pubs.acs.org/Langmuir © 2010 American Chemical Society
Novel Biocompatible DNA Gel Particles M. Carmen Moran,*,† M. Rosa Infante,‡ M. Grac-a Miguel,† Bj€orn Lindman,†,§ and Ramon Pons‡ †
‡
Chemistry Department, Rua Larga, Coimbra University, 3004-535 Coimbra, Portugal, Institut de Quı´mica Avanc-ada de Catalunya, IQAC- CSIC, c/Jordi Girona 18-26, 08034 Barcelona, Spain, and § Physical Chemistry 1, Lund University, P.O. Box 124, 22100 Lund, Sweden Received February 25, 2010. Revised Manuscript Received May 12, 2010
Surfactants with the cationic functionality based on an amino acid structure have been used to prepare novel biocompatible devices for the controlled encapsulation and release of DNA. We report here the formation of DNA gel particles mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with two different single-chain amino acidbased surfactants: arginine-N-lauroyl amide dihydrochloride (ALA) and NR-lauroyl-arginine-methyl ester hydrochloride (LAM). The degree of DNA entrapment, the swelling/deswelling behavior, and the DNA release kinetics have been studied as a function of both the number of charges in the polar head of the amino acid-based surfactant and the secondary structure of the nucleic acid. Analysis of the data indicates a stronger interaction of ALA with DNA, compared with LAM, mainly attributed to the double charge carried by the former surfactant compared to the singly charged headgroup of the latter species. The stronger interaction with amphiphiles for ssDNA compared with dsDNA suggests the important role of hydrophobic interactions in DNA. Data on the microstructure of the complexes obtained from small-angle X-ray scattering (SAXS) of the particles strongly suggests a hexagonal packing. It was found that, the shorter the lattice parameter, the stronger the surfactant-DNA interaction and the slower the DNA release kinetics. Complexation and neutralization of DNA on the DNA gel particles was confirmed by agarose gel electrophoresis measurements.
Introduction A major research thrust in the pharmaceutical and chemical industry is the development of controlled release systems for drugs and bioactive agents. Many of these delivery systems in use and under development consist of a drug dispersed within a polymeric carrier. Drug carrier materials are for the most part insoluble due to the presence of chemical cross-links. These chemicals provide the network structure and physical integrity, but they are typically toxic. In addition, problems encountered in reaching this goal are related not only to the preparation technology, but also to the intrinsic nature of polymers. Indeed, the encapsulation technologies imply the use of organic solvents and high energy sources, thus leading to a significant degradation of the encapsulated molecule during the course of the polymer hydrolysis. Oppositely charged surfactants and polyelectrolytes have a strong propensity to bind to one another.1-7 Furthermore, when surfactant/polyelectrolyte attraction overcomes their solubility, associative phase separation occurs.3,4,7-10 This results in the formation of concentrated liquid, gel, or precipitate phases in *Corresponding author. E-mail:
[email protected]. (1) Goddard, E. D. Interactions of Surfactants with Polymers and Proteins; CRC Press: Boca Raton, 1992. (2) Goddard, E. D. Colloids Surf. 1986, 19, 301–329. (3) Piculell, L.; Lindman, B. Adv. Colloid Interface Sci. 1992, 41, 149–178. (4) Thalberg, K.; Lindman, B. Interactions of Surfactants with Polymers and Proteins; CRC Press: Boca Raton, 1993. (5) Hansson, P.; Almgren, M. Langmuir 1994, 10, 2115–2124. (6) Hansson, P. Langmuir 2001, 17, 4167–4180. (7) Ober, C. K.; Wegner, G. Adv. Mater. 1997, 9, 17–31. (8) Carnalli, J. Langmuir 1993, 9, 2933–2941. (9) Wang, Y. L.; Kimura, K.; Huang, Q.; Dubin, P. L.; Jaeger, W. Macromolecules 1999, 32, 7128–7134. (10) Wang, Y. L.; Kimura, K.; Dubin, P. L.; Jaeger, W. Macromolecules 2000, 33, 3324–3331. (11) Swanson-Wethamuthu, M.; Dubin, P. L.; Almgren, M.; Li, Y. J. Colloid Interface Sci. 1997, 186, 414–419. (12) Ilekti, P.; Martin, T.; Cabane, B.; Piculell, L. J. Phys. Chem. B 1999, 103, 9831–9840.
