Noncovalent Self-Assembling Nucleic Acid-Lipid Based Materials

Biomacromolecules 2009, 10, 221–228

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Noncovalent Self-Assembling Nucleic Acid-Lipid Based Materials Wirasak Smitthipong,†,‡ Thorsten Neumann,†,§ Surekha Gajria,§ Youli Li,† Arkadiusz Chworos,| Luc Jaeger,*,†,§ and Matthew Tirrell*,†,‡ Materials Research Laboratory, Department of Chemical Engineering, Department of Chemistry and Biochemistry, and Department of Physics, University of California, Santa Barbara, California 93106 Received June 27, 2008; Revised Manuscript Received November 22, 2008

We detail a method originally described by Okahata et al. (Macromol. Rapid Commun. 2002, 23, 252-255) to prepare noncovalent self-assembling films by exchanging the counter-ions of the nucleic acid phosphate moieties with those of cationic lipid amphiphiles. We are able to control the strength and surface properties of these films by varying the composition between blends of DNA of high molecular weight and RNA of low molecular weight. X-ray and AFM results indicate that these films have a lamellar multilayered structure with layers of nucleic acid separated by layers of cationic amphiphile. The tensile strength of the blended films between DNA and RNA increases elastically with DNA content. The length as well as the molecular structure of nucleic acids can affect the topology and mechanical properties of these films. We suspect that the permeability properties of these films make them good candidates for further biological applications in ViVo.

Introduction In the last two decades, materials science has developed into an interdisciplinary field that encompasses organic, polymeric, and biological components. Multicomponent composites make it possible to combine two or more desirable properties or to provide additional stability for otherwise functional biomolecules or biomolecular assemblies. Noncovalent interactions are increasingly being used in the molecular self-assembly of welldefined supramolecular structures and materials. Such interactions are important in the field of materials where electrostatic and other reversible interactions lead to useful characteristics such as molecular recognition, directionality, addressability, programmability, and tunability of interaction strength.1-8 These are characteristics that represent great potential for the design and development of new material architectures with tailor-made properties. Electrostatic interaction between oppositely charged polyelectrolytes can lead to self-assembled multilayered films9 made from polyanions with positively charged polycations. Langmuir-Blodgett (LB) and layer by layer deposition (LbL) are widely used techniques to generate homogeneous films. Besides these commonly used techniques, it was reported that the selfassembly of counter-charged molecules leads to multilayered films as well.10-13 Sequential anionic/cationic polyelectrolyte interactions have important consequences for flocculation and are therefore of interest in developing large-scale biomaterial properties such as drug delivery,14 gene delivery,15 biosensors,16 and electronics.17 Nucleic acid is a perfect biomaterial candidate for new materials due to its ease of electrostatic complexation with cationic agents, its variety of secondary structure and its inherent * To whom correspondence should be addressed. Tel.: +18058933628 (L.J.); +18058933141 (M.T.). Fax: +18058934210 (L.J.); +18058938124 (M.T.). E-mail: [email protected] (L.J.); [email protected] (M.T.). † Materials Research Laboratory. ‡ Department of Chemical Engineering. § Department of Chemistry and Biochemistry. | Department of Physics.

biocompatibility.5,8,15,18 There are many opportunities to explore the integration of nucleic acid based materials with the elements of more traditional macromolecular science, including the use of nucleic acids to generate tailor-made, novel synthetic materials.19 Although nucleic acids are water soluble, they will not be appropriate for many applications without adding complexing agents in solution to allow for better processing and structure formation. Okahata and co-workers1,20 developed a method to prepare nucleic acid/cationic lipid films based on their electrostatic interaction. While the structure of amphiphilic lipid nucleic acid complexes in aqueous solution is well understood,23 much less is known about the structure of the dry film. It is well-known that the nature of cylindrical lipids like DDAB drives the structure of the complexes toward a lamellar shape in aqueous solution with separate layers of lipids and DNA.21 However, the relationship between the resulting structures and properties of nucleic acid/lipid films remains to be investigated. The film preparation based on Okahata’s method9 to make nucleic acid/cationic lipid films was slightly modified to facilitate their manipulation. We demonstrate herein the formation of self-standing cast films using nucleic acids with different blends of RNA (short chain) and DNA (long chain), with the cationic amphiphile didodecyldimethylammonium bromide (DDAB). The structure of the nucleic acid film has been studied by X-ray diffraction, XPS, AFM, tensile test, and fluorescence spectrometry. We have generated hybrid blended complex films of long DNA and shorter RNA mixed with DDAB, which allow us to control the mechanical and surface properties of these films.

