Transfection Efficiency of DNA Enhanced by Association with Salt

Jul 18, 2013 - Controlling the Capture and Release of DNA with a Dual-Responsive Cationic Surfactant. Lu Xu , Lei Feng , Jingcheng Hao , and Shuli Don...
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Transfection Efficiency of DNA Enhanced by Association with SaltFree Catanionic Vesicles Lu Xu, Lei Feng, Renhao Dong, Jingcheng Hao, and Shuli Dong* Key Laboratory of Colloid and Interface Chemistry & Key Laboratory of Special Aggregated Materials, Shandong University, Ministry of Education, Jinan 250100, China ABSTRACT: The interaction of DNA with salt-free tetradecyltrimethylammonium hydroxide and lauric acid lamellar vesicles with positive charges was investigated to probe potential applications of vesicles in DNA transfection. The aggregation morphology of the vesicles changes greatly with the addition of DNA due to the dissociation of anionic surfactants, as indicated by 1H nuclear magnetic resonance, and the expelled surfactant molecules self-assemble into micelles at high concentrations of DNA. Salt-free cationic and anionic (catanionic) vesicles have a much higher binding saturation point with DNA at R = 2.3 (the ratio of DNA to the excess positive charge in vesicles) than formerly reported saltcontaining systems, implying high transfection efficiency. DNA retains its native stretched state during the interaction process. This very interesting result shows that catanionic vesicles could help transport undisturbed and extended DNA molecules into the target cells, which is of great importance in gene delivery, nanomedicine field, and controlling the formation of certain morphological aggregates.

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INTRODUCTION Interactions between self-assembled surfactants and biomacromolecules, such as proteins and nucleic acids, have aroused much interest because of potential applications in biochemistry and medicine fields,1,2 especially for gene therapy based on transporting functional DNA into target cells.3,4 However, cell membranes are not easily crossed by biopolymers for conformational or electronic reasons.5,6 To solve such problems, originally, synthetic liposomes were used,7,8 but these structures are difficult to prepare and to store due to their low stability.7,9−11 One of the main challenges today is to explore the novel transfection techniques and systems with dynamic and adaptive properties that emulate biological systems. To date, investigations on interaction between biopolymers and surfactant aggregates have been extensively reported, including vesicles, micelles, and so on due to their high stability, easy preparation, and low cytotoxic properties.12 Different systems have been carried out to prepare vesicles, including the catanionic surfactant mixtures in nonstoichiometric amounts.13−16 The interactions between DNA and catanionic vesicles have been recently investigated, and one of the numerous advantages is their lack of toxicity as transfection agents compared with their monovalent counterparts.17−19 Recently, it was found that “true”, salt-free catanionic vesicles could be constructed by using extraction, dialysis, or ion exchange to remove the inorganic salts formed by small counterions from mixtures of different surfactants.20−24 These types of salt-free vesicles differ from the formerly reported saltcontaining catanionic surfactant systems, as evidenced from our previous freeze fracture transmission electron microscopy (FF© 2013 American Chemical Society

TEM) observations and small-angle X-ray scattering (SAXS) measurements. Salt-free catanionic surfactant systems have been widely investigated by scientists from different groups, and in which many meaningful results have been achieved.25−32 Herein, we focused on the interaction between DNA and salt-free catanionic tetradecyltrimethylammonium hydroxide/ lauric acid (TTAOH/LA, in this case, TTAOH + LA → TTAL + H2O) vesicles, in which TTAOH is in excess at low concentration, to explore the aggregation behavior, binding character, and interaction mechanism of DNA with the vesicles, and so on. The interaction between the components has a pronounced effect on the physicochemical properties of the system as well as on the structure of complexes. Cryogenic transmission electron microscopy (cryo-TEM), fluorescence spectroscopy, and small-angle X-ray scattering (SAXS) were used to characterize the aggregation behavior of DNA and vesicles.13,14 Moreover, some conventional measurements for dynamics and electrochemistry such as zeta potential and circular dichroism (CD) measurements facilitated the measurement of the conformation of DNA and the binding saturation point of DNA with vesicles.15,16 The powerful 1H NMR was also introduced to help us better understand the interaction mechanism on the nucleus level. For a salt-free TTAOH/LA mixture system, pH is a critical parameter for biomolecule studies. If the pH is 12, the hydrogen bonds between the double helix of DNA will be damaged, the double-helix structure will be destroyed, and the Received: April 30, 2013 Revised: July 13, 2013 Published: July 18, 2013 2781

