA−GelStar at different cis-1 concen- trations, as well as the results of all studied systems, are also provided bromide (DTAB), which have been extensively investigated. in Figures S1 and S2, respectively. Concentration ratios were The obtained data should shed light on the potential of chiral expressed as a function of the surfactant μM concentration divided cyclobutane β-amino acid-based surfactants to be used as by ppm of DNA. In addition, results are also presented as a function nonviral vectors in gene therapy. of the charge ratio (Zhp), defined as the ratio between the molar 7035 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article Figure 1. Fluorescence intensity of DNA−surfactant complexes normalized to DNA solutions in the absence of surfactant, as a function of surfactant concentration, for surfactants possessing (a) 12 carbons in the hydrocarbon chain: cis- and trans-1 and DTAB; (b) 16 carbons in the hydrocarbon chain: cis- and trans-2 and CTAB. The concentration of surfactant is given as both μM/ppm and Zhp. The DNA concentration was 2.0 μg/mL, and the excitation and emission wavelengths were 493 and 540 nm, respectively. concentration of charges from the surfactants, that is, NH3+ for cis- in the vicinity of an oppositely charged polyelectrolyte is called and trans-1 and -2, assuming that all the surfactants are protonated, the critical association concentration (CAC). and N(CH3)3+ for CTAB and DTAB, and the molar concentration of In this work, DEA experiments were performed to estimate DNA nucleotides (PO4−, p). the CAC values for the DNA−surfactant complexes. The Electrophoretic Mobility Shift Assay (EMSA). 20 μL samples were prepared by adding 5 μL of a 20 μg mL−1 DNA solution, 5 μL of a results obtained for the novel cis- and trans-1 and -2 surfactants surfactant solution at different concentrations, and 10 μL of Tris-HCl were compared with those of commercial DTAB and CTAB, buffer and incubating for 1 h. To compare the results with those which have been extensively studied as DNA-compacting obtained from the DEA, the concentration of surfactant will be agents. Further, the obtained results were complemented with expressed as a function of the charge ratio (Zhp). The samples were EMSA experiments that also explore the degree of complex- loaded in a 0.8% agarose gel (SeaKemRLE agarose from Lonza, ation of DNA by the cationic surfactants, while CD Rockland, ME USA) and run for 20 min at 100 V. Afterward, the gels spectroscopy was used to monitor the variation of the DNA were visualized under UV light using a ChemiDoc instrument. Circular Dichroism (CD) Spectroscopy. CD spectra were obtained structure upon surfactant self-assembly. AFM was used to in a Jasco-715 spectropolarimeter. 350 μL solutions of 0.333 μM (1 probe the geometry of the complexes formed at intermediate mg mL−1) Salmon Sperm DNA in the absence and presence of known surfactant concentrations. concentrations of surfactants were prepared, left to equilibrate for a Accessibility of a Fluorescent Dye to DNA in the couple of minutes, and placed into a 1 mm length-path cuvette. The Presence of Surfactants. As described in the experimental CD spectra of the solutions were recorded. part, the fluorescence emission spectra of DNA−GelStar Atomic Force Microscopy (AFM). Samples were prepared by complexes were measured for increasing concentrations of all mixing 80 μM solutions of the respective surfactants with an 8 μg the surfactants under study. All the studied systems were found mL−1 solution of DNA. After 1 h of incubation, 10 μL of the resultant solution was transferred to a freshly cleaved 5 mm diameter mica disk. to have a maximum of emission at 540 nm, and thus, to more Then, the sample surface was rinsed rapidly with pure water (Milli-Q) easily analyze the results, the intensity of each sample was to obtain a clean surface and then dried under a stream of N2 followed normalized with respect to the intensity of the DNA−GelStar by vacuum-drying at a pressure of 1.3 × 10−4 Pa for 5 h.48 The complex (absence of surfactants) and represented as a function imaging was performed in a Multimode 8 from Bruker using of the surfactant concentration (Figure 1). The general trend ScanAsyst mode in air and employing ScanAsyst-air cantilevers with shows that an increase in cationic surfactant amount leads, at nominal spring constants of 0.4 N/m. low concentrations, to a small increase in the emission ■ RESULTS AND DISCUSSION Several techniques were used to study the surfactant features intensity, followed by a significant decrease in fluorescence. Some of the systems show a further increase in fluorescence intensity with increasing surfactant concentrations. The reason favoring the interactions between these and DNA and for the initial increase in fluorescence is not clear but could be concomitant DNA condensation. due to an extension of the DNA chain, which makes it more The binding of cationic surfactants to DNA and surfactant available to the fluorescent dye upon the addition of small self-assembly