pubs.acs.org/Langmuir © 2010 American Chemical Society
Interaction between DNA and Cationic Amphiphiles: A Multi-Technique Study Pietro Di Profio, Raimondo Germani, Laura Goracci, Roberto Grilli, Gianfranco Savelli,* and Matteo Tiecco CEMIN, Center of Excellence on Innovative Nanostructured Materials, Department of Chemistry, University of Perugia, Via Elce di Sotto 8, I-06123 Perugia, Italy Received December 18, 2009. Revised Manuscript Received January 21, 2010 The interaction of cationic amphiphiles with calf thymus DNA has been investigated by physicochemical techniques (surface tension, conductometry, UV spectroscopy, thermal denaturation) and morphological microscopies (AFM and TEM). The cationic molecules were the amphiphiles cetyltrimethylammonium and cetyltributylammonium bromides (CTAB and CTBAB, respectively), compared to the nonamphiphilic tetramethyl- and tetrabutylammonium bromides (TMAB and TBAB, respectively) and, as a transfection-efficient comparison, a commercial poliethyleneimine (PEI). As a result, well below their critical micelle concentrations (cmc), CTAB and CTBAB showed a peculiar, nonlinear adsorption profile with the nucleic acid, which showed a correlation with the melting temperatures and morphological changes observed with AFM and TEM microscopies. On the other hand, TMAB and TBAB interact much less with the DNA duplexes and do not induce any modifications of the structures. The same behavior was observed with PEI; however, CTAB and CTBAB proved much less effective in condensing the nucleic acid.
Introduction The reversible condensation of nucleic acids is a process that is naturally accomplished by histones, which are positively charged, barrel-shaped proteins.1 This compaction is also actively studied for its relevance in the transport of DNA and other nucleic acids (notably, siRNAs) through the cell membrane for in vitro and in vivo gene delivery.2-4 With DNA being a polyanion, the study and application of transfection carriers have been focused mainly upon cationic polymers5-7 and cationic amphiphiles, or surfactants,8,9 which act by partially neutralizing the surface charge of DNA, thus facilitating the transport of the DNA-carrier complex through the lipid bilayer. An important aspect of nucleic acid transfection is the ability to release the transfected DNA fragment within the cell, as a strong binding to the carrier may disturb its further processing by the nuclear machinery. Within this growing research effort, we have already published a paper concerning the use of a pH-sensitive probe (Hoechst 33258) to characterize the binding of pH-sensitive amphiphiles, dodecyldimethylamine oxide (DDAO) and p-dodecyloxybenzyldimethylamine oxide (pDoAO), as promising carriers which can be easily released by *Tel./Fax: (þ39)075-5855538. E-mail:
[email protected]. (1) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 5th ed.; W.H. Freeman & Co, 2008. (2) Lasic, D. D.; Templeton, N. S. Adv. Drug Delivery Rev. 1996, 20, 221–226. (3) Zhang, Y.; Reimer, D. L.; Zhang, G.; Lee, P. H.; Bally, M. B. Pharm. Res. 1997, 14, 190–196. (4) Chittimalla, C.; Zammut-Italiano, L.; Zuber, G.; Behr, J. P. J. Am. Chem. Soc. 2005, 127, 11436–11441. (5) Subramanian, M.; Holopainen, J. M.; Paukku, T.; Eriksson, O.; Huhtaniemi, I.; Kinnunen, P. K. J. Biochim. Biophys. Acta 2000, 1466, 289–305. (6) Barreleiro, P. C. A.; Lindmann, B. J. Phys. Chem. B 2003, 107, 6208–6213. (7) Byk, G.; Dubertret, C.; Escriou, V.; Frederic, M.; Jaslin, G.; Rangara, R.; Pitard, B.; Crouzet, J.; Wils, P.; Schwartz, B.; Scherman, D. J. Med. Chem. 1998, 41, 224–235. (8) Marchetti, S.; Onori, G.; Cametti, C. J. Phys. Chem. B 2005, 109, 3676–3680. (9) Zhao, X.; Shang, Y.; Hu, J.; Liu, H.; Hu, J. Biophys. Chem. 2008, 138, 144– 149. (10) Goracci, L.; Germani, R.; Savelli, G.; Bassani, D. ChemBioChem 2005, 6, 197–203.