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SAXS Measurements. SAXS experiments were carried out at Beamline 4B9A at the Beijing Synchrotron Radiation Facility at 298 K using a SAXS apparatus constructed at the station. The incident X-ray wavelength was 1.54 Å, and the imaging plate was a Mar 345 with a resolution factor of 3450 × 3450. The obtained 2-D scattering pattern of SAXS consisted of concentric circles, and the intensity (I) versus scattering vector (q) profile was independent of the azimuthal angle. The range of the scattering vector was chosen from 0.05 to 4 nm−1 (q) [4π sin(θ/2)]/λ, where θ and λ are the scattering angle and the wavelength, respectively. The distance from the sample to the detector was 2003 mm, and the data accumulation time was 600 s for each sample. Electrical Conductivity. A DDSJ-308A analyzer was used to perform electrical conductivity experiments. The electrical resistance of each solution was measured at 1.0 and 10.0 kHz three times. The Pyrex glass measuring cell was placed in a water bath at 25.0 ± 0.3 °C. Measurements were conducted by adding certain amounts of DNA to TTAL vesicles by a transfer pipet under mild stirring and recorded 10 min after each addition. Turbidity Measurements. Turbidity of the supernatants for DNA/vesicle complexes was examined by a U-4100 UV/visible spectrometer using 10 mm path length quartz cell at a wavelength of 400 nm. Zeta-Potential Measurements. The zeta potential of the dispersions was measured with a Zeta PALS potential analyzer instrument (Brookhaven, USA) with parallel-plate platinum black electrodes spaced 5 mm apart and a 10 mm path length rectangular organic glass cell. All samples were measured using a sinusoidal voltage of 80 V with a frequency of 3 Hz. Each sample was measured three times. Dynamic Light Scattering (DLS) Measurements. The average particle size of aggregates formed by DNA/TTAL vesicle complexes was measured by a BI-200SM instrument (Brookhaven) at a constant scattering angle of 90° at 25 °C. The measurements were performed by adding certain amounts of DNA to the vesicle solutions, then stirring the resulting mixtures for ∼20 min, thereafter, removing the supernatants of the resulting suspensions for measurements. Circular Dichroism Measurements. A JASCO J-810 spectropolarimeter was used to perform CD spectroscopy. Samples were located in 10 mm path length cells, and the scanning speed was controlled to 200 nm/min with the measuring range of 200−320 nm. Each sample was measured three times. Cryo-TEM Observations. A 4 μL drop of sample solution was dropped on a microgrid under a high humidity environment to minimize water loss, and two pieces of blotting paper were used to remove the excess solution. Then, the microgrid was quickly plunged into liquid ethane at its melt temperature. The vitrified sample was then transported to a holder (Gatan 626) and inserted into a JEOL JEM-1400 TEM under liquid nitrogen to avoid the formation of ice. The TEM was operated at 120 kV. A Gatan multiscan CCD was used to record every image. 1 H NMR Measurements. All of the samples were dissolved in D2O (Aldrich product, ≥99%). 1HNMR spectra were recorded on a Bruker Avance 400 spectrometer equipped with pulse-field gradient module (Z axis) using a 5 mm BBO probe at 400.13 MHz, and the 1H chemical shifts are reported relative to DHO at 4.70 ppm as internal standard.

target cell cannot survive. In this study, the typical vesicle samples were prepared at a relatively low concentration of 20 mmol L−1 TTAOH and 19 mmol L−1 LA interacting with varying amounts of DNA. In our systems, the final pH is between 7.7 and 9.5, which is suitable for preserving the DNA and living cells. The aim of this work is not only to report some novel experimental results, theoretical considerations, and simulations of the competition mechanism of DNA interactions with saltfree vesicles but also to stress future possible biochemical and biotechnological applications of DNA and salt-free vesicles, such as nanomaterial fabrication, DNA manipulation, and gene regulation.