Experimental Section Materials. RNA from yeast (approximately 15 nucleotides, as determined by polyacrylamide gel electrophoresis and HPLC) was purchased from Roche. These small RNA molecules are the result of degradation of natural RNA, especially tRNA. tRNA from yeast (70 nucleotides, as determined by polyacrylamide gel electrophoresis) was purchased from USB corporation. DNA from salmon testes (2000 base pairs, as determined by agarose gel electrophoresis) was purchased from

10.1021/bm800701a CCC: $40.75  2009 American Chemical Society Published on Web 12/18/2008

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Biomacromolecules, Vol. 10, No. 2, 2009

Figure 1. Chemical structure of nucleic acids (A) RNA and DNA. For DNA the 2′-OH group is replaced with an -H group. The negatively charged phosphate group can interact with the cationic lipid headgroup (B) didodecyldimethylammonium bromide (DDAB).

Figure 2. Preparation method of self-standing films via electrostatic interaction between the polyanionic nucleic acid (DNA or RNA) and the cationic amphiphile (DDAB). The nucleic acids are dissolved in buffer and mixed with a solution of DDAB in water. The formed complex is washed, dried under vacuum, and then dissolved in a 4:1 chloroform-ethanol mixture. The dissolved complex is cast in a glass mold to obtain a self-standing film after slow evaporation in a desiccator.

Sigma Aldrich. Didodecyldimethylammonium bromide (DDAB) was purchased from TCI America. The chemical structures of the materials are presented in Figure 1. TBE buffer (10×, Omnipure) was purchased from EMD and certified as RNase free. The water was obtained by a Milli-Q system that was certified as RNase free as well. All prepared buffers were filtered with GH Polypro 0.2 µm filters before their usage. Ethanol (Merck) and chloroform (Merck) were used as purchased. Glass molds and glass slides were washed with Milli-Q water and ethanol and dried under nitrogen before use. Preparation of the Self-Standing Film. Figure 2 shows the procedure for obtaining a nucleic acid film from nucleic acid and lipids.22 The nucleic acid was dissolved in TBE 1× buffer solution (0.089 M Tris, 0.089 M borate, and 0.002 M EDTA) and was then mixed with an aqueous solution containing 1.1 mol equivalents of the cationic amphiphile DDAB. The reaction was first shaken at room temperature for 3 h. The water-insoluble complexes were then centrifuged, washed three times with water, and finally lyophilized. The dry complexes obtained were soluble only in organic solvents such as chloroform and ethanol. The solution of the complex in a solvent mixture of 4:1 chloroform/ethanol was cast either on a glass mold or on a glass slide and allowed to slowly dry in a desiccator at room temperature for 5 days. The resulting translucent films were several microns thick. The film thickness can be controlled by varying the amount of complex dissolved in organic solvent. Finally, the selfstanding films were removed from the glass mold using tweezers. We have developed this method to prepare not only RNA or DNA films, but also to prepare any charge-complex polymer film.23 The synthesized films were formed in a one-to-one stoichiometry of the polyanion and DDAB, which was confirmed by elemental analysis. Small- and Wide-Angle X-ray Scattering (SAXS and WAXS). Transmission SAXS and WAXS measurements were performed with Cu KR radiation using a custom built X-ray diffractometer with a MAR180 (SAXS) and MAR345 (WAXS) image plate detectors and a Rigaku (UltraX18HF) rotating anode X-ray generator.18 The sample to detector distance was 758 mm for SAXS and 235 mm for WAXS. The integrated diffraction data were plotted as a function of q ) (4π/ λ) sin(θ), where λ is the wavelength of the beam (λ ) 0.154 nm) and