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a DNA molecule following their deprotonation switch by a pH change within the physiological range.10 In general, most recent studies point to the importance of a multidisciplinary approach for achieving a deeper understanding of the carrier-mediated transfection processes, and several physical, chemical, and biomedical techniques are being increasingly combined to obtain a broader, more integrated range of information.11-15 Herein, we report the results of a multitechnique study on the interaction of eukaryotic DNA with cationic surfactants (CTAB and CTBAB), nonamphiphilic quaternary ammonium salts (TMAB and TBAB), and a transfection-efficient, commercial polyethyleneimine (PEI; ExGen 500, Fermentas). Surface tension, conductivity, UV characteristic absorption, and DNA melting measurements were supplemented with morphological studies by atomic force microscopy (AFM) and transmission electron microscopy (TEM). In the design of the experiments, particular care was taken to select experimental conditions which could be adopted basically unchanged for all techniques, thus adding to the relevance of the comparisons among various approaches. The ultimate goal was to achieve a significant set of correlations among the various physicochemical behaviors of the DNA-carrier complexes and the structural changes induced by such complex formation.
Experimental Section Materials and Methods. CTAB and CTBAB surfactants,
TMAB and TBAB salts, and SSC (sodium chloride - sodium citrate buffer) solution were commercial (Sigma-Aldrich); ExGen 500 was obtained from Fermentas. Double-stranded calf thymus (11) Bhattacharya, S.; Mandal, S. S. Biochemistry 1998, 37, 7764–7777. (12) Mel’nikova, Y.; Lindmann, B. Langmuir 2000, 16, 5871–5878. (13) Wang, L.; Yoshida, J.; Ogata, N.; Sasaki, S.; Kajiyama, T. Chem. Mater. 2001, 13, 1273–1281. (14) Sun, X.; Cao, E.; Zhang, X.; Liu, D.; Bai, C. Inorg. Chem. Commun. 2002, 5, 181–186. (15) Marchetti, S.; Onori, G.; Cametti, C. J. Phys. Chem. B 2006, 110, 24761– 24765.
Published on Web 02/10/2010
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Figure 1. Surface tension measurements of CTAB in the presence (circles) and absence (squares) of DNA. DNA was from Sigma-Aldrich; its concentration was determined spectrophotometrically by considering the molar extinction coefficient of DNA bases to be equal to 6600 M-1 cm-1 at 260 nm.16 The concentration of DNA is expressed in bM (base Molar) that corresponds to the concentration of base pairs, which equals the concentration of phosphate groups. The water used was ultrapure (Milli-Q). Surface Tension Measurements. Surface tension was measured by using a Fisher 20, du No€ uy type, tensiometer in a thermostated bath (298.1 ( 0.1 K). To a DNA solution (concentration determined spectrophotometrically) in SSC, specific amounts of ammonium salt were added. After each addition, the solution was stirred for 2 min, then left for 5 min before reading the surface tension values. Conductivity Measurements. Conductivity was measured at 298.1 K ((0.1 K) in an Analytical Control conductivity meter (model 120) equipped with a platinum cell (cell constant = 1.05 cm-1). To a DNA solution (concentration determined spectrophotometrically), specific amounts of ammonium salt were added by syringe. After each addition, the solution was stirred for 30 s, then left for 1 min before reading the conductivity value. UV Spectroscopy. Absorbance at 260 nm was measured with an Agilent 8453 spectrophotometer equipped with Peltier temperature control Agilent 89089A, at a temperature of 298.1 K ((0.1 K). The quartz cells had an optical path length of 1 cm. To a DNA solution (concentration determined spectrophotometrically) in SSC, specific amounts of ammonium salt were added by syringe. DNA Melting Measurements. Absorbance at 260 nm was measured with the above spectrophotometer, controlling the temperature with a Peltier temperature control Agilent 89089A ((0.1 K). Quartz cells are as above. The starting temperature was set at 303.1 K and the final temperature at 363.1 K, with 2 min of thermal stabilization between each increment of 1 K. The instrument software calculates automatically the melting temperature; however, all the data were double-checked with independent, commonly used data processing software. Atomic Force Microscopy (AFM) Measurements. A Solver-Pro (NT-MDT) atomic force microscope equipped with a NSG10 (NT-MDT) cantilever (resonant frequency 190325 kHz, force constant 5.5-22.5 N/m, curvature radius 10 nm) was used to record topography and phase images. The measure(16) Sambrook, E. F.; Fritsch, E. J.; Maniatis, T. Molecular Cloning: A; Laboratory Manual; Cold Spring Harbor Laboratory Press: New York, 1989.