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EXPERIMENTAL SECTION

Materials. The surfactant LA, CH3(CH2)8CH2CH2COOH, was purchased from Shanghai Shiyi Chemicals Reagent and used without further purification. TTAOH solution was prepared through anion exchange (Ion exchanger III, Merck) from TTAB (tetradecyltrimethylammonium bromide, purchased from Merck and used without further purification). The method was previously described.24,26,27,32 Salmon testes double-stranded DNA Na salt was purchased from Acros (Fairlawn, NJ). Its molecular weight was between 100 and 250 base pairs, as determined by agarose gel electrophoresis (AGE), and its concentration was determined through considering the molar extinction coefficient of DNA bases to be 6600 mol−1 cm−1 at 260 nm.33 The absorbance ratio of DNA stock solution was 1.8 to 1.9 at 260 and 280 nm, which suggests that no protein was present. Sample Preparation. The salt-free catanionic vesicles were prepared by adding a selected amount of LA solid into 20 mmol L−1 TTAOH stock solution in a tube and equilibrating for about 1 month. Proper sonication and centrifugation were used to help the solution disperse homogeneously, and the resulting solution turned bluish. The typical stock vesicle solution of 20 mmol L−1 TTAOH and 19 mmol L−1 LA, with 1.0 mmol L−1 excess TTA+, was prepared and used to interact with DNA. As we know, the high negatively charged DNA can hardly interact with negatively charged or electrically neutral surfactant aggregates.13 A stock DNA solution of 3 mmol L−1 was also prepared. All DNA/ TTAL vesicle complex solutions were prepared by mixing the desired amount of both DNA and vesicle solution to a fixed volume in plastic centrifuge tubes at different required charge ratio R values, where the R refers to the charge ratio [DNA]/[TTA+] (where [TTA+] means the excess concentration of cationic TTAOH, that is, [TTA+] = cTTAOH − cLA). Herein, the charge ratio, R, is defined to trace the variation of DNA/vesicle complexes with different amounts of DNA for convenience.13−15 All solutions were left to equilibrate at room temperature for a few days. Then, the complex solutions were centrifuged at 11 000 rpm for 60 min to remove as much supernatant as possible. Thrice-distilled water was used to prepare all sample solutions except when D2O was used for 1HNMR instead. All experiments were conducted at 25.0 ± 0.1 °C. Fluorescence Microscopy (FM) Observations. Samples of DNA-TTAL precipitates were prepared for fluorescence tests as follows: first, DNA stock solution containing a fluorescent dye 4′,6diamidino-2-phenylindole (DAPI), 0.5 mmol L−1, was prepared and kept for 2 days to reach a binding equilibrium. The resulting solution was then gently mixed with the vesicle solution at a desired ratio, allowed to equilibrate for several days, and then centrifuged. The precipitate was then removed carefully from the solution. A small amount of precipitates was then dispersed homogenously on the object stage and illuminated with a UV-mercury lamp: Fluorescence images of DNA molecules in the precipitate were observed using an IX81FVAF microscope equipped with a 60× oilimmersed objective lens and digitized on a personal computer through a high-sensitivity EMCCD and an image processor (Cascade 512B).

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RESULTS AND DISCUSSION DNA/TTAL vesicle complexes were prepared with different charge ratios, R (= [DNA]/[TTA+]). In this case, the phase behavior of TTAL vesicles with the addition of DNA was investigated, indicating the phase-transition process of TTAL vesicles and the variation of microstructure of DNA/TTAL vesicle complexes with an addition of different amounts of DNA. One can clearly observe the formation of precipitate phase, which could be a consequence of flocculation and sedimentation of DNA/TTAL vesicle complexes due to a 2782

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supernatant reached a minimum at around R = 2.3, then slowly increased again due to the excess DNA. A break point at R = 2.3 is evident. Self-Assembled Structure of DNA/TTAL Vesicle Complexes in Supernatants. Cryo-TEM images of DNA/TTAL vesicle complex supernatants markedly demonstrated the existence of vesicles, as shown in Figure 2a−c. Disc-like micelles (arrows) coexist with uni- and bilamellar vesicles in TTAOH and LA mixture at [TTA+] = 1 mmol L−1, and the average size of the vesicles is between 130 and 230 nm (Figure 2a). When mixing with DNA at R = 0.6, huge, nonspheroid and condensed multilamellar DNA/TTAL complexes were detected, as shown in Figure 2b, and some are even larger than 900 nm. The appearance of such complexes could be attributed to the strong interaction between DNA and the positively charged TTAL vesicles. No doubt, the decrease in charge density in DNA/TTAL complexes with the addition of DNA also plays an important role. DNA molecules may serve as one kind of anionic glue and induce the tight adhesion and stacking of vesicles.34 At high R, the vesicles even fuse to form much larger multi-lamellar vesicles (Figure 2c). Close-up images are shown in Figure 2d,e, in which some DNA coils (arrows) adsorbed on the surface of vesicles can be traced, indicating that the DNA molecules do not change their native conformation during the interaction with vesicles. DLS measurements may provide important information on the average size changes with the addition of varying amounts of DNA, which provides a clear idea of what is going on. As shown in Figure 3a, the

strong interaction between DNA and positively charged vesicles. Phase-Separation Behavior. Positively charged TTAL vesicles demonstrated a strong phase separation with DNA at very low amounts of DNA, forming precipitates at all net charge ratios (R) used in this work. Meaningful differences in separation behavior were observed at different R values. When R < 1.0, the whole system presents a homogenously emulsionlike turbid solution, but with an increase in R value, a precipitate began to appear that remained suspended on top of the solution. This phenomenon could be attributed to the low density of the precipitates, in which the molar weight of TTAOH is lower than its relevant salt, TTABr, and the density of LA is even lower than pure water. Characterization of the Supernatant of Complexes. Phase separation occurred in all samples investigated. After centrifugation, we noted that slight differences in the morphology of the supernatants with the increase in R value. For R < 1.9, the supernatants were turbid and bluish, with some precipitates still suspended in them; however, for other samples at high R, the supernatants were clear, as shown in Figure 1