Smitthipong et al. θ is the Bragg angle. The experimental data were corrected for background scattering and sample attenuation.24 X-ray Photoelectron Spectroscopy (XPS). XPS measurements were performed on a Kratos Axis Ultra using a monochromatic Al KR source. Film surfaces were investigated at 90° (normal detection) and 30° (60° off-normal) between sample film and detector. Tilting the sample increases the distance that electrons heading in the direction of the detector axis must travel to exit the sample surface, making the XPS experiment more surface sensitive. When a sample is flat (90° with respect to the X-ray beam), electrons can escape from a depth of about 100 Å. When the sample is tilted at 60° with respect to the beam, electrons can only escape from a depth of about 20 Å. Decomposition of high-resolution spectra, which have been normalized by fixing the carbon peak at 285.0 eV, is performed using a least-squares fitting program. The nitrogen spectra of the different species are decomposed at different binding energies: quaternary nitrogen (positively charged nitrogen) at 402.3 eV, aliphatic nitrogen at 400.2 eV, and aromatic nitrogen at 398.9 eV. Atomic Force Microscope (AFM). The AFM experiments were performed using a MultiMode microscope equipped with Nanoscope IIIA controller (Veeco, Santa Barbara, CA). The AFM images were recorded in tapping mode under ambient conditions using moderate force ratio to avoid contact loss. Experiments were performed using a commercial silicon tip (NSC12) with resonance frequency f ) 150-200 kHz and spring constant k ) 4-8 N/m. A typical scan range of 1 and 5 µm with resolution of 512 × 512 pixels was used for image recording. Each film sample was typically cast three times and analyzed in multiple regions. Images were processed and analyzed using NanoScope software. Raw data was leveled by a first and second order of plane fit correction to remove the sample tilt. Tensile Test. The mechanical properties of the films were recorded on a standard tensile testing machine (Instron). The tensile test was carried out on dumbbell specimens with a crosshead speed of 0.5 in/ min. The dumbbell specimens were produced from the obtained selfassembling films with a thickness of 20-50 µm. Each of the films was tested at least three times. Fluorescence Spectrometer. Fluorescence emission measurements of ethidium bromide intercalated within the films, excited at 530 and emitted at 610 nm, with excitation and emission slit widths of 10 nm, were performed on a Varian fluorescence spectrophotometer (Cary Eclipse) under ambient conditions. The intercalation process was done with DNA/DDAB and RNA/DDAB films, which were immersed into an aqueous solution of 0.1 mg/mL ethidium bromide. A reference experiment was done by first intercalating ethidium bromide into the native nucleic acid, which was then complexed with the lipid followed by the casting procedure described above.

Results and Discussion To better understand the structure of pure 100% and blended nucleic acid/lipid complexes in the dry and wet states, we studied complex samples obtained after the lyphilization process as well as in buffer medium (Figure 3A-D) by SAXS. The data present similar harmonic peaks (00 L), suggesting a lamellar structure with layers of nucleic acid separated by lipid layers of DDAB in wet as well as dry environments. The SAXS profiles of certain complexes of DNA with double-chain cylindrical surfactants (e.g., DDAB) suspended in water have previously been identified as a multilayer structure with alternating lipid bilayers and DNA monolayer.25 The X-ray profile of DNA/DDAB powder suspended in TBE 1× buffer solution (Figure 3B) also showed the in-plane correlation between parallel DNA helices, qDNA, which is in good agreement with the literature data of a similar complex in aqueous solution. Indeed, calf thymus DNA/lipid complexes as reported in the literature have a similar qDNA value