in its vicinity is known to occur at concentrations amounts of surfactant. Such a chain extension at low much below the CMC of the DNA-free surfactant solutions.49 concentrations of condensing agent (polycations) has been The binding of the monovalent surfactant itself is not expected observed using coarse-grained systems and Monte Carlo to affect the DNA conformation; however, the formation of simulations.53 Upon further addition of surfactant, the surfactant aggregates in the vicinity of the DNA, driven by fluorescence intensity decreases due to the exclusion of the hydrophobic interactions between surfactant tails and counter- dye from the DNA molecules. The increase in fluorescence ion release, leads to attractions between DNA chains and/or intensity observed at larger surfactant concentrations for some within a single chain, leading to the condensation of DNA.50,51 of the systems is likely due to the formation of overcharged Accordingly, binding isotherms of cationic surfactants to DNA DNA−surfactant complexes, which leads to an expansion of show a sigmoidal shape, indicating cooperativity.52 The the complex and consequently an increase in the accessibility concentration at which the surfactant starts forming micelles of the DNA to the dye.54 7036 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article Panels a and b in Figure 1 show the results obtained for well as the corresponding charge ratio, Zhp‑2. CCI is thus surfactants possessing 12 (1 and DTAB) and 16 (2 and defined as the lower surfactant concentration in which all (or CTAB) carbons in the hydrocarbon chain, respectively. most) DNA molecules are condensed. In addition, ΔZhp Starting with an overall assessment, it can be observed (Figure values, calculated as Zhp‑2 − Zhp‑1, are also listed in Table 1 1a) that the behavior of cis- and trans-1 is very similar, as they allow one to evaluate the cooperativity of the suggesting that cis/trans stereochemistry does not play a surfactant-induced DNA condensation, that is, the tendency significant role in the interaction of the surfactants with the of the surfactant to associate to a DNA molecule already DNA in these systems. Both surfactants are found to be much occupied by surfactants.36 Thus, the lower the ΔZhp, the higher more efficient than DTAB in inducing DNA condensation, that will be the cooperativity and the strength of the interaction is, the decrease of fluorescence intensity occurs at a lower between the DNA and the surfactant aggregates. Finally, the concentration of surfactant. In contrast, large differences are CMC of the surfactants is also presented. found between the more hydrophobic trans-2 and cis-2 In more detail and starting with the surfactants with the surfactants; trans-2 behaves similarly to CTAB while cis-2 is shortest alkyl chain, it can be observed in Table 1 that both cis- clearly less efficient in inducing DNA condensation (Figure and trans-1 present a similar behavior (CAC of 7 μM, CCI of 1b), pointing out the influence of stereochemistry for these 100 μM) and that they are much more efficient in condensing surfactants. DNA than DTAB (CAC of 50 μM, CCI of 2000 μM). Since the In all studied systems, the higher normalized intensity values alkyl chain of the surfactants is the same, the difference must have larger error bars than those with lower intensities. This originate in the headgroup. The lower CAC indicates that the fact can be attributed to several factors related to the different attractive interactions between surfactant headgroups are species present in the solution. At high intensities, DNA is not stronger for the β-amino acid-based surfactants than for the condensed and has many degrees of freedom presenting tetramethylammonium surfactants. This could be due to the different coil conformations, each one with different formation of H-bonds between the headgroups (more accessibility for the GelStar. In addition, the cooperative effect favorable than those with water) and/or the more hydrophobic of the binding process, highlighted by the sigmoid shape, also nature of the headgroups due to the chiral cyclobutane. This accounts for the larger errors; further binding of the reasoning is supported by the fact that the CMC of the cis- and compacting agent to the partially compacted DNA is preferred, trans-1 surfactants is much lower than that of the DTAB. It is and the coexistence of more compact and more extended also interesting to note that the CAC of the cis- and trans-1 is DNA−surfactant complexes is likely to contribute to the large apparently the same even though the CMC of trans-1 is half of error bars.24 that of cis-1. It has been shown that the CMCs of cis- and trans- Since the decrease in fluorescence intensity is associated 1 are about the same at low pH conditions45 where most of the with the lower accessibility of GelStar, due to the formation of surfactants are protonated, which suggests that the interaction surfactant aggregates at the surface of DNA, it is tempting to with DNA enhances the ionization of the