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equilibrium with a dilute solution.3,4,7,9-13 The polyelectrolyte chains can assume different conformations, either expanded, as in a solution or a hydrogel, or collapsed, such as around a surfactant aggregate as precipitate. Control of these transitions between different states allows exploitation of surfactant and polyelectrolyte mixtures in a wide array of commercial applications, such as drug delivery, cosmetic, and medical formulations.14-18 A general understanding of the interactions between DNA and oppositely charged agents, and in particular the phase behavior, has given us a basis for developing novel DNA-based materials, including gels, membranes, and gel particles.19 We have recently prepared novel DNA gel particles based on associative phase separation and interfacial diffusion. By mixing solutions of DNA (either single- (ssDNA) or double-stranded (dsDNA)) with solutions of different cationic agents, such as surfactants, proteins, and polysaccharides, the possibility of formation of DNA gel particles without adding any kind of cross-linker or organic solvent has been confirmed.20-23 The strength of association, which is tuned by varying the structure of the cationic agent, (13) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M. G.; Lindman, B. Macromolecules 1999, 32, 6626–6637. (14) Lapitsky, Y.; Kaler, E. W. Colloids Surf., A: Physicochem. Eng. Aspects 2004, 250, 179–187. (15) Lapitsky, Y.; Eskuchen, W. J.; Kaler, E. W. Langmuir 2006, 22, 6375–6379. (16) Lapitsky, Y.; Kaler, E. W. Colloids Surf., A: Physicochem. Eng. Aspects 2006, 282, 118–128. (17) Lapitsky, Y.; Kaler, E. W. Soft Matter 2006, 2, 779–784. (18) Greenfield, M. A.; Hoffman, J. R.; Olvera de la Cruz, M.; Stupp, S. I. Langmuir 2010, 26, 3641–3647. (19) Costa, D.; Moran, M. C.; Miguel, M. G.; Lindman, B. Cross-linked DNA Gels and Gels Particles, in DNA Interactions with Polymers and Surfactants, Dias, R. S., Lindman, B., Eds.; Wiley Interscience: New York, 2008. (20) Moran, M. C.; Miguel, M. G.; Lindman, B. Langmuir 2007, 23, 6478–6481. (21) Moran, M. C.; Miguel, M. G.; Lindman, B. Biomacromolecules 2007, 8, 3886–3892. (22) Moran, M. C.; Laranjeira, T.; Ribeiro, A.; Miguel, M. G.; Lindman, B. J. Dispers. Sci. Technol. 2009, 30, 1494–1499. (23) Moran, M. C.; Ramalho, A.; Pais, A. A. C. C.; Miguel, M. G.; Lindman, B. Langmuir 2009, 25, 10263–10270.
Published on Web 06/01/2010
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allows control of the spatial homogeneity of the gelation process, producing either a homogeneous DNA matrix or different reservoir devices. This allows for various applications regarding the controlled encapsulation and release of ssDNA and dsDNA, with clear differences in the mechanism.23 Cationic surfactants have offered a particularly efficient control of the properties of DNA-based particles,20 but they are typically toxic. While toxicity certainly applies for most classical cationic surfactants, we are currently focusing on novel surfactants with much improved biocompatibility. These include surfactants with the cationic functionality based on an amino acid structure.24 Previous studies in our laboratory have demonstrated that single-chain arginine-based surfactants constitute a novel class of biobased materials of low toxicity, with excellent surface properties, and wide antimicrobial activity. They selectively disrupt bacteria membranes at submicellar concentrations, but not erythrocytes or skin cell membranes.25 Furthermore, the cationic arginine-based surfactant arginine-N-lauroyl amide dihydrochloride (ALA) has the ability alone to compact DNA. ALA gives, in combination with anionic surfactants, spontaneously stable vesicles, and special attention was given to the association of these catanionic vesicles, with a net positive charge, to DNA.26,27 Toxicity studies were performed by measuring the capacity of mouse B-16 V cells or humans HeLa cells to reduce the molecule of resazurin after incubation with ALA (data to be published elsewhere). Cell viabilities around 100% were observed for both cell lines tested. The aim of this work was to prepare novel DNA gel particles in which surfactants with the cationic functionality based on amino acid structure can interact with nucleic acids to form biocompatible devices for the controlled encapsulation and release of DNA. In this paper, the formation of DNA gel particles prepared by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with two different single-chain amino acid-based surfactants, arginine-N-lauroyl amide dihydrochloride (ALA) and NR-lauroylarginine-methyl ester hydrochloride (LAM), is presented. Results on the encapsulation of DNA and its release are presented, using the surfactant headgroup charge and the DNA conformation as controlling parameters. We also investigated the DNA-surfactant systems more closely, looking at the microstructure using small-angle X-ray scattering (SAXS), that demonstrates the stronger ALA-ssDNA association.