Noncovalent Self-Assembling Nucleic Acid Based Materials

Figure 3. SAXS profiles of (A) DNA/DDAB powder and (C) RNA/ DDAB powder obtained after the drying step. The spectra of the nucleic acid DDAB powder suspended in TBE buffer are shown in (B) for DNA/DDAB and for (D) RNA/DDAB. The repetition unit of the DDAB complex increases for both types of nucleic acid. Table 1. SAXS Results for the Blended Films of DNA and Both Short Chain RNA (15 nucleotides) and Long Chain RNA (70 nucleotides) at Varying Concentrations q001 d q002 ratio (nm-1) (nm) (nm-1) q002/q001

film sample DNA (2000 bp) + RNA (15 nt) 100% DNA/DDAB film 80% DNA + 20% RNA/DDAB film 50% DNA + 50% RNA/DDAB film 30% DNA + 70% RNA/DDAB film 10% DNA + 90% RNA/DDAB film 5% DNA + 95% RNA/DDAB film 1% DNA + 99% RNA/DDAB film 100% RNA/DDAB film

2.10 2.09 2.14 2.14 2.14 2.15 2.15 2.15

2.99 3.01 2.94 2.94 2.94 2.92 2.92 2.92

4.19 4.18 4.30 4.31 4.33 4.31 4.30 4.33

2.00 2.00 2.01 2.01 2.02 2.00 2.00 2.01

Table 2. Percentage of Different Nitrogen Species on the Film Surfaces as Revealed by XPS DNA/DDAB film

RNA/DDAB film

nitrogen species (N)

90°

30°

90°

30°

aromatic N (%) aliphatic N (%) quaternary N or N+ (%)

21 19 59

16 14 70

33 30 37

30 29 41

Table 3. Mechanical Properties of Different Types of Blended Films film sample 100% DNA/DDAB film 80% DNA + 20% RNA/DDAB film 30% DNA + 70% RNA/DDAB film 10% DNA + 90% RNA/DDAB film 1% DNA + 99% RNA/DDAB film 0.2% DNA + 99.8% RNA/DDAB film 100% RNA/DDAB film

tensile strengtha tensile straina (MPa) (%) 4.96 ( 0.05a 4.44 ( 0.08b 3.21 ( 0.11c 2.69 ( 0.08d 2.15 ( 0.05e 1.78 ( 0.08f 0.68 ( 0.04g

214 ( 4a 170 ( 11b 175 ( 60b 141 ( 5c 45 ( 4d 26 ( 0.5d 24 ( 0.4d

a Means of three replicates + standard deviations. Any means in the same column followed by the same letter are not significantly different (P > 0.05) by Bonferroni’s multiple range test.

of around 1.42 nm-1. Based on this result the structure of the complex should not be affected by the sequence or origin of the DNA.26 However, we did not find qDNA for the dried DNA/DDAB powder (Figure 3A). The peak width in the dry powder of DNA/ DDAB complex (Figure 3A) is much broader than the complex in the solution (Figure 3B). The lack of correlation spacing for the DNA (qDNA) in the dried state suggests that the dried material