Figure 3. (a) DLS results of hydrodynamic radius (Rh) change of TTAL vesicles with an addition of varying amounts of DNA at different charge ratio R. (b) CD spectra of DNA aqueous solution and complexes of DNA/TTAL mixtures at R = 0.6, 2.4 in the range of 220 to 320 nm.

Figure 1. Photographs of DNA/TTAL vesicle complex supernatants at different R: 0.2, 0.6, 1.0, 1.4, 1.8, 1.9, 2.0, 2.3, 2.6, 3.0, respectively (top). Turbidity data of TTAL/DNA mixtures as a function of R at λ = 400 nm (bottom).

hydrodynamic radius (Rh) of the DNA/vesicle complexes increases with the amount of DNA and reaches a maximum around R = 2.3. This variation is a consequence of the lipoplex formation and the aggregation of vesicles induced by the

(top). Turbidity was then measured by the optical absorbance at 400 nm, and the turbiditiy data of supernatants as a function of R are plotted in Figure 1 (bottom). The turbidity of the

Figure 2. Cryo-TEM images: disc-like micelles, uni- and multilamellar vesicles of TTAL mixture with [TTA+] = 1.0 mmol L−1 (a) and multilamellar vesicles of DNA/TTAL complex at R = 0.6 (b) and 2.0 (c). Magnified images (d,e) of the rectangle parts in panel c. 2783

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DNA.16 The DLS results supported the cryo-TEM observations, and CD measurements can also provide the evidence of the conformation change of the DNA. Conformation of Adsorbed DNA. Conventionally, cationic surfactants improve the DNA transfection efficiency by compacting stretched rigid coil DNA into a globular state.35,36 The mechanism, however, for salt-free catanionic vesicles may be quite different, hence we needed to determine the conformational change of DNA during aggregation. To investigate the possible change of secondary structure of DNA in solution, the powerful tool of CD measurement was applied in the range of 220−320 nm. In this region, the spectrum of DNA in water shows a negative band at 249 nm and a positive one at 275 nm, with a nodal point at ∼258 nm37 because of the helicity and the base stacking. CD spectra of DNA upon the interaction with TTAL vesicles at different R are given in Figure 3b. Bonincontro et al.37 studied the interaction between DNA and nonionic dodecyldimethylamine oxide (DDAO) that can be turned into cationic DDAOH+ by changing the pH. DDAOH+ interacts with DNA to form DNA/DDAO+ complexes, resulting in the conformation changes to DNA molecule. The corresponding CD spectrum showed an obvious red shift and a strong decrease in intensity. However, for the present salt-free catanionic vesicles, the overall spectra was almost unchanged in both the negative and positive band positions, with only some decrease in peak intensity, whether at low or high R values. The decrease in peak intensity may be attributed to the lower concentration of free DNA in solution due to the precipitation of bound DNA after phase separation.15,16 Consequently, we assume that the DNA retained its native conformation of a linear double helix after aggregation with TTAL vesicles. Similar results were also found in some DNA/salt-containing catanionic vesicle systems.15,16,37 Properties of Complexes in the Precipitates. An interesting phenomenon was observed when all of these complex solutions were centrifugated at higher speed for 1 h: the suspended precipitates turned to spread spontaneously as films on the surface of the solution, as shown in Figure 4. The

by FM are shown in Figure 5a,b. Although a strong electrostatic attraction with TTAL vesicles exists, no obviously compacted

Figure 5. Fluorescence microscope images of precipitated films of TTAL/DNA complex at R = 3.0 (a). By comparing the image in panel a to that of pure 3 mmol L−1 DNA aqueous solution (b), one can find the DNA molecules adopted a concentrated coil conformation due to the higher concentration, in contrast with the compact “global” state of DNA in cationic TTAOH solution (c).