Experimental Section Materials. The sodium salt of deoxyribonucleic acid (DNA) from salmon testes of an average degree of polymerization of about 2000 base pairs was purchased from Sigma and used as received. The DNA concentrations were determined spectrophotometrically considering that, for an absorbance of 1, at 260 nm, a solution of dsDNA has a concentration of 50 μg/mL and a solution of ssDNA has a concentration of 40 μg/mL.28 All DNA concentrations are presented in molarity per phosphate group, i.e., molarity per negative charge. The ratios in absorbance at 260 and 280 nm of the stock solutions were found to be between 1.8 and 1.9, which suggested the absence of proteins.29 The
Figure 1. Chemical structure of the arginine-N-lauroyl amide dihydrochloride (ALA) and NR-lauroyl-arginine-methyl ester hydrochloride (LAM) derivatives. arginine-N-lauroyl amide dihydrochloride (ALA) and NR-lauroylarginine-methyl ester hydrochloride (LAM) (Figure 1) were synthethized previosly in our lab.30,31 N,N,N0 ,N0 -tetramethylacridine3,6-diamine (acridine orange (AO)) was supplied by Molecular Probes (Invitrogen). All experiments were performed using Millipore Milli-Q deionized water (18.2 MΩ cm resistivity). Particle Preparation. dsDNA stock solutions were prepared in 10 mM NaBr in order to stabilize the DNA secondary structure in its native B-form conformation. ssDNA stock solutions were prepared by thermal denaturation of dsDNA stock solution at 80 °C for 15 min and then immediately dipping into ice for fast cooling, to prevent renaturation. Surfactants were dissolved in Milli-Q water. DNA solutions were added dropwise via a 22-gauge needle into gently agitated surfactant solutions (1 mL). Under optimal conditions, droplets from DNA solutions instantaneously gelled into discrete particles upon contact with the surfactant solution. Thereafter, the particles were equilibrated in the solutions for a period of 2 h. After this period, the formed particles were separated by filtration through a G2 filter and washed with 5 8 mL volumes of Milli-Q water to remove the excess of salt and surfactant. Determination of DNA Entrapment Degree. The degree of entrapment was determined by quantifying both the nonbound DNA in the supernatant solution and the bound DNA in the gel particles. The entire quantity of supernatant surfactant solution containing the nonbound DNA was removed to be quantified by spectrophotometry. Thereafter, the particles were washed with Milli-Q water as described in the previous section. The particles were magnetically stirred in pH 7.6, 10 mM Tris HCl buffer to promote swelling and breakup of the structure. The resulting mixture, containing skins of the particles, was filtered, and then, the filtrates were quantified by a spectrophotometer. The amount of DNA present in the obtained skins was estimated considering the initial amount of DNA added. Loading capacity (LC) and loading efficiency (LE) were determined by the following equations: LCð%Þ ¼ ½ðtotal amount of DNA - nonbound DNAÞ= weight of particles 100
(24) Moran, M. C.; Pinazo, A.; Perez, L.; Clapes, P.; Angelet, M.; Garcı´ a, M. T.; Vinardell, M. P.; Infante, M. R. Green Chem. 2004, 6, 233–240. (25) Moran, M. C.; Clapes, P.; Comelles, F.; Garcı´ a, M. T.; Perez, L.; Vinardell, M. P.; Mitjans, M.; Infante, M. R. Langmuir 2001, 17, 5071–5075. (26) Rosa, M.; Infante, M. R.; Miguel, M. G.; Lindman, B. Langmuir 2006, 22, 5588–5596. (27) Rosa, M.; Moran, M. C.; Miguel, M. G.; Lindman, B. Colloids Surf., A: Physicochem. Eng. Aspects 2007, 301, 361–375. (28) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: a laboratory manual; Cold Spring Harbor Laboratory Press: New York, 1989; Vol. 3, App. C.1. (29) Saenger, W. Principles of Nuclei Structure; Springer-Verlag: NewYork, 1984.