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is less well-ordered or the domain size is smaller than the hydrated complex. In the case of RNA/DDAB powder in TBE buffer solution (Figure 3D), short RNA fragments were randomly distributed within the complex so that they did not show the in-plane correlation between parallel RNA strands.18 The DNA molecules show liquid-like in plane structure (amorphous), whereas RNA molecules show the liquid crystalline-like structure in the RNA layer. The peak width observed for RNA/DDAB complex, in both dried and wet environments, was typically narrower than that of DNA/DDAB complex, implying a higher degree of order in the lamellar structure.18 We can therefore think of the RNA-DDAB film as being composed of small scattered crystal islands. RNA is shorter and can crystallize more easily because it is on approximately the same scale as DDAB compared to DNA, which becomes randomly ordered when complexed with a small molecule such as DDAB. The repeat distance (d) of the lipid DDAB alone was determined from the relation d ) 2π/q001, where q001 is a q value of the first peak (a peak at the lowest q).22 The obtained d value of lipid DDAB is consistent with the theoretical calculation for lipid bilayers with interdigitated lipid tails (≈3 nm).27-29 Interestingly, the d value of DNA/DDAB complex (Figure 3A and B) is slightly higher than that of RNA/ DDAB complex in the same environment (Figure 3C and D), presumably due to the difference in diameter between DNA and RNA molecules. We believe that this RNA sample is mostly single stranded according to gel electrophoresis (data not shown). For the dried complex (Figure 3A and C), the d values are less than the ones of the wet complex by 25-30% (Figure 3B and D), possibly due to the exclusion of water. This well-defined lamellar multilayered structure can be found in the pure nucleic acid/lipid film as well as blended films containing mixtures of DNA and RNA by using X-ray diffraction (Figure 4). All the film samples were obtained by dissolving the complex in a mixture of 4:1 CHCl3/EtOH and then casting the solution in a glass mold (see Experimental Section). Figure 4B and C present an enlarged view of the second scattering peak, q002, and the broad peak of DNA/DDAB and RNA/DDAB films, respectively. The broad peak seen in WAXS data from all films (around q ≈ 14 nm-1) confirmed that the lipid DDAB chains are in the disordered fluid state, as expected from LR phase (lamellar structure). A schematic representation of the film structure in aqueous solution was presented in our previous work.22 This result agrees with the literature that hydrophobic interactions among the double tails of DDAB molecules led to the formation of lipid bilayers in the complexes when suspended in water.30 The electrostatic interaction between nucleic acid and cationic lipid plays an essential role in forming complexes in which the cationic lipids are concentrated in the vicinity of the nucleic acid backbone.27 The effects of electrostatic and hydrophobic interactions on the structural formation and transition of other nucleic acid/lipid complexes have been examined in terms of the charge density, hydrophobicity, backbone flexibility of nucleic acid chains, and the surfactant tail length.31,32 Our data suggest that significant differences exist between the structures of the nucleic acid/lipid complex in aqueous solution and in the cast film. The structure of the dried film is under ongoing investigation. The effect of RNA chain size on the structure of DNA and RNA blended films was also studied by SAXS and WAXS (Table 1, Table S1). Based on these data, there are no significant differences between the structures of the blended films of DNA and both short chain RNA (15 nt) and long chain RNA (70 nt). This means that the structure of the DNA and RNA blended

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Figure 4. SAXS and WAXS profiles for films made with blends of RNA and increasing amounts of DNA. (A) A multilamellar structure was also indicated by SAXS for all the film samples independent of DNA content. (B) and (C) present WAXS profiles of pure RNA and DNA lipid films including the second scattering peak, q002, and the broad peak of DNA/DDAB and RNA/DDAB films. All the films showed a broad peak around q ∼ 14 nm-1, indicating that the lipid DDAB chains are in a disordered fluid state.

Figure 5. SAXS profiles of the films after annealing at different temperatures: (A) DNA/DDAB films and (B) RNA/DDAB films. There are no significant differences between the structures of DNA/DDAB and RNA/DDAB films over the course of heating (see also Table 2), thus indicating that the films have a thermostable structure in the range from 20 to 90 °C.

complex films is dominated by DDAB. It should be mentioned that the repeat distance (d) increased slightly with higher DNA content (>80%) in the DNA and RNA blended complex films (Table 1), although the reasons for this remain unclear. The effect of thermal annealing on the structure of DNA and RNA films was also investigated by SAXS (Figure 5 and Table S2). All the films were heated at 75 or 90 °C for 1 h and then cooled down to room temperature. This heating and cooling cycle was repeated three times before analysis using SAXS. No major differences in the WAXS profile could be seen between the nontreated and the temperature-treated films as long as the temperature does not exceed 90 °C. Small differences in