DNA structures can be distinguished. The extended DNA molecules adopted concentrated unfolded coil conformation due to the relatively high DNA concentration, indicating that DNA molecules kept almost the same elongated “coil” conformation as the pure DNA samples.38,39 For the sake of comparison, the compacted “globule” state of DNA molecules induced by cationic TTAOH is also displayed in Figure 5c. The results strongly suggest that for salt-free catanionic vesicle/ DNA systems, DNA molecules still maintained their uncompacted structures in precipitate, despite the phase separation due to the strong electrostatic attraction between the opposite charged entities. This finding is in good agreement with the conclusions obtained by cryo-TEM images and the CD data of the supernatant. SAXS patterns can also give much information on the microstructure of the DNA/TTAL vesicle complexes in the precipitates. A typical SAXS diffractogram of the precipitate at R = 3.0 presented two Bragg peaks at 0.167 (q1) and 0.334 Å−1 (q2), as shown in Figure 6. An additional weak and broad peak

Figure 4. Films of precipitate from TTAL/DNA complex at R = 2.0, 2.3, 2.8, and 3.0 from left to right. Figure 6. Typical SAXS pattern of the DNA/TTAL precipitates at R = 3.0.

average thickness of the films is from 150 to 200 μm. Depending on the R values, the films possessed different features. At lower R (2.3), the films were tighter and denser. FM images can conveniently provide the first-hand evidence of the stretched state of DNA molecules in the precipitates. Illustrative micrographs of the vesicle/DNA sediment obtained

corresponding to the DNA molecules aligned in a liquid crystalline fashion can also be observed. The relative position of the two typical peaks is q1/q2 = 1:2, implying the existence of lamellar DNA/TTAL complex microstructures in the precipitate. According to the formula d = 2π/q, a repeat distance of 3.76 nm for the lamellar structure was calculated. Interaction Dynamics and Binding Saturation Condition. When DNA molecules are adsorbed on the vesicles, the electrostatic attraction between the negative charges on the 2784

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Figure 7. Electrical conductivity κ (a) and zeta potential ζ (b) of suspensions of DNA/TTAL vesicles at different R. R = 2.3 was determined as the binding saturation point of DNA/vesicle complexes. T = 25.0 ± 0.1 °C.

Here τ refers to the thickness of the electric double layer of the DNA/TTAL vesicles complexes; ε0 is the vacuum dielectric constant, and ε is the solvent permittivity. According to eq 2, ζ is related to the electric moment of each unit area. When ζ reaches or approaches zero, the στ also reaches zero.15 When DNA interacts with vesicles and reaches the binding saturation point, the surface charge density σ of the whole aggregate is equal to zero. Accordingly, the binding saturation point of these complexes can be obtained. The zeta potentials, ζ, of the supernatants of DNA/TTAL vesicle complexes are plotted as a function of R in Figure 7b. One can find that the ζ decreases with an increase in R, indicating that the positive charges in the vesicles are reduced by the addition of DNA. At R = 2.3, the ζ reaches zero within the error, which corresponds to near a charge neutralization limit. This result is in good agreement with the findings reported in the Electrical Conductivity and Turbidity Measurement sections with the same break point. Thus, the binding saturation point of these salt-free TTAL vesicle/DNA complexes can be confirmed at R = 2.3. In addition, the extra unbound DNA molecules with small hydrodynamic radius in supernatants may contribute to the decrease in the average particle size;16 as a consequence, the DLS results reach a maximum around the binding saturation point. Zeta potential can also help to estimate the stability of the system. The high ζ values, usually ζ > 30 mV or ζ < −30 mV, often predicate the high stability of a system;15 while the intermediate values usually indicate a thermodynamic instability and the formation of aggregates. Close to the binding saturation point, from R = 1.9 on, with the increase in the amount of DNA, the surface charge density reduces and favors the flocculation and sedimentation of the complexes. As a result, the supernatants become clear, and the turbidity in Figure 1b reaches its minimum at R = 2.3. It is worth noting that the obtained binding saturation point, R = 2.3, is much higher than that in some formerly investigated salt-containing systems.13−16 Significantly, this finding suggests that the salt-free TTAL vesicles might have a higher potential ability to absorb more DNA and hence a higher transfection efficiency. 1 H NMR Measurements. NMR technique can serve as a good analytical tool to trace the composition changes33 as well as the aggregation behavior of the supernatant, which changes with R values. To better understand the phase separation of TTAL/DNA complexes at different R, 1H NMR measurements were performed, which provide extensive information on the electronic microenvironment, numbers of chemically distinct hydrogens, as well as information on the size and asymmetry of