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ð1Þ
LEð%Þ ¼ ½ðtotal amount of DNA - nonbound DNAÞ= total amount of DNA 100
ð2Þ
(30) Clapes, P.; Moran, M. C.; Infante, M. R. Biotechnol. Bioeng. 1999, 63, 333–343. (31) Infante, M. R.; Garcia Dominguez, J. J.; Erra, P.; Julia, M. R.; Prats, M. Int. J. Cosmet. Sci. 1984, 6, 275–282.
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Three batches of particles of each system were prepared, and the results are presented as average and standard deviation. Swelling and Dissolution Behavior of the Particles. Studies were conducted in pH 7.6, 10 mM Tris HCl buffer. Particles (around 100 mg) were exposed to dissolution media at an agitation rate of 40 rpm using the ST 5 CAT shaking platform. At specific time intervals, the entire quantity of dissolution medium was removed and particles placed in the container were weighed. Then, new solution was added in order to maintain a clean environment. This procedure was repeated until particles were completely dissolved. The data were then transformed to the relative weight loss using the following equation: relative weight ðRWÞ ¼ Wt =Wi
ð3Þ
Where Wi stands for the initial weight of the particles and Wt for the weight of the particles at time t. DNA Release from the Particles. Simultaneously to the studies of swelling/dissolution behavior, DNA release studies were carried out. Hence, at defined time intervals, the supernatant was collected and particles were resuspended in fresh solution. DNA released into the supernatant solutions was quantified by measuring the absorbance at 260 nm with a spectrophotometer (UV/vis spectrophotometer UV-2450 Shimadzu). Fluorescence Microscopy Imaging. Particle integrity and DNA conformational state were determined using the acridine orange fluorescent assay. Freshly prepared particles were stained for 10 min with Acridine orange (0.3 mg/mL), washed in distilled water, and immediately examined with a Olympus BX51 M microscope equipped with a UV-mercury lamp (100W Ushio Olympus) and a filter set type MNIBA3 (470-495 nm excitation and 505 nm dichromatic mirror). Images were digitized on a computer through a video camera (Olympus digital camera DP70) and were analyzed with an image processor (Olympus DP Controller 2.1.1.176, Olympus DP Manager 2.1.1.158). All observations were carried out at 25 °C. Polarized Light Microscopy. A microscope (Reichert Polyvar 2) equipped with a heating stage was used to observe the texture of the mixtures during the heating process. Particles kept between coverslips were heated at 25 °C and their changes were monitored. The mixtures were illuminated with linearly polarized light and analyzed through a crossed polarizer. To capture images, a video camera and a PC running Leica IM 500 software were used. X-ray Scattering. Small-angle X-ray scattering (SAXS) measurements were carried out by using a S3-MICRO (Hecus X-ray systems GMBH Graz, Austria) coupled to a GENIX-Fox 3D X-ray source (Xenocs, Grenoble), which provides a detector focused X-ray beam at Cu KR line with more than 97% purity and less than 0.3% KR. The linear detector was PSD-OED 50 M-Braun and the temperature controller was a Peltier KPR AP PAAR model. Particles were inserted between two Mylar sheets with a 1 mm separation. The SAXS scattering curves are shown as a function of the scattering vector modulus q ¼
4π sin ðθ=2Þ λ
ð4Þ
where θ is the scattering angle and λ = 0.1542 nm the wavelength of the radiation. The q values with our setup ranged from 0.1 nm-1 to 6.0 nm-1. Using the Bragg’s law with d = 2πn/q and n the order of reflection, the corresponding distances were between 60.0 and 1.1 nm. The system scattering vector was determined by measuring a standard silver behenate sample. The obtained scattering curves are smeared mainly by the detector width; by comparison to some 2D images taken at the same spot, no significant differences in the peak positions encountered in the present samples are present; however, the width of the peaks (which appear at q ≈ 0.15 nm-1) increases by 10% because of the smearing present. 10608 DOI: 10.1021/la100818p
Figure 2. Representative images of the studied DNA gel particles.