the signal intensity at 90 °C might be due to the removal of residual water or solvent in the film. In addition to the SAXS and WAXS data indicating a lamellar structure, the structure of the lipid nucleic acid films was explored by other surface characterization techniques such as XPS which quantifies the chemical composition of the film. In an angle-dependent mode, XPS measurements allow the determination of the chemical composition at different depths in the material. By lowering the angle between sample and detector from 90° (normal) to 30° (60° off-normal) the analysis depth into the DNA/DDAB and the RNA/DDAB films could be varied from approximately 100 Å down to 20 Å respectively. The chemical composition of the uppermost layer (or surface layer), as well as the layers beneath it, can therefore be determined. All XPS data obtained were correlated with the NIST XPS database. Nitrogen was chosen as the control to measure with XPS because it was the only atom with a different chemical environment in both the nucleic acid and the lipid. While the headgroup of the lipid DDAB contains a quaternary amine (Figure 1B), the nucleic acids only contain aromatic and aliphatic nitrogen atoms (Figure 1A). The difference in chemical binding of nitrogen atoms was reflected in the binding energy. The amount of quaternary nitrogen increased when we tilted the sample from 90° to 30° for DNA as well as RNA DDAB films (Table 2), which indicates that the upper layer is formed by the DDAB lipid layer. However, the assembly within the RNA film appears to contain more chemically heterogeneous layers than the assembly within the DNA film because the intensity of aromatic and aliphatic nitrogen peaks in RNA is close to the quaternary nitrogen peaks (Figure 6). This, we expect, is due to the fact that RNA is approximately one hundred times shorter than DNA. In addition perhaps this clustering of short RNA molecules (also seen in the AFM and X-ray data) allows DDAB to interpenetrate the layers more easily. RNA and DDAB are on approximately the same length scale compared to DNA and so can populate the surface more equally. AFM experiments were used to investigate the surface structure of the films (Figure 7). Comparing the 5 × 5 µm



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Figure 6. Decomposed high-resolution XPS spectra of the different nitrogen species by changing the angle between sample and detector (A) DNA/DDAB film 90° (normal with respect to the X-ray beam), (B) DNA/DDAB film 30° (60° off normal with respect to the X-ray beam), (C) RNA/DDAB film 90° (normal), and (D) RNA/DDAB film 30° (60° off normal). These angles correspond to the escape depths of approximately 100 Å at normal and 20 Å at 60° off normal.

images, we can see that there are step-like structures present within the DNA/DDAB films, but not in the RNA/DDAB films. This suggests that the RNA/DDAB film has apparently a more heterogeneous structure compared to the DNA/DDAB film. However, on increasing the resolution of analysis to a scale of 1 µm × 1 µm (insert images), we can observe small irregular cavities in the RNA films instead of the steps seen in the DNA films. Based on the hydrophilic character of the cavities (Figure S1), we postulate them to be exposed nucleic acids in the first layer of the complex. Thus, step-like layers are formed with both types of films, but the lengths of the strands are responsible for the organization of layers within the film. Longer DNA molecules form more homogeneous layers with widespread plateaus, while shorter RNA chains form small domains several hundred nanometers in diameter. The AFM measurement of the step height for DNA/DDAB film is 2.8 ( 0.2 nm and for RNA/ DDAB is 2 ( 0.5 nm. In the DNA/DDAB film no large distinct patches of hydrophobic and hydrophilic areas can be observed, and so the step structure we measured was largely hydrophobic, according to the phase image. However, in the RNA/DDAB film we measured the step height between a hydrophobic area on top (light area) and a hydrophilic area (dark area) below. This indicates that the patches in the RNA/DDAB film do not represent the total repeat unit of RNA/DDAB but merely the upper lipid layer. The lamellar structure and step height measurements from AFM experiments are in reasonable agreement with the results obtained by X-ray and XPS. We also studied the mechanical properties and permeability of the films for further use in materials applications such as implants in vivo. First we prepared a set of DNA (2000 base pairs) and RNA (15 nucleotides) blended films to investigate the relationship between mechanical and morphological properties. Tensile strength and elongation values for each type of film were determined in triplicate. Statistics on a completely randomized design were determined using Statgraphic software (release 5.0, Statgraphic Corp.) to calculate analysis of variance (ANOVA). Bonferroni’s multiple range test was used to determine significantally different averages at a 95% confidence interval.33 We found that the tensile strength of DNA and RNA blended films increased with DNA concentration (Figure 8 and

Figure 7. AFM height images 5 × 5 µm of (A) DNA/DDAB film (2000 base pairs) and (B) RNA film (15 nucleotides). AFM height images 1 × 1 µm of the same films are given in the blue insert. The Z range of all images is equal to 50 nm.