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Figure 2. Surface tension profiles of CTBAB in the presence (circles) and absence (squares) of DNA. ments were carried out in semicontact mode imaging in air and at room temperature. Images were processed by flattening to remove the background slope, and the contrast and brightness were adjusted. Twenty-five μL of the DNA/surfactant solution at the desired concentration ratio was deposited onto a freshly cleaved mica surface and allowed to dry for 24 h in a drybox containing silica gel. Herein, the sample was washed with ultrapure water (Milli-Q) and dried again for 24 h. Electronic Transmission Microscopy (TEM). Three drops of surfactant/DNA solutions in ultrapure water (prepared calculating the DNA concentration spectrophotometrically) were dripped on the surface of an AGAR S120 film (200 mesh). No contrast agents were added. Imaging was carried out on a Philips CM100 microscope at 80 kV. The images reported are scans of photographic films and were obtained by selecting from different images on different samples of the same mixtures.
Results and Discussion Surface Tension Measurements. Figure 1 shows the surface tension vs [CTAB] with (circles) and without (squares) 1.28 10-4 bM calf thymus DNA in 0.01 SSC. Surfactant alone features a monotonic decrease of surface tension up to a critical point, the cmc, where it stops decreasing due to the formation of micellar aggregates.17 The addition of DNA gives rise to a more complex profile, where a first, decreasing region is followed by a sudden further decrease of the surface tension, which is steeper than that observed with surfactant alone and, after passing through a minimum, rises to reach the curve of the surfactant without DNA, almost overlapping it thereafter. The first critical point on the curve occurs at a [CTAB] to [DNA] ratio of ca. 0.53. Incidentally, herein we refer to the molar ratio of [surfactant]/[DNA], instead of reporting the molar concentrations, for two main reasons: first, it is easier in this way to quantify the interaction of surfactant molecules with the phosphate groups of the DNA polyanion. Second, the use of that ratio allows to compare consistent numbers among duplicate and triplicate experiments, which typically may contain slightly different concentrations of DNA. When the same experiment is conducted with CTBAB as a surfactant, a profile which is very similar to that (17) Mukerjee, P.; Mysels, K. J. Critical micelle concentration of aqueous surfactant systems; NSDS-National Bureau of Standards, 1971.
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Figure 4. A260 adsorption profile of CTAB and DNA system in 0.01 SSC.
Figure 3. A260 vs [CTAB]/[DNA].