surfactants upon estimate the CAC values from the concentration of surfactant interaction. This is induced by the decrease in the electrostatic at which the intensity starts to decrease. These are presented in repulsions between the headgroups of the surfactants on the Table 1 using both surfactant molar concentration and charge aggregates, as has been shown for protein− and nanoparticle− polyelectrolyte systems using experiments and molecular Table 1. Summary of the Obtained CAC and CCI Values, modeling.60−62 The longest surfactants (2), on the other Corresponding to the Charge Mixing Ratios Zhp‑1 and Zhp‑2, hand, show a different behavior with the cis isomer having a Respectively, Their Difference (ΔZhp), and the CMC Values larger CAC and CCI (estimated to be 60 and 300 μM, for the Surfactants under Study respectively). The CAC of trans-2 was found to be the same as surfactant CACa (Zhp‑1) CCIa (Zhp‑2) ΔZhp CMCb surfactants 1 and CTAB, while the CCI was even lower than CTAB, highlighting its strong cooperativity and rendering it cis-1 7 (1) 100 (15) 14 0.87 the most efficient surfactant for DNA condensation of those trans-1 7 (1) 100 (15) 14 0.42 studied in this work, that is, low concentrations of surfactant DTAB 50 (7.5) 2000 (300) 292.5 16 are required to both start DNA condensation and to render it cis-2 60 (9) 300 (45) 36 0.14 inaccessible to small molecules. An increase in the hydro- trans-2 7 (1) 20 (3) 2 0.15 phobic surfactant chain is known to increase the cooperativity CTAB 7 (1) 50 (7.5) 6.5 0.98 of the binding to DNA.63 The cis-2 surfactant, on the other a μM. bmM. CMC values, in mM at natural pH, were taken from refs hand, shows the largest CAC of all surfactants under study, 45 (for cis- and trans-1 and -2), 58 (for DTAB), and 59 (for CTAB). including all three possessing a shorter chain length. The CCI and ΔZhp are however lower than that of DTAB, showing a ratio between surfactant headgroups and DNA phosphate stronger cooperative binding than the commercial surfactant. It groups, here designated by Zhp‑1. The CAC of CTAB in the should be mentioned that cis-2 has a low solubility, and presence of DNA has been reported to be between 4 and 10 surfactant crystals were observed at concentrations of 2.5 mM μM,51,55−57 depending on the used technique and conditions, using cryogenic transmission electron microscopy,45 which which is in good agreement with the obtained result. Since the could be the reason behind the surprisingly high CAC. hydrophobic part of the surfactant is shorter, DTAB shows a Electrophoretic Mobility Experiments. The condensa- higher CAC than CTAB, but the value (50 μM) is somewhat tion of DNA induced by the cationic surfactants was lower than the reported value of 80 μM obtained using additionally studied using EMSA experiments, and the results fluorescence microscopy.51 are shown in Figure 2. The complexation of the surfactant to Table 1 also includes the surfactant concentration at which the DNA can be seen by the decrease in the intensity of the the normalized fluorescence intensity reaches its minimum, band corresponding to the naked DNA (due to the lower defined here as the concentration of critical intensity, CCI, as accessibility of the dye), the lower mobility of the bands, and/ 7037 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article not trivial. The decrease in the dimensions of the DNA molecules (see AFM results below) increases its electro- phoretic mobility. On the other, both the neutralization of the DNA charges and the increase in the molecular weight of the complexes (due to the presence of the surfactant) lead to a decrease in the electrophoretic mobility. The retention of the DNA in the wells at high surfactant concentrations occurs due to the neutralization of the negative charge of DNA and/or the formation of DNA−surfactant complexes that are larger than the mesh size of the gel.64 The images show very clearly the difference in the DNA condensation ability of the different cationic surfactants. While it is difficult to assess when condensation begins, the concentration at which no free DNA is observed can be compared to the Zhp‑2 value obtained from the DEA. Except for the DTAB, the concentration of free DNA is reduced to 0 within the studied surfactant concentration ranges, showing total DNA complexation induced by the surfactant aggregates. We can see that trans-2 is the most efficient surfactant showing complete DNA complexation for Zhp = 3 in excellent agreement with the results of the DEA. CTAB, cis-1, and cis- 2 show similar complexation profiles with the band corresponding to the bare DNA molecules disappearing for Figure 2. Electrophoretic mobility shift assay of 25 μg/mL DNA with Zhp values between 15 and 31. While cis-1 is in reasonable increasing surfactant concentrations. The first lane in each gel agreement with the DEA, CTAB required a much larger contains the ladder, and the lanes marked with DNA refer to DNA in the absence of surfactant. The surfactant concentration for each surfactant concentration