Table 3). This result is in good agreement with earlier report for the blended films between DNA and both poly(adenylic acid) (poly A) and poly(adenylic acid: uridylic acid) (poly AU).23 However, in the present case, the elongation at break or tensile strain started to change at around 10 wt % of DNA concentration (Figure 8 and Table 3). The tensile properties of these blended films could be rationalized by considering the molecular structure of DNA and RNA films. Moreover, the length of DNA can account for the elastic tensile properties of these self-assembling films, without needing to invoke the rod-like structure of DNA. Our observations do not conform to simple laws of mixture. Nonlinear mixing rules are often found in blending polymers of differing molecular weights.34,35 It should be noted that stress-strain curve of DNA/DDAB film looks like a typical cold drawing elastic polymer.36 During the experiment, the individual DNA chains in the film tended to align due to viscous flow, which gave rise to the late increase in stress after initial yielding. We recognize the influences of the large difference in molecular weight between the RNA and the DNA used on the mechanical properties; however, we believe these are much more important than the chemical differences. In general, the mechanical properties of polymer blends are highly dependent on the state of the morphology,37 so the surface morphology of these blended films was then studied using AFM

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Figure 8. (A) Stress-strain curves of pure DNA/DDAB and RNA/ DDAB films as well as DNA and RNA blended films. The tensile strength of DNA and RNA blended films increased with DNA concentration, however, the elongation started to change at around 10 wt % of DNA concentration. 3D red-cyan stereo projection of AFM images of (B) 100% DNA/DDAB film, (C) 10% DNA + 90% RNA/ DDAB film, (D) 1% DNA + 99% RNA/DDAB film, and (E) 100% RNA/ DDAB film. The film morphology also starts to change at 10 wt % of DNA. Beyond this concentration, the “step” feature was found.

(Figure 8B-E shows 3D AFM images of these films). The morphology of DNA and RNA blended films also starts to change at 10 wt % of DNA concentration, and step-like lamellar features were also found above this concentration. The multilayer step structure of DNA film probably makes the film more stretchable due to the possible independent sliding effect of each layer, which is why it presents the highest strain at break. However, the topology of short RNA films looks more amorphous, which means they are less prone to mechanical elongation or that these films are easier to break. These results illustrate the understanding of correlation of nucleic acid morphological structure to the mechanical properties of these self-assembling films.

Smitthipong et al.

The strength and extendability of a film depend on the length of the molecules; however, the secondary structure, like the double helix formed by hydrogen bonds, should also have an effect on the properties of the films. It has been proposed by Okahata et al.1 that the staining of nucleic acid/lipid films by ethidium bromide is indicative of the retention of the helical structure of DNA within the film. Ethidium bromide is a dye that has been widely used for staining nucleic acid: its primary (and generally stronger) mode of binding, where a part of the ethidium ion sandwiches between adjacent base pairs of the double helix, is known as “intercalation”.11,38 The maximum degree of intercalation is one molecules of ethidium bromide for every five base pairs.39 It is still unclear how this interaction is affected by the complexation with a cationic lipid. In this experiment, the films were cast on the glass slide instead of in the glass mold. The films with the glass slide were used to prevent the films from collapsing in ethidium bromide solution. The films must be usually removed from the glass mold for characterization, which may threaten their stability after having been immersed in the solution. Films supported by a glass slide can be measured the fluorescence directly without detaching them from the substrate, thus avoiding the problem. When the DNA and RNA blended films were immersed in the ethidium bromide solution for 24 h, the films changed to a red color due to ethidium bromide penetration (Figure 9A). We found that the fluorescence signal decreases with increasing amount of DNA (Figure 9B). Two plausible reasons can be given for this phenomenon. The DNA molecules used in this study are about 100 times longer than the RNA molecules. Shorter molecules like the RNA are more flexible in terms of motion and allow higher penetration rate of molecules such as ethidium bromide into the film. This flexibility could also lead to the rough and unordered surface seen in the AFM image. Longer more inflexible DNA molecules form a well-ordered sheet structure on the surface, as seen by AFM, which reduces the penetration of ethidium bromide into the film. We expect that this is the reason why the DNA/DDAB film shows a lower fluorescent signal than the RNA/DDAB film. However when native DNA and RNA molecules are labeled beforehand with ethidium bromide and then used to form lipid complexes and cast as films, we can see the opposite intensity relation. DNA/ DDAB films show a higher fluorescent signal than RNA/DDAB films (Figure 9C) as the DNA used is longer and has a higher content of base pairs to entrap ethidium bromide. When we use the same argument as before then we can reason that the ethidium bromide molecules are within the DNA/DDAB film and cannot leach out as easily as with the RNA/DDAB film, which has many permeable patches on its surface. This is why we obtain a higher signal for DNA/DDAB films than RNA/ DDAB films when the native DNA was labeled with ethidium bromide than when we immerse the film into an aqueous solution of ethidium bromide. However, it must be noted that we are comparing an intercalation process in an aqueous medium to a prelabeled nucleic acid complex cast from isopropanol. At this point, we are not able to demonstrate the double stranded nature of nucleic acid within the film. We plan to continue our investigations of the structure of the dried film and the state of the dried nucleic acids.