DNA and the positive charges facing outward from the vesicles plays a key driving role in the interaction.13−16,40 Electrochemical methods such as electrical conductivity and zeta potential, ζ, measurements can provide useful information and better understanding of the interaction dynamics and binding saturation condition of DNA/TTAL vesicle systems.15,41 Conductivity measurements were performed on the suspensions of DNA/TTAL vesicle complexes at different R values, as shown in Figure 7a. Two linear segments are observed versus R, and the turning point lies at R = 2.3. As we know, for a salt-free catanionic system, the electrical conductivity values are much lower than those for saltcontaining ones because of the very low concentration of counterions. When DNA interacts with TTAL vesicles, a few counterions such as Na+ from DNA are released into the bulk solution, which could cause an increase in the electrical conductivity of the suspensions. 15 Therefore, the first conductometric stage may be associated with the continuous binding of DNA molecules to vesicles, while the counterions, namely, Na+ ions (from DNA) and OH− (from the excess TTAOH in vesicles) are released into the solution due to the electrostatic interactions. The second stage reflects an increase in conductivity ascribed to a partial release of Na+ to the bulk solution due to the excess DNA in solution after binding saturation is reached.15,16 The slope of the first stage, 35.6, is larger than that of the second one, 18.9. The zeta-potential measurements were further used to determine the binding saturation point of DNA/TTAL vesicle complexes. According to the classical models,40−43 colloid particles usually contain two regions, the Stern and diffuse layers. In the Stern layer, counterions are strongly bound and move with the particles as a whole dynamic entity. The others move freely in the bulk and maintain dynamic equilibrium with those in the Stern layers. When these particles lie in an electric field, the two regions would have a relative displacement to form a slipping plane, and the electrokinetic potential of anywhere in this plane is defined as zeta potential, ζ, which is a good way to evaluate the surface charge density of DNA/TTAL complexes. The surface charge density, σ, can be calculated through the formula below:15 σ=

ζεε0 4πτ

(1)

or: στ =

ζεε0 4π

(2) 2785

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from complexes, and the 1H signal peaks of anionic L− expelled from the complexes could be clearly distinguished. Meanwhile, because of the interaction between DNA and cationic TTA+ of vesicles, one type of new aggregates will be formed, and the hydrophobic part of cationic surfactants will be packed in the inner segment of DNA/TTAL vesicle aggregates with bound DNA, which explains why most signals of cationic hydrophobic chains cannot be traced in the supernatant, although R is big enough, up to R > 2.3. L− monomolecules expelled from vesicles can move freely in the aqueous solution, and their signal peaks are much easier to identify. Interestingly, to the best of our knowledge, this is the first time we observe that an anionic surfactant has been observed to be expelled from catanionic vesicles at R = 0.8, far before the binding saturation point (2.3), and it is quite different from previous saltcontaining systems,15,16 in which the anionic surfactants were expelled from vesicles above the saturation point. It has been well-established that in aqueous solution of amphiphilic molecules, the physicochemical environment of surfactant depends on their concentration or aggregate behavior. Single molecules in solution exist in a hydrophilic environment, while within a micelle, surfactant hydrophobic tails exist in a low polarity environment. As a result, for a surfactant aqueous solution, the 1H NMR signal peak of the terminal group −CH3 is the strongest, and its chemical shift is the most sensitive to slight environment changes as micelles form. It should be noted that for 1H NMR the exchange rate of protons is quite rapid between monomolecule and aggregate states. Therefore, despite the coexistence of individual molecules and micelles, the observed chemical shift (δ) is a weighted average value of the monomeric shift (δmon) and of the micelle shift (δmic). The relationship of the chemical shift (δ) of a given group of surfactant hydrocarbon chain, such as −CH3, and the concentration of the surfactant can be described by a two-step model:46,47

aggregates through related line-broadening effects.14,44,45 Typical spectra of TTAL vesicle/DNA complex solutions at different R are collected in Figure 8; the chemical structure, 1H NMR spectrum, and peak assignment of the anionic surfactant LA are also shown for comparison.

Figure 8. 1H NMR spectra of aqueous solutions of pure TTAOH, SL (sodium laurate). Because LA is minimally soluble in water, we here present the spectrum of SL instead of LA (which is the same as that of LA), TTAL vesicle, pure DNA, and supernatants of DNA/TTAL complex aqueous solutions at different R, as noted from bottom to top.