to reach full DNA condensation and sample is shown in the gel image as charge ratios (Zhp). cis-2 required less. It is not clear to us why this is so. The discrepancy between the results obtained using EMSA and DEA has been observed previously for the DNA−CTAB or complete retention of the bands in the wells as the system and justified by the difference in the experimental surfactant concentration increases. The impact of the setup.47,64 In short, these are the presence of the electric field association of cationic surfactants to DNA on its mobility is and the impact of the gel network on the complexes, the Figure 3. Circular dichroism spectra of (a) salmon sperm DNA-(cis-1), (b) DNA-(trans-1), (c) DNA-(cis-2), and (d) DNA-(trans-2) complexes at different Zhp values. 7038 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article Figure 4. Intensity of CD spectra of the DNA−surfactant complexes at (a) 248 nm and (b) 277 nm normalized to the intensity of the band for DNA alone. Figure 5. AFM images of (a) free DNA and DNA complexed with (b) cis-1 and (c) trans-1 during the compaction process at Zhp ∼ 6. Panel (d) shows extended and partially compacted DNA chains obtained at Zhp ∼ 6. dilution of the complexes when these are loaded in the gel, and more pronounced in the case of the longer-chained surfactants, the different buffer used in the gel electrophoresis (to keep the and these also show a stronger ability to complex DNA. buffering capacity at higher temperatures). However, they do Circular Dichroism of DNA−Surfactant Complexes. not seem to affect all samples equally. It was also surprising The variation of the DNA conformation upon surfactant addition was monitored by CD spectroscopy.65−68 As shown in that a Zhp above 62 was required to induce full complexation of Figure 3, the CD spectrum of DNA is affected by the addition DNA using trans-1, making it the least efficient surfactant in of different amounts of surfactant. this assay with the exception of DTAB. While these results The black dashed curves in Figure 3 refer to DNA control were in lesser agreement with the DEA data, some of the samples (without surfactant) and show the characteristic trends are the same. Namely, the effect of stereochemistry is features of the B form of DNA given by a negative signal at 245 7039 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article nm, a positive band at 274 nm, and a crossover near 260 nm.69 radius (∼80 nm) of T2-DNA (164 kbp)−CTAB complexes.71 When one starts with cis- and trans-1 surfactants (Figure 3a,b, DNA compaction by the 12 carbon-long surfactants under respectively), it is seen that, up to Zhp = 0.78, the intensity of study is thus shown to be very effective and suitable for both positive and negative bands slightly decrease without an biological studies. Furthermore, extended DNA chains and appreciable shift in their position. At Zhp of 1.12 and above, partially compacted chains were also observed in these sample significant changes are observed in the CD spectra of the DNA. as shown in panel (d), confirming that the large error bars in Besides the large decrease in the signal, the negative band at the DEA experiments are related to the coexistence of different around 246 nm, attributed to the helicity of the DNA chain,66 species in solution due to the cooperative binding of the becomes positive; the positive band at 274 nm shifts to slightly surfactant molecules. larger wavelengths, and a small increase in the intensity of the band at 220 nm is observed with a further increase in surfactant concentration. Similar, albeit less extreme, changes ■ CONCLUSION DNA condensation by β-amino acid-based surfactants have been observed for intermediate concentrations of gemini possessing different chain lengths and headgroups with cis/ cationic surfactants.66 trans stereochemistry was studied using a range of techniques cis- and trans-2 surfactants seemingly induce analogous and compared with the well-studied surfactants DTAB and changes to the CD spectrum of DNA, but the variations are CTAB. All considered surfactants were shown to induce DNA more gradual (Figure 3c,d). In addition, a larger concentration condensation, which is expected to provide DNA with of cis-2 surfactant is required to induce the large variations in protection against DNases.64 The effect of the cis/trans the CD spectrum of DNA. stereochemistry was found to be most relevant for a surfactant To best visualize the differences between the surfactants, the with the longest alkyl chain with cis-2 showing the highest normalized intensity of the peaks at 248 and 277 nm is shown CAC according to dye exclusion assays. We speculate that this as a function of the ratio of charge, Zhp, in Figure 4. may be related to the lower solubility of this surfactant. trans-2, It is interesting to note that the abrupt variation in intensity on the other hand, was shown to be the most efficient of the observed for both cis-1 and trans-1 surfactants occurs at Zhp ∼ studied surfactants with a CAC similar to that of