of CTAB is obtained (Figure 2), and the critical point marking the start of a steep decrease of surface tension is around [CTBAB]/[DNA] = 0.57. Incidentally, the above critical phenomena all occur well below the cmc’s of CTAB and CTBAB, i.e., the interactions giving rise to the observed behaviors are not to be ascribed to micelles. We may reason that the first region of surface tension values is characterized by an almost quantitative binding of surfactant molecules to the DNA polyanion, such that the concentration of amphiphile at the water surface is negligible, as suggested by the low surface activity. After the critical point is reached at [surfactant]/[DNA] = 0.53 to 0.57, the sudden decrease of surface tension, which is faster than with surfactant alone, may be ascribed to a sort of saturation of the binding sites on the polyanion, whereupon further addition of surfactant restores its distribution on the water surface, and an additional contribution to surface tension decrease may be due to a moderate surface transfer of a “lipophilized” nucleic acid. Recent experiments with 2D Langmuir balance and AFM have pointed to the importance of the surfactant hydrocarbon tails in determining the binding to DNA and the resulting physicochemical properties of the resulting complexes.3,18 To confirm the important role of a hydrophobic moiety in the interactions of amphiphiles with nucleic acids, we performed the same surface tension measurements with TMAB and TBAB, which may be considered analogues of CTAB and CTBAB, respectively, except for lack of a tail. Neither of these ammonium salts results in any significant surface activity differences in the absence and presence of DNA (Figure 1 in Supporting Information). Interestingly, the same experiments carried out in water instead of 0.01 SSC gave strikingly similar results, and this represents an important validation to the comparison with the results of conductometry. UV Spectroscopy. Absorbance measurements at 260 nm (maximum wavelength of absorption of nucleic acids of DNA) were conducted at a fixed DNA concentration and increasing surfactant concentrations in 0.01 SSC. As shown in Figure 3, absorbance at 260 nm (A260) decreases with a constant slope as [CTAB] increases up to [CTAB]/[DNA] = ca. 0.63. Afterward, the rate of absorbance decrease changes to a second, less negative value. (18) Chen, X.; Wang, J.; Liu, M. J. Colloid Interface Sci. 2005, 287, 185–190.
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The same qualitative and quantitative behavior was observed with CTBAB, which shows a discontinuity on the ratio [CTBAB]/[DNA] = 0.51. On the other hand, only a modest, monotonic decrease of A260 was obtained with DNA in the presence of both TMAB and TBAB (Figure 2 in Supporting Information). When the experiments were repeated in pure water (no SSC), the resulting A260 curves were qualitatively similar to those in 0.01 SSC (Figure 4). It has been reported that double-stranded DNA denaturates to a single-stranded form without the proper ionic strength in aqueous solutions;10 therefore, one may question why a similar sigmoidal decrease of A260 vs [surfactant] is observed for both SSC and pure water. In other words, it seems that the interaction of a cationic surfactant and DNA;which is a sum of contributions of electrostatic interactions and hydrophobic interactions due to the alkyl chains;is not dramatically dependent on the association state of the nucleic acid, i.e., double- or single-stranded, as far as the UV characteristic absorption and surface tension behaviors are considered. This is apparently in contrast to a recent report, which shows that DNA-cationic surfactant interactions are different for ds- and ss-DNA.19 Conductometry. As a matter of course, conductometric measurements were performed only in water, as 0.01 SSC would give such a high conductivity as to hide the subtle variations due to DNA-amphiphile interactions. First, a conductometric profile of CTAB alone was obtained up to a surfactant concentration well above its cmc, whereupon a value of 8.6 10-4 M was obtained as the cmc at the point where the slope changes to a less positive value due to micellar aggregate formation.17 When the same conductivity profile is obtained for the system CTAB þ DNA, the observed cmc is increased to 9.39 10-4 M (Figure 5). Then, if we zoom in on the leftmost portion of this profile (Figure 5 inset), on a CTAB concentration range which is 1 order of magnitude lower than its cmc, we observe that the initial slope of the conductivity profile is lower. Then, the slope returns to its “control” value (i.e., without DNA) after a certain CTAB concentration which corresponds to a molar ratio to DNA of ca. 0.56.20,21 (19) Rosa, M.; Dias, R.; Miguel, M.; Lindmann, B. Biomacromolecules 2005, 6, 2164–2171. (20) Tiecco, M.; Savelli, G.; Goracci, L.; Di Profio, P. Proceedings of the 9th Conference on Colloid Chemistry; Siofok, Hungary, 2007. (21) Di Profio, P.; Germani, R.; Goracci, L.; Savelli, G.; Tiecco, M. Proceedings of the 9th Conference on Colloid Chemistry; Siofok, Hungary, 2007.