δ = pδmon + (1 − p)δmic

(3)

where δmon and δmic refer to the chemical shift of monomeric molecules and micelles, respectively; p is the percent of monomeric molecules. Where c < cmc, surfactants exist in solution as monomeric molecules:

As shown in Figure 8, at low R, such as 0.2 and 0.6, the NMR spectrum is quite similar to that of vesicles, with only a small decrease in the whole intensity. This phenomenon may be due to the low concentration of both the vesicles and DNA in the supernatant. When R reaches 0.8, the peaks corresponding to the vesicles disappear. Various peaks corresponding to three components can be traced. From a comparison of the different spectra, the signal peaks assigned to the anionic surfactant (LA) are especially evident, although the chemical shifts of different groups along the hydrocarbon backbone underwent some changes. For DNA and cationic surfactant TTAOH, only partial resonance signals can be seen. The peaks at 1.08 and 3.58 ppm positions can be as assigned to DNA, and methyl groups for hydrophilic part −N (CH3)3 (3.05 ppm) and methene groups in α, β, and γ positions (3.21, 1.68, and 1.28 ppm) of TTAOH can also be distinguished. With an increase in R up to 3.0, the whole 1H NMR spectrum remains similar to that at R = 0.8, with only some changes in chemical shift at different positions of the anionic LA hydrocarbon chain taking place. As negatively charged DNA molecules are added into a catanionic vesicle solution, the interaction point would tend to be between DNA and the cationic surfactants on the vesicles. As a consequence, as the amount of DNA increases, up to R = 0.8, the anionic surfactant molecules, L−, might be expelled

δ = δmon

(4)

With the increase in concentration, when c > cmc, molecules exist in monomeric and micellar states. For systems involving only one surfactant, above the cmc, the chemical shift of −CH3 group of a hydrocarbon surfactant is given by eqs 5 and 6, with the assumption that the monomer concentration is approximately equal to the cmc. That is, when c is above cmc, we have: cmc p= cT (5) and: δ = δmic +

cmc (δmon − δmic) cT

(6)

where cmc is the critical micelle concentration; cT is the total surfactant concentration, that is, the sum of the amphiphile concentration in both the monomeric and the micellar states; and δmon and δmic are the chemical shifts of −CH3 in the monomeric and micellar states, respectively. 2786

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From Figure 8, one can clearly find that with the increase in R, that is, the increase in DNA, anionic L− can be precisely recognized in the 1H NMR spectra. Then, one could wonder: in what state does the anionic L− molecules exist in solution after extrusion from the catanionic vesicles after the addition of DNA? According to eqs 4 and 6, a plot of Δδ versus c−1 T is linear above the cmc, and below the cmc δ corresponds to δmon. The break point gives the cmc.46−48 The 1H chemical shift (Δδ) of −CH3 of L− as a function of R is plotted in Figure 9, in which R replaces the reciprocal concentration on the horizontal axis.

interact with cationic TTA+ in catanionic vesicles to reach a saturated state, and a further increase in the amount of DNA could not cause the release of more L− into solution. Combining the 1H NMR and CD results with the observations by cryo-TEM, we propose a novel interaction mechanism for salt-free catanionic TTAL vesicles interacting with DNA, as shown in Figure 10. As we know, the electrostatic attraction is the main driving force between the high negatively charged DNA backbone and positively charged catanionic TTAL vesicles in the presence of excess TTAOH. Taking into account that the elongated coil DNA backbones remain unchanged during the interaction, whether in the supernatant or in the precipitate state, one can deduce that in the first stage, as a small amount of DNA molecules was added to vesicles at lower R, the DNA molecules adsorbed on the vesicles. Acting as one kind of electrostatic glue, DNA molecules induce the vesicle bilayers (disc-like micelles and unilamellar vesicles) to aggregate and stack, forming large multilamellar vesicles, which can be clearly seen by the image at R = 0.6 (Figure 2b). On the basis of previous results,16,49 the excess TTA+ tends to distribute on the outer layers of vesicles and regions of lower curvature. With the increased number of rigid DNA molecules, in addition to competition for the electrostatic interaction between DNA and anionic surfactants, the electrostatic attraction between the rigid DNA and excess TTA+ on vesicles surfaces induces the vesicles to spread with lower curvature either, but on the contrary, the short chain length L− tends to distribute on regions with relatively higher curvature to better contact with DNA, and a small number of L− ions were therefore expelled from the catanionic vesicles into solution. In this stage, the expelled L− exists in a monomer state, surrounded by water molecules. Upon further increasing the amount of DNA, at high R close to 2.0, the expelled anionic L− reach a high enough concentration to self-assemble to form micelles in solution, as evidenced by 1H NMR. At the same time, the charge density as well as the curvature of vesicles changes a lot, inducing vesicles to rearrange and form substantially large, flat, fused microstructures. When the DNA molecules interact with catanionic vesicles near the saturation point, no more anionic L− will be expelled. Ultimately, the attraction between cationic and anionic surfactants is stronger than that between DNA and cationic surfactants.15,16 In addition, DNA molecules induce the tight adhesion, aggregation, or even fusion of vesicles; simultaneously, the specific surface area of vesicles decreases greatly, and the specific surface charge density increases accordingly, which is why the salt-free catanionic vesicles possess a much higher binding saturation point at the charge ratio of 2.3 than those of

Figure 9. 1H chemical shift (Δδ) of the −CH3 group of L− in TTAL/ DNA in D2O versus R. T = 25.0 ± 0.1 °C.