CTAB and a 1, identified as the CAC values (Zhp‑1) for these surfactants very short coexisting region of condensed and extended DNA based on the DEA experiments. On the other hand, trans-2, molecules. Surprisingly, the β-amino acid-based surfactants which was deemed to be the surfactant with the most with the shortest chain length (C12) were found to be as cooperative binding to DNA, is found to give rise to a efficient in condensing the first DNA molecules as CTAB smoother conformational transition, though seemingly starting (C16), considered a reference of efficiency. These results point at lower surfactant concentrations, at least in what concerns out the singularity of the headgroups in these surfactants that the negative band. Finally, cis-2 shows, again, a very smooth show stronger attractive interactions than for the tetramethy- decrease in intensity. Interestingly, the smoother conforma- lammonium ones, thus favoring the formation of surfactant tional transitions observed with CD were obtained for the aggregates. This is probably due to hydrogen bonding between longer-chained surfactants (cis- and trans-2) although the the headgroups and/or the enhanced hydrophobicity of these reason for this remains unclear. surfactants induced by the chiral cyclobutane unit. The relation Although it is difficult to compare the results of the different between the CAC of the DNA−surfactant systems and the techniques directly, the results obtained using CD spectrosco- CMC of the surfactants was found to be less straightforward py are consistent with those of DEA and EMSA experiments. than usual. The CMC varies with the pH of the solution and, These results suggest that the length of the DNA chain (2 or 4 although the work was performed in a buffer solution at pH kbp) has a low influence on the condensation process and on 7.4, the presence of DNA is likely to affect the protonation/ the CAC value. deprotonation of the surfactants. Considering the potential of Atomic Force Microscopy. It has been observed using pH-sensitive lipid formulation in the delivery of nucleic previously that cis- and trans-1 surfactants form, above the acids, a more in-depth study of these mechanisms is deemed CMC and at neutral pH, vesicles and mixtures of vesicles and important, as extrapolating the efficiency of a lipid formulation fibers, respectively. The longer-chained surfactants, on the based on its CMC only may not be sufficient. other hand, were found to form precipitates and crystals.45 The very low concentrations of β-amino acid-based Even though these studies are performed with surfactant surfactant required to condense DNA (except for cis-2), the concentrations below the CMC, it was interesting to verify the obtained DNA−surfactant aggregates with diameters of about impact of the stereochemistry of the surfactant in the 55−110 nm, and the absence of cytotoxicity45 identify these morphology of the formed complexes. To this end, DNA− cationic surfactants as promising candidates for efficient DNA surfactant complexes formed with cis- and trans-1 were delivery. ■ analyzed by AFM. To verify the impact of surfactant concentration on the complex geometry, a concentration of ASSOCIATED CONTENT surfactant of Zhp ∼ 6, between Zhp‑1 and Zhp‑2, was considered. * Supporting Information sı Figure 5a shows an AFM image of a DNA molecule with a The Supporting Information is available free of charge at contour length of about 1.35 μm as one would expect from a https://pubs.acs.org/doi/10.1021/acsabm.1c00683. 4145 bp-long molecule. Panels (b) and (c) show images of the DNA−surfactant complexes for cis- and trans-1, respectively. Details for the DEA experiments: experimental param- Clearly, these surfactants compact DNA into very small eters of the measurements of the emission spectra of the complexes with diameters ranging from about 55 to 110 nm. DNA−GelStar−surfactant mixtures (Table S1), emis- This is in a similar size range to DNA−surfactant complexes sion spectra of DNA−GelStar complexes at different precipitated from chloroform (diameters of 25−70 nm using concentrations of cis-1 (Figure S1), and emission spectra 300−500 bp long DNA molecules)70 and the hydrodynamic of DNA−GelStar complexes at different surfactant 7040 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article concentrations for all studied surfactants (Figure S2) Auricchio, A.; High, K. A.; Bennett, J. Safety and Efficacy of Gene (PDF) Transfer for Leber’s Congenital Amaurosis. N. Engl. J. Med. 2008, 358, ■ 2240−2248. (5) Kaplitt, M. G.; Feigin, A.; Tang, C.; Fitzsimons, H. L.; Mattis, P.; AUTHOR INFORMATION Lawlor, P. A.; Bland, R. J.; Young, D.; Strybing, K.; Eidelberg, D.; Corresponding Authors During, M. J. Safety and Tolerability of Gene Therapy with an Adeno- Rita S. Dias − Department of Physics, The Norwegian Associated Virus (AAV) Borne GAD Gene for Parkinson’s Disease: University of Science and Technology, NO-7491 Trondheim, An Open Label, Phase I Trial. Lancet 2007, 369, 2097−2105. Norway; orcid.org/0000-0001-9600-0934; (6) Yang, Z. R.; Wang, H. F.; Zhao, J.; Peng, Y. Y.; Wang, J.; Guinn, Email: rita.dias@ntnu.no B.