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Figure 5. Conductivity profile of CTAB in the presence of DNA (4.73 10-5 bM). Figure 7. Conductivity profiles of TBAB (triangles) and TMAB (squares) in the presence of DNA. Table 1. Melting Temperatures of Additive-DNA Systems [CTAB]/[DNA]
Tm (K)
[TMAB]/[DNA]
Tm (K)
Only DNA 0.015 0.03 0.15 0.3
327.84 327.84 329.59 329.95 332.19
Only DNA 0.015 0.03 0.15 0.3
327.84 327.58 328.30 329.02 329.73
Table 2. Van’t Hoff Calculations of Free Energy Variations in the Additive-DNA Systems [CTABr]/ [DNA] Only DNA 0.015 0.03 0.15 0.3
Figure 6. Conductivity profile of CTBAB in the presence of DNA.
Performing the same experiments with CTBAB and DNA results in qualitatively similar values to those with CTAB, with a “critical” slope change value of ca. 0.51 (Figure 6). Then, we measured the conductivity profiles of TMAB and TBAB both with and without DNA, as a control for estimating the role of the hydrophobic chain of the above surfactants on the interaction with nucleic acids, as already discussed. Figure 7 shows the case for TBAB and TMAB, and it is clearly seen that the profiles do not show discontinuities. In particular, the profiles of TBAB-DNA and TMAB-DNA are strictly linear and do not feature any slope changes as those observed with the surfaceactive analogues. When we compare the results obtained with the first three techniques as reported above, the main observation that can be made is about the spotting of a “critical” molar fraction range of [surfactant]/[DNA] of 0.5 to 0.6, which is remarkably narrow given the inherent, large differences among the physical parameters investigated by each technique. Within that range, the surfactant-DNA complex shows (i) a steep increase of surface 7888 DOI: 10.1021/la9047825
ΔG (J/mol) ΔH (J /mol) ΔS (J/mol K) Keq
T=327.84K
T=327.84K
T=327.84K
1 0 0.876 362.497 0.752 776.71 0.631 1257.52 0.279 3486.27
188408.51 224631.87 172706.76 204187.31 187930.38
574.68 684.08 524.44 618.98 562.58
ΔG (J/mol)
ΔH (J/mol) ΔS (J/mol K)
[TMABr]/ [DNA]
Keq
T=327.84K
T=327.84K
T=327.84K
Only DNA 0.015 0.03 0.15 0.3
1 1.083 0.794 0.642 0.508
0 -217.93 627.93 1210.81 1849.24
188408.51 280147.16 285604.24 219921.72 237044.06
574.68 855.20 869.22 667.12 717.41
activity, (ii) an increase of bulk conductivity slope, and (iii) an inflection point on the characteristic UV absorption curve, respectively. Thermal Denaturation (Melting) of DNA. We also conducted preliminary studies (limited to CTAB and TMAB) of DNA denaturation by heat, or melting, which consists of the dissociation of DNA duplexes due to a temperature increase. The temperature corresponding to a 50% increase of maximum hyperchromicity is the melting (transition) temperature (Tm). In Table 1 are reported the melting temperatures at four different [additive]/[DNA] ratios (0.015, 0.03, 0.15, 0.30, with additive being CTAB and TMAB, respectively) and with DNA alone as a control (in Supporting Information Figures 3 and 4 are reported the melting curves). Langmuir 2010, 26(11), 7885–7892
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Figure 8. AFM images of DNA (A), [CTAB]/[DNA] = 0.3 (B), [CTAB]/[DNA] = 1 (C), [TMAB]/[DNA] = 1 (D).