What is interesting is that this curve shows the characteristic feature of a typical aqueous solutions of ionic hydrocarbon surfactant;46,48 that is, at lower R, the chemical shift of −CH3 remains almost constant, where δmon is identified with δ (due to the more complicated conditions in a DNA/TTAL vesicle complex than those in a pure surfactant aqueous solution, the chemical shifts in the first stage show somewhat fluctuations), then a sharp increase in Δδ follows at higher R. Theoretically, the Δδ versus c−1 T curve can be linearly fitted by two lines, where the intersection point corresponds to the cmc in a surfactant system solution. Therefore, we can confidently deduce that with the increase in R, more DNA molecules interact with the cationic TTA+ in catanionic vesicles, and meanwhile, more anionic L− are expelled into solution to form micelle-like aggregates at the intersection point, corresponding to the cmc*, R = 2.0. Furthermore, as R increases to 2.3, the chemical shift Δδ becomes constant again, which is completely consistent with the binding saturation point determined by the zeta potential, electrical conductivity, and turbidity measurements. We can understand that at this point, DNA molecules

Figure 10. Proposed mechanism for the interaction process of TTAL vesicles upon addition of different amounts of DNA. 2787

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ACKNOWLEDGMENTS This work was financially supported by the NSFC (grant nos. 21033005 & 21273136) and the National Basic Research Program of China (973 Program, 2009CB930103). We thank Dr. Pamela Holt for editing the manuscript.

reported salt-containing systems, for example, CTAB/SOS/ DNA at 1.8 and DDAB/SDS/DNA at 0.8.15,16 Taking into account the reciprocal size of DNA and the fused huge vesicles, the real reason DNA preserves its conformation may underlie in the steric hindrance effect. In the present investigation, the catanionic vesicles in the absence of DNA have an average size of ∼130 to 230 nm; the DNA molecule essentially retains a linear configuration. If each base pair (bp) has a size of 0.34 nm,38 the DNA persistence length is on the order 85 nm (∼250 bps). It is reasonable to assume that one DNA molecule may interact with more than one vesicle, thus inevitably inducing the aggregation of huge vesicle/DNA complexes, and compared with simple cationic surfactantbound DNA systems, the change of surface charge density of vesicle-bound DNA may be small due to the steric effect; therefore, it cannot induce the compaction of the DNA doublehelix structure.

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REFERENCES

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CONCLUSIONS In the present work, a multiperspective study on the interaction between DNA and salt-free catanionic vesicles has been presented using different experimental approaches. A strong phase-separation phenomenon occurred when a small amount of DNA was added. The binding saturation at a net charge ratio R of 2.3 is determined, at which the turbidity of the supernatant reaches its minimum. From CD and FM measurements, we conclude that DNA molecules retain the elongated linear configuration in both supernatant and precipitate due to the steric hindrance. Multilamellar DNA/vesicles complex microstructures were observed through cryo-TEM. The results are ascribed to DNA molecules, which play a key role in the formation of DNA/vesicle complexes because the DNA can serve as a glue to attract more vesicles to form large fused microstructures. Other experimental measurements including electric conductivity, zeta potential, and SAXS support the above findings. 1H NMR results indicate that the anionic L− may be expelled from DNA/vesicles long before the binding saturation occurs. The expelled L− may form small micelle-like aggregates at R = 2.0; above R = 2.3, the vesicles are saturated with DNA molecules and further increases in the amount of DNA could not cause the release of more L− into solution. The efficiency of binding DNA with TTAL vesicles is much higher than those reported for salt-containing catanionic vesicle systems, implying a higher transfection efficiency of DNA. Serving as a vector, salt-free catanionic vesicles could help linear extended DNA molecules enter or exit cells depending on similarity and compatibility between bilayers of amphiphilic molecules and phospholipids; under some appropriate conditions, the vesicles could be disrupt, allowing a pure, undisturbed, extended DNA molecule to be transported into the target cells. This strategy could be applied in gene delivery and therapy as well as nanomedicine; in the meantime, the morphology change of aggregates with R values can guide the synthesis of aggregates with certain morphologies.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +86-531-88564750. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2788

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