-A.; Huang, L. Q. Recent Developments in the Use of Adenoviruses Rosa M. Ortuño − Departament de Química, Universitat and Immunotoxins in Cancer Gene Therapy. Cancer Gene Ther. 2007, Autònoma de Barcelona, 08193 Cerdanyola del Vallès, 14, 599−615. Barcelona, Spain; orcid.org/0000-0001-7635-7354; (7) Friedmann, T., Dunlap, J. C., Goodwin, S. F., Eds. Advances in Email: Rosa.Ortuno@uab.cat Genetics; Elsevier Inc.: Oxford, 2015; Vol. 89. (8) Niven, R.; Pearlman, R.; Wedeking, T.; Mackeigan, J.; Noker, P.; Authors Simpson-Herren, L.; Smith, J. G. Biodistribution of Radiolabeled Bernat Pi-Boleda − Departament de Química, Universitat Lipid-DNA Complexes and DNA in Mice. J. Pharm. Sci. 1998, 87, Autònoma de Barcelona, 08193 Cerdanyola del Vallès, 1292−1299. Barcelona, Spain; Present Address: Kao Corporation, S.A., (9) Mintzer, M. A.; Simanek, E. E. Nonviral Vectors for Gene Puig dels Tudons, 10, 08210 Barberà del Vallès, Delivery. Chem. Rev. 2009, 109, 259−302. (10) Gigante, A.; Li, M.; Junghänel, S.; Hirschhäuser, C.; Knauer, S.; Barcelona, Spain Schmuck, C. Non-Viral Transfection Vectors: Are Hybrid Materials Sravani Ramisetty − Department of Physics, The Norwegian the Way Forward? MedChemComm 2019, 10, 1692−1718. University of Science and Technology, NO-7491 Trondheim, (11) Stekar, J.; Nössner, G.; Kutscher, B.; Engel, J.; Hilgard, P. Norway; Present Address: University of Southern Synthesis, Antitumor Activity, and Tolerability of Phospholipids California Health Sciences Campus, Zilkha Neurogenetic Containing Nitrogen Homologues. Angew. Chem., Int. Ed. Engl. 1995, Institute, 1501 San Pablo Street, Los Angeles, CA 90033, 34, 238−240. USA. (12) Zhi, D.; Bai, Y.; Yang, J.; Cui, S.; Zhao, Y.; Chen, H.; Zhang, S. Ona Illa − Departament de Química, Universitat Autònoma de A Review on Cationic Lipids with Different Linkers for Gene Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain; Delivery. Adv. Colloid Interface Sci. 2018, 253, 117−140. orcid.org/0000-0001-7390-4893 (13) Damen, M.; Groenen, A. J. J.; van Dongen, S. F. M.; Nolte, R. J. Vicenç Branchadell − Departament de Química, Universitat M.; Scholte, B. J.; Feiters, M. C. Transfection by Cationic Gemini Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Lipids and Surfactants. MedChemComm 2018, 9, 1404−1425. Barcelona, Spain; orcid.org/0000-0003-3480-1669 (14) Wettig, S. D.; Verrall, R. E.; Foldvari, M. Gemini Surfactants: A New Family of Building Blocks for Non-Viral Gene Delivery Systems. Complete contact information is available at: Curr. Gene Ther. 2008, 8, 9−23. https://pubs.acs.org/10.1021/acsabm.1c00683 (15) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. A Versatile Vector for Gene Notes and Oligonucleotide Transfer into Cells in Culture and in Vivo: The authors declare no competing financial interest. ■ Polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (16) Van Bruggen, C.; Hexum, J. K.; Tan, Z.; Dalal, R. J.; Reineke, ACKNOWLEDGMENTS T. M. Nonviral Gene Delivery with Cationic Glycopolymers. Acc. Financial support from Ministerio de Economía y Compet- Chem. Res. 2019, 52, 1347−1358. itividad (MINECO), Spain (CTQ2016-77978-R AEI/FEDER, (17) Veeralakshmi, S.; Nehru, S.; Sabapathi, G.; Arunachalam, S.; UE), is gratefully acknowledged. Venuvanalingam, P.; Kumar, P.; Anusha, C.; Ravikumar, V. Single and ■ Double Chain surfactant-cobalt(III) Complexes: The Impact of REFERENCES Hydrophobicity on the Interaction with Calf Thymus DNA, and Their Biological Activities. RSC Adv. 2015, 5, 31746−31758. (1) Cavazzana-Calvo, M. Gene Therapy of Human Severe (18) Buhleier, E.; Wehner, W.; Vögtle, F. Cascade- and Nonskid- Combined Immunodeficiency (SCID)-X1 Disease. Science 2000, Chain-like Syntheses of Molecular Cavity Topologies. Synthesis 1978, 288, 669−672. 1978, 155−158. (2) Manno, C. S.; Pierce, G. F.; Arruda, V. R.; Glader, B.; Ragni, M.; (19) Dufes, C.; Uchegbu, I. F.; Schatzlein, A. G. Dendrimers in Gene Rasko, J. J. E.; Rasko, J.; Ozelo, M. C.; Hoots, K.; Blatt, P.; Konkle, B.; Delivery. Adv. Drug Delivery Rev. 2005, 57, 2177−2202. Dake, M.; Kaye, R.; Razavi, M.; Zajko, A.; Zehnder, J.; Rustagi, P. K.; (20) Martin, M. E.; Rice, K. G. Peptide-Guided Gene Delivery. Nakai, H.; Chew, A.; Leonard, D.; Wright, J. F.; Lessard, R. R.; AAPS J. 2007, 9, E18−E29. Sommer, J. M.; Tigges, M.; Sabatino, D.; Luk, A.; Jiang, H.; Mingozzi, F.; Couto, L.; Ertl, H. C.; High, K. A.; Kay, M. A. Successful (21) Chen, J.; Guan, X.; Hu, Y.; Tian, H.; Chen, X. Peptide-Based Transduction of Liver in Hemophilia by AAV-Factor IX and and Polypeptide-Based Gene Delivery Systems. Top. Curr. Chem. (Z) Limitations Imposed by the Host Immune Response. Nat. Med. 2017, 375, 32. 2006, 12, 342−347. (22) Jiménez Blanco, J. L.; Benito, J. M.; Ortiz Mellet, C.; García (3) Boyd, A. C., Ed. Gene and Stem Cell Therapy; Karger: Basel: New Fernández, J. M. Molecular Nanoparticle-Based Gene Delivery York, 2006. Systems. J. Drug Delivery Sci. Technol. 