It is observed that both alkylammonium salts give rise to an increase of Tm, as is known for salts in general, but CTAB shows a higher stabilization of the DNA duplex as compared to TMAB, given by the respective increases of Tm (4.35 and 1.89 K for CTAB and TMAB, respectively; see Table 1). If we measure a melting curve increasing the concentration of the surfactant ([CTAB]/ [DNA] up to 2), no denaturation is observed at all up to 368 K, which is not the case for TMAB (data not shown). Melting curves allow us to determine the thermodynamic parameters of DNA denaturation and quantify the stabilization caused by ammonium salt additives.22 During a thermal denaturation experiment as followed by absorbance measurements at 260 nm, the fraction of single-stranded DNA molecules at a given temperature T are given by ð1Þ θT ¼ ðAT - Amin Þ=ðAmax - Amin Þ where AT, Amin, and Amax are the absorbance values read at a temperature T, at the beginning of the experiment, and at full denaturation (>368 K), respectively. In this respect, the melting temperature (Tm) is the temperature at which Γ = 0.5. Assuming that the equilibrium between double- and single-stranded forms is a two-state one, a van’t Hoff approach may be used to estimate the thermodynamic parameters of denaturation, i.e., the standard enthalpy and entropy of the process, ΔH° and ΔS°: ΔH° ¼ 4RðTm Þ2 ðdθ=dTÞTm
ð2Þ
ΔS° ¼ ΔH°=Tm
ð3Þ
(22) Guglielmi, L.; Gianfranceschi, G. L.; Venanzi, F.; Polzonetti, A.; Amici, D. Mol. Biol. Rep. 1978, 4, 195–201.
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From these values of enthalpy and entropy variations, the Helmoltz free energy (ΔG) of the denaturation can be calculated. In particular, ΔG = 0 at the Tm for the ct-DNA without additives under the experimental conditions adopted, and therefore values of ΔG for the same DNA and conditions, but in the presence of CTAB and TMAB, gives a quantitative estimate of the stabilization of the DNA molecule by these ammonium salts. Table 2 reports the results of van’t Hoff calculations and the free energy variations obtained according the above method (Keq = (θT/1 - θT). The variations of ΔH° and ΔS° with increasing additive ratios are nonmonotonic, and the highest variations relative to DNA alone were observed with the lowest concentration of additives (CTABr]/[DNA] = 0.015 and [TMABr]/[DNA] = 0.03). Even if we recognize that structural inferences based on enthalpy and entropy data should be taken cautiously, we may propose that a possible structural effect on the secondary and/ or tertiary structure of DNA induced by the two ammonium salts appears stronger at the lowest additive concentrations, and this effect seems much more remarkable with CTAB than TMAB. It should be added that this structural effect at low additive concentration is barely visible according to AFM and TEM imaging (see below). In terms of the free energy XG, however, this thermodynamic approach shows that CTAB at [CTAB]/[DNA] = 0.3 gives a twofold stabilization of the DNA duplex as compared to TMAB at the same ratio (Table 2), and this is in good agreement with the morphological changes observed (for CTAB) with the AFM and TEM microscopies (below). Atomic Force Microscopy (AFM) Studies. Tapping-mode AFM imaging was performed on dilute samples of DNA or DOI: 10.1021/la9047825
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Figure 11. UV absorpion profile of ExGen 500 with DNA in 0.01 SSC.
Figure 9. TEM images of DNA (A), [CTAB]/[DNA] = 0.3 (B), [CTAB]/[DNA] = 1 (C), [CTAB]/[DNA] = 2 (D).
Figure 12. Melting measurements of the ExGen-DNA systems.
Figure 10. TEM image of [TMAB]/[DNA]bM = 1.
DNA-additive complexes which were deposited on mica and dried as reported in the Experimental Section. We recognize that removal of water may cause aggregative phenomena leading possibly to artifacts; nevertheless, a careful selection of conditions for sample preparation allowed us to obtain topographies which can be related well to the phenomena observed with the other (23) Krasnoslobodtsev, A. V.; Shlyakhtenko, L. S.; Lyubchenko, Y. L. J. Mol. Biol. 2007, 365, 1407–1416.