Electrophoresis in Agarose Gel. Agarose (0.7 g) was dissolved in TBE buffer (100 mL) by heating until boiling and was afterward allowed to cool down. Five μL of 10,000x solution of Gelstar was added to the agarose solution which was then poured into a 10 20 cm gel tank. Possible air bubbles were removed with a pipet tip and gel comb was added to the gel which was allowed to set. DNA solution (19.8 mg/mL) and individual DNA gel particles were added to each well. The electrophoresis was carried out in a horizontal tank containing TBE buffer and was run at 90 V for 45 min. Gels were imaged using a UV transilluminator.
Results and discussion Particle preparation. Gel particles were prepared using solutions with a concentration such that the DNA to surfactant molar ratios, R = [DNA]/[Sþ], were equal to 1. The DNA concentration was set to 60 mM. The choice of DNA concentration reflects the fact that DNA produces high-viscosity solutions, which makes it a convenient system for the preparation of stable DNA gel particles.20 The formation of the DNA gel particles was initially studied using mixtures of DNA and the surfactant LAM. Droplets of DNA solutions instantaneously gelled into discrete particles upon contact with the surfactant solution. Thereafter, the formation of solid gel-like particles was observed. The size of the resulting particles reflects the size of the parent drop, the mean particle diameter being 1-2 mm. The corresponding experiments were carried out using ALA. Under these conditions, droplets from DNA solutions instantaneously gelled into discrete particles upon contact with the surfactant solution. Interestingly, after a few minutes, the particles prepared with ALA visibly reduced their size (to 99%), compared with 97% obtained for particles containing the LAM derivative. No significant differences were observed as a function of the initial secondary structure of the polyelectrolyte. A similar trend was observed in the determination of the entrapped DNA as a function of the weight of the particles (LC values). The lowest LC values (∼4%) were obtained in particles containing LAM, whereas the amount of encapsulated DNA using ALA as cationic agent was almost twice as large. Some features involved in the formation of the DNA gel particles can be elucidated from these results. Since the surfactants and DNA are oppositely charged, it may be assumed that the driving force of particle formation has an electrostatic origin. ALA is a divalent cation, whereas LAM is a monovalent one. Hence, for the same hydrophobic contribution (twelve carbon atoms in the alkyl chain) a more charged cation provides both higher efficiency and capacity characteristics. This is offset to a degree that is not well understood by the lower tendency of a divalent surfactant to self-assemble; note that the surfactant always associates to DNA in a self-assembled state. Structural studies of gel-like particles formed by mixing a solution of a cationic cellulose derivative with oppositely charged catanionic surfactant solutions composed of sodium perfluorooctanoate (FC7) and cetyltrimethylammonium bromide (CTAB) have suggested that the structure of the resulting particles is governed by the gelation kinetics, i.e., surfactant diffusion, gel formation, structural rearrangements (gel collapse), and solvent release.14 Surfactant penetration into the drops is primarily controlled by the surfactant concentration and surfactant permeability into the gel.14 Structural features have also been observed in deswelling of polyacrylate and hyaluronate gels induced by oppositely charged alkyltrimethylammonium bromide surfactants.32 When these gel are placed in a solution containing oppositely charged surfactant of a concentration exceeding the critical association concentration (cac), but of equal ionic strength, the surfactant monomers will diffuse into the gel and there form micelles. This allows the release of