2017, 42, 18−37. (4) Maguire, A. M.; Simonelli, F.; Pierce, E. A.; Pugh, E. N.; (23) Zhao, N.; Yan, L.; Zhao, X.; Chen, X.; Li, A.; Zheng, D.; Zhou, Mingozzi, F.; Bennicelli, J.; Banfi, S.; Marshall, K. A.; Testa, F.; X.; Dai, X.; Xu, F.-J. Versatile Types of Organic/Inorganic Surace, E. M.; Rossi, S.; Lyubarsky, A.; Arruda, V. R.; Konkle, B.; Nanohybrids: From Strategic Design to Biomedical Applications. Stone, E.; Sun, J.; Jacobs, J.; Dell’Osso, L.; Hertle, R.; Ma, J.-X.; Chem. Rev. 2019, 119, 1666−1762. Redmond, T. M.; Zhu, X.; Hauck, B.; Zelenaia, O.; Shindler, K. S.; (24) Dias, R. S., Lindman, B., Eds. DNA Interaction with Polymers Maguire, M. G.; Wright, J. F.; Volpe, N. J.; McDonnell, J. W.; and Surfactants; John Wiley & Sons, Inc: Hoboken, NJ, 2008. 7041 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article (25) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. Cyclobutane β-amino Acid-based Cationic Amphiphiles: Possible R.; Anderson, D. G. Non-viral Vectors for Gene-based Therapy. Nat. Candidates for Use in Gene Therapy. J. Mol. Liq. 2020, 297, 111856. Rev. Genet. 2014, 15, 541−555. (46) Balzer, S.; Kucharova, V.; Megerle, J.; Lale, R.; Brautaset, T.; (26) Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. Size- Valla, S. A comparative analysis of the properties of Regulated dependent Internalization of Particles Via the Pathways of Clathrin- Promoter Systems Commonly Used for Recombinant Gene and Caveolae-mediated Endocytosis. Biochem. J. 2004, 377, 159−169. Expression in Escherichia coli. Microb. Cell Fact. 2013, 12, 26. (27) Guo, X.; Huang, L. Recent Advances in Nonviral Vectors for (47) Ainalem, M.-L.; Bartles, A.; Muck, J.; Dias, R. S.; Carnerup, A. Gene Delivery. Acc. Chem. Res. 2012, 45, 971−979. M.; Zink, D.; Nylander, T. DNA Compaction Induced by a Cationic (28) Wang, W.; Li, W.; Ma, N.; Steinhoff, G. Non-Viral Gene Polymer or Surfactant Impact Gene Expression and DNA Delivery Methods. Curr. Pharm. Biotechnol. 2013, 14, 46−60. Degradation. PLoS One 2014, 9, No. e92692. (29) Husale, S.; Grange, W.; Karle, M.; Bürgi, S.; Hegner, M. (48) Maurstad, G.; Danielsen, S.; Stokke, B. T. Analysis of Interaction of Cationic Surfactants with DNA: A Single-Molecule Compacted Semiflexible Polyanions Visualized by Atomic Force Study. Nucleic Acids Res. 2008, 36, 1443−1449. Microscopy: Influence of Chain Stiffness on the Morphologies of (30) Cheng, C.; Ran, S.-Y. Interaction between DNA and Trimethyl- Polyelectrolyte Complexes. J. Phys. Chem. B 2003, 107, 8172−8180. Ammonium Bromides with Different Alkyl Chain Lengths. Sci. World (49) Zhu, D.-M.; Evans, R. K. Molecular Mechanism and J. 2014, 2014, 1−9. Thermodynamics Study of Plasmid DNA and Cationic Surfactants (31) Dias, R.; Rosa, M.; Canelas Pais, A.; Miguel, M.; Lindman, B. Interactions. Langmuir 2006, 22, 3735−3743. DNA-Surfactant Interactions. Compaction, Condensation, Decom- (50) Mel’nikov, S.; Khan, M. O.; Lindman, B.; Jönsson, B. Phase paction and Phase Separation. J. Chin. Chem. Soc. 2004, 51, 447−469. Behavior of Single DNA in Mixed Solvents. J. Am. Chem. Soc. 1999, (32) Pérez, N.; Pérez, L.; Infante, M. R.; García, M. T. Biological 121, 1130−1136. Properties of Arginine-Based Glycerolipidic Cationic Surfactants. (51) Dias, R.; Mel’nikov, S.; Lindman, B.; Miguel, M. G. A DNA Green Chem. 2005, 7, 540−546. Phase Behavior in the Presence of Oppositely Charged Surfactants. (33) Infante, M. R.; Pérez, L.; Morán, M. C.; Pons, R.; Mitjans, M.; Langmuir 2000, 16, 9577−9583. Vinardell, M. P.; Garcia, M. T.; Pinazo, A. Biocompatible Surfactants (52) Shirahama, K.; Takashima, K.; Takisawa, N. Interaction from Renewable Hydrophiles. Eur. J. Lipid Sci. Technol. 2010, 112, between Dodecyltrimethylammonium Chloride and DNA. Bull. 110−121. Chem. Soc. Jpn. 1987, 60, 43−47. (34) Pinazo, A.; Pons, R.; Pérez, L.; Infante, M. R. Amino Acids as (53) Dias, R. S.; Pais, A. A. C. C.; Miguel, M. G.; Lindman, B. Raw Material for Biocompatible Surfactants. Ind. Eng. Chem. Res. Modeling of DNA Compaction by Polycations. J. Chem. Phys. 2003, 2011, 50, 4805−4817. 119, 8150−8157. (35) Chandra, N.; Tyagi, V. K. Synthesis, Properties, and (54) Carlstedt, J.; Lundberg, D.; Dias, R. S.; Lindman, B. Applications of Amino Acids Based Surfactants: A Review. J. Condensation and Decondensation of DNA by Cationic Surfactant, Dispersion Sci. Technol. 2013, 34, 800−808. Spermine, or Cationic Surfactant - Cyclodextrin Mixtures: Macro- (36) Foley, P.; Kermanshahi, A.; Beach, E. S.; Zimmerman, J. B. scopic Phase Behavior, Aggregate Properties, and Dissolution Derivation and Synthesis of Renewable Surfactants. Chem. Soc. Rev. Mechanisms. Langmuir 2012, 28, 7976−7989. 2012, 41, 1499−1518. (55) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. Discrete Coil- (37) Fielden, M. L.; Perrin, C.; Kremer, A.; Bergsma, M.; Stuart, M. Globule Transition of Large DNA Induced by Cationic Surfactant. J. C.; Camilleri, P.; Engberts, J. B. F. N. Sugar-Based Tertiary Amino Am. Chem. Soc. 1995, 117, 2401−2408. Gemini Surfactants with a Vesicle-to-Micelle Transition in the (56) Dias, R. S.; Magno, L. M.; Valente, A. J. M.; Das, D.; Das, P. K.; Endosomal pH Range Mediate Efficient Transfection in Vitro. Eur. Maiti, S.; Miguel, M. G.; Lindman, B. Interaction between DNA and J. Biochem. 