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techniques. Moreover, Krasnoslobodtsev, Lyubchenko, and Shlyakhtenko23 have shown that, under certain conditions, DNA molecules keep their morphologies and withstand the rinsing-drying steps required in the dry sample preparation procedure. Figure 8A shows AFM images of DNA alone deposited from a dilute solution of 1.91 10-6 bM, where the observed filaments may be ascribed to a certain coiled form of the nucleic acid duplexes. When CTAB is added to [CTAB]/[DNA] = 0.3, the AFM topography changes to more irregular features, and the filaments observed without CTAB seem to unwind into a web-like formation (Figure 8B). Increasing the concentration of the surfactant up to [CTAB]/DNA] = 2, this phenomenon is even more remarkable, and the original filaments seem to evolve into a heavily unwound form (Figure 8C). It is worth noting that TMAB does not cause any such modifications to the original DNA filaments, as clearly observed in Figure 8D where no major modifications to the structures of Figure 8A are apparent with up to [TMAB]/[DNA] = 1. Electron Transmission Microscopy (TEM) Studies. Figure 9A,B shows TEM photomicrographs of DNA without additives and [CTAB]/[DNA] = 0.3, prepared as described in the Experimental Section. The rod-like shapes obtained in the absence of surfactant barely change when the surfactant/DNA ratio is increased to 0.3, but an unfolding of the rods starts to be Langmuir 2010, 26(11), 7885–7892
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Figure 13. AFM images of ExGen 500 (A), [ExGen]/[DNA] = 0.2 (B), [ExGen]/[DNA] = 0.8 (C).
apparent when this ratio reaches 1 (Figure 9C), and it is still more pronounced at [CTAB]/[DNA] = 2 (Figure 9D). Again, TMAB does not seem to induce any unwinding of the TEM images of DNA, as is clear from Figure 10. We can now make a comparison between the two “microscopies” adopted in the present study to obtain a structural basis for the physicochemical phenomena observed by spectrophotometry, surface tension, and conductometry. In the case of the amphiphilic molecules (such as CTAB), the nonlinear adsorption profiles observed correspond to morphological changes of DNA as clearly indicated by melting temperatures and AFM and TEM images. In fact, after the critical points observed, the DNA appears to be in a web-like, unfolded structure, instead of the rod-like and coiled shape when no additives were added. With respect to TMAB, no major structural changes are apparent at all [TMAB]/[DNA] ratios, as expected from the absence of critical points in the conductivity, surface tension, and spectrophotometry profiles. ExGen 500 Polyethyleneimine (PEI) Studies. The same experimental approach used with CTAB, CTBAB, and the nonamphiphilic analogues ammonium salts TMAB and TBAB was used with ExGen 500, a commercial polyethyleneimine transfection agent.24,25 All measurements were carried out in water at pH = 7, where about 20% of the amino groups are protonated. UV absorption, melting temperature, and AFM measurements were performed. Conductivity was not tested considering the low conductivity of the polymer due to the low mobility arising from its large size. Surface tension profiles were also not carried out since the polymer does not have any surface activities. In Figure 11 is reported the UV absorpion profile at 260 nm vs [ExGen]/[DNA] ratio in 0.01 SSC. The concentration of the carrier was considered in terms of the protonated amino groups to compare the results with the ammonium salts as above. ExGen 500 polyethyleneimine transfection carrier has the same UV absorption behavior of the surfactants CTAB and CTBAB. Again, a nonlinear profile is observed, and the rate of absorbance changes to a second, less negative value after reaching a [ExGen]/[DNA] ratio about 0.45. In correspondence to this “break” in the physicochemical property, morphological changes of DNA were investigated with melting measurements and AFM imaging. In Figure 12, A260 vs T profiles of DNA, at [ExGen]/ [DNA] = 0.18 and 0.40, are reported. These melting profiles demonstrate that morphological changes of DNA are observed corresponding to the break of [Carrier]/[DNA] = 0.4. Indeed, for higher ratios the DNA does not melt, and the interaction with the carrier possibly changes the DNA structure to supercoiled to explain the absence of melting, (24) Huh, S.; Do, H.; Lim, H.; Kim, D.; Choi, S.; Song, H.; Kim, N.; Park, J.; Chang, W.; Choung, H.; Kim, J. Biologicals 2007, 35, 165–171. (25) Remy, J.; Abdallah, B.; Zanta, M. A.; Boussif, O.; Behr, P.; Demeneix, B. Adv. Drug Delivery Rev. 1998, 30, 85–89.