Conclusions Noncovalent electrostatic interaction is a fascinating approach for the fabrication of supramolecular self-supporting



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Figure 9. (A) DNA/DDAB film turns red when immersed in ethidium bromide solution for 24 h. The postlabeled films were analyzed by fluorescence spectroscopy. The fluorescence emission spectra of DNA/DDAB, RNA/DDAB films and DNA+RNA/DDAB blended films are shown in (B). The penetration of ethidium bromide molecules in the RNA/DDAB film is faster than in the DNA/DDAB film, as the fluorescent intensity decreased with the increase of DNA content in the blended films. (C) Evolution of maximum fluorescent intensity and the related diffusion limitation of ethidium bromide to tensile strain as a function of DNA content in RNA/DDAB films. (D) Fluorescence emission spectra of DNA/DDAB and RNA/DDAB films with initially ethidium bromide treated native nucleic acid molecules before complexation with DDAB.

films.9,40 This technique provides the ability to manufacture nucleic acid based materials that can have both considerable mechanical strength, and useful responsiveness to conditions such as heat and an aqueous environment. These DNA and RNA films have a thermostable structure from 20 to 90 °C. Interestingly, the properties of the film depend on the length as well as the molecular structure of the nucleic acids. This study provides a useful model to investigate the correlation of nucleic acid structure on the formation and properties of macroscale materials from the bottom-up. For future work, we will focus on the degradability property of the films in the different environments as well as their biocompatibility with living cells. One can anticipate that these new biomaterials could have interesting applications as a biocompatible functional material for delivery of a drug or other therapeutic agents due to their high permeability for small molecules. Potentially active DNA or RNA molecules such as siRNA can be embedded into the film structure and used after implementation for controlled delivery. The mechanical properties of nucleic acid/surfactant can also be tuned to match the elasticity required for artificial skin. Here a few important properties for such application are found in one material: potential drug release, mechanical strength, and biodegradability. We anticipate that these new biomaterials could have significant potential applications as a functional material, a drug delivery medium, a biosensor, or a matrix for cell growth or tissue regeneration. Acknowledgment. We gratefully acknowledge Dr. Thomas Mates for the useful discussion of XPS results, Dr. Rungsima Chollakup for the statistic calculation of mechanical tests, and Mr. Morito Divinagracia for the helpful advice on X-ray

experiments. This work is supported by the MRSEC Program of National Science Foundation (DMR05-20415) and College of Engineering at UCSB. Wirasak Smitthipong has been supported by these sources as a postdoctoral researcher at UCSB. Surface characterization experiments were performed in the laboratory of Dr. Helen G. Hansma and supported by NSF (MCB 0236093). We thank the reviewers for their excellent suggestions in improving this paper. Supporting Information Available. AFM experiments of height, amplitude, and phase images for DNA/DDAB and RNA/ DDAB films. This material is available free of charge via the Internet at http://pubs.acs.org.

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