2001, 268, 1269−1279. Cationic Surfactants: Effect of DNA Conformation and Surfactant (38) Bordes, R.; Tropsch, J.; Holmberg, K. Role of an Amide Bond Headgroup. J. Phys. Chem. B 2008, 112, 14446−14452. for Self-Assembly of Surfactants. Langmuir 2010, 26, 3077−3083. (57) Husale, S.; Grange, W.; Karle, M.; Bürgi, S.; Hegner, M. (39) Bracic, M.; Hannson, P.; Pérez, L.; Zemlijic, L. F.; Kogej, K. Interaction of Cationic Surfactants with DNA: a Single-molecule Interaction of sodium hyaluronate with a biocompatible cationic Study. Nucleic Acids Res. 2008, 36, 1443−9. surfactant from lysine: a binding study. Langmuir 2015, 31, 12043− (58) Causi, S.; DeLisi, R.; Milioto, S.; Tirone, N. Dodecyltrimethy- 12053. lammonium Bromide in Water-urea Mixtures. Volumes, Heat (40) Bodnár, K.; Fegyver, E.; Nagy, M.; Mészárós, R. Impact of Capacities and Conductivities. J. Phys. Chem. 1991, 95, 5664−5673. polyelectrolyte Chemistry on the Thermodynamic Stability of (59) Okuda, H.; Imae, T.; Ikeda, S. The Adsorption of Oppositely harged Macromolecule/ Surfactant Mixtures. Langmuir Cetyltrimethylammonium Bromide on Aqueous Surfaces of Sodium 2016, 32, 1259−1268. Bromide Solutions. Colloids Surf. 1987, 27, 187−200. (41) Wu, M.; An, N.; Li, Y.; Sun, J. Layer-by-layer Assembly of (60) Cakara, D.; Chassagne, C.; Gehin-Delval, C.; Borkovec, M. Fluorine-free olyelectrolyte-Surfactant Complexes for the Fabrication Protonation of Carboxyl Latex Particles in the Presence of a Strong of Self-healing uperhydrophobic Films. Langmuir 2016, 32, 12361− Cationic Polyelectrolyte. Colloids Surf., A 2007, 294, 174−180. 12369. (61) da Silva, F. L. B; Lund, M.; Jonsson, B.; Akesson, T. On the (42) Pi-Boleda, B.; Sorrenti, A.; Sans, M.; Illa, O.; Pons, R.; Complexation of Proteins and Polyelectrolytes. J. Phys. Chem. B 2006, Branchadell, V.; Ortuño, R. M. Cyclobutane Scaffold in Bolaamphi- 110, 4459−44. philes: Effect of Distereoisomerism and Regiochemistry on their (62) Stornes, M.; Blanco, P. M.; Dias, R. S. Polyelectrolyte- Surface Activity Aggregate Structure. Langmuir 2018, 34, 11424− Nanoparticle Mutual Charge Regulation and its Influence on their 11432. Complexation. Colloids Surf., A 2021, 628, 127258. (43) Sorrenti, A.; Illa, O.; Pons, R.; Ortuño, R. M. Chiral (63) Hayakawa, K.; Santerre, J. P.; Kwak, J. C. The Binding of Cyclobutane β-amino Acid-based Amphiphiles: Influence of cis/ Cationic Surfactants by DNA. Biophys. Chem. 1983, 17, 175−181. trans Stereochemistry on Solution, Self-aggregation and Recognition. (64) Dias, R. S.; Svingen, R.; Gustavsson, B.; Lindman, B.; Miguel, Langmuir 2015, 31, 9608−9618. M. G.; Åkerman, B. Electrophoretic Properties of Complexes Between (44) Sorrenti, A.; Illa, O.; Pons, R.; Ortuño, R. M. Chiral yclobutane DNA and the Cationic Surfactant Cetyltrimethylammonium Bromide. β-amino Acid-based Amphiphiles: Influence of cis/trans Stereo- Electrophoresis 2005, 26, 2908−2917. chemistry on Condensed Phase and Mono-layer Structure. Langmuir (65) Mohanty, A.; Dey, J. Effect of the Headgroup Structure on the 2016, 32, 6977−6984. Aggregation Behavior and Stability of Self-Assemblies of Sodium N- (45) Pi-Boleda, B.; Bouzas, M.; Gaztelumendi, N.; Illa, O.; Nogués, [4-(n-Dodecyloxy)benzoyl]-L-Aminoacidates in Water. Langmuir C.; Branchadell, V.; Pons, R.; Ortuño, R. M. Chiral pH-Sensitive 2007, 23, 1033−1040. 7042 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043 ACS Applied Bio Materials www.acsabm.org Article (66) Grueso, E.; Kuliszewska, E.; Roldan, E.; Perez-Tejeda, P.; Prado-Gotor, R.; Brecker, L. DNA Conformational Changes Induced by Cationic Gemini Surfactants: The Key to Switching DNA Compact Structures into Elongated Forms. RSC Adv. 2015, 5, 29433−29446. (67) Khouri, S. J.; Buss, V. Circular Dichroism and Derivative Spectra Study of the Excitonic Aggregation of Pinacyanol by Aerosol- OT. Open J. Phys. Chem. 2012, 2, 34−40. (68) Chang, Y.-M.; Chen, C. K.-M.; Hou, M.-H. Conformational Changes in DNA upon Ligand Binding Monitored by Circular Dichroism. Int. J. Mol. Sci. 2012, 13, 3394−3413. (69) Berova, N., Polavarapu, P. L., Nakanishi, K., Woody, R. W., Eds. Comprehensive Chiroptical Spectroscopy, Applications in Stereochemical Analysis of Synthetic Compounds, Natural Products and Biomolecules; John Wiley & Sons, Inc: Hoboken, NJ, 2012. (70) Komolov, A.; Mel’nikov, A. B.; Schaumburg, K.; Ryumtsev, E.; Lezov, A. Sizes and Conformation of the Molecules of DNA- Surfactant Complexes in Dilute Solutions and on the Atomic Smooth Substrates. Colloid J. 2002, 64, 155−159. (71) Dias, R. S.; Innerlohinger, J.; Glatter, O.; Miguel, M. G.; Lindman, B. Coil-globule Transition of DNA Molecules Induced by Cationic Surfactants: A Dynamic Light Scattering Study. J. Phys. Chem. B 2005, 109, 10458−10463. 7043 https://doi.org/10.1021/acsabm.1c00683 ACS Appl. Bio Mater. 2021, 4, 7034−7043