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Table 3. Melting Temperature and Van’t Hoff Calculations on [ExGen]/[DNA] = 0.18 [ExGen]/ [DNA]
Tm (K)
ΔG (J/mol) ΔH (J/mol) ΔS (J/mol K) Keq
Only DNA 327.84 1 0.18 338.74 0.2458
T=327.84K
T=327.84K
T=327.84K
0 3827.57
188408.51 71833.34
574.68 207.41
as is also clear from AFM images (see Figure 13). In Table 3 are reported melting temperature and Van’t Hoff calculation of free energy variation of [ExGen]/[DNA] = 0.18.
Conclusions A multitechnique approach was developed to study the DNA-amphiphile systems, with the aim to achieve a composite method to study the relationships among the structural features of cationic amphiphiles and the induced morphological changes on the nucleic acid. All the physicochemical techniques used in this investigation (surface tension, UV spectroscopy, conductometry) showed peculiar nonlinear profiles at specific [Surfactant]/[DNA] ratios that are consistent with functional and morphological changes in the DNA, as observed from melting temperatures and AFM and TEM images. Surface tension measurements showed discontinuities in the profiles at [Surfactant]/[DNA] ratios 0.53 and 0.57 for CTAB and CTBAB, respectively; UV spectroscopy showed breaks at 0.63 for CTAB and 0.60 for CTBAB; and conductivity profiles change their slopes at 0.56 for CTAB and 0.51 for CTBAB. All the techniques used in this study seem to point to the importance of the amphiphilic behavior of the additive. The same experiments conducted on the nonamphiphilic analogues (TMAB and TBAB) in fact did not show any discontinuities on the profiles or structural changes on the DNA. The electrostatic interaction between the headgroup of the surfactant and the phosphate group of the nucleic acid, as well as the hydrophobic interaction forces due to the tails of the surfactants, may be the major factors controlling the morphological changes induced. It is worth noting that these changes are not to be ascribed to micellar aggregates, because the observed phenomena arise within a concentration range of the surfactants which is far below their critical micellar concentration.26,27 The headgroup size of the surfactant (trimethyl and tributyl for CTAB and CTBAB, respectively) seems to have little relevance on the [Surfactant]/[DNA] ratios observed.28 Another important finding is that all the morphological changes in the DNA occur at a [Additive]/[DNA] value lower than 1, which indicates that these (26) Wang, Y.; Dubin, P. L.; Zhang, H. Langmuir 2001, 17, 1670–1673. (27) Rodriguez-Pulido, A.; Aicart, E.; Junquera, E. Langmuir 2009, 25, 4402– 4411. (28) Dias, R. S.; Magno, L. M.; Valente, A. J. M.; Das, D.; Das, P. K.; Maiti, S.; Miguel, M.; Lindmann, B. J. Phys. Chem. B 2008, 112, 14446–14452.
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phenomena do not imply a complete neutralization of the phosphate groups. The same studies conducted on ExGen 500, an efficient commercial transfection agent, showed this carrier to be more efficient as a DNA compaction and transfection agent. Incidentally, the experiments conducted with PEI showed the same behavior observed with CTAB and CTBAB, thus indicating a common fundamental mechanism of interaction, which could form a basis for designing and testing novel molecules as DNA carriers.
7892 DOI: 10.1021/la9047825
Di Profio et al.
Acknowledgment. This work was funded by MUR (Ministero dell’Universita e della Ricerca Scientifica) Italy. Supporting Information Available: Surface tension profiles of TMAB and TBAB in the presence and in the absence of DNA, A260 adsorption profile of CTAB and DNA system in 0.01 SSC, structures of the additives investigated. This material is available free of charge via the Internet at http:// pubs.acs.org.
Langmuir 2010, 26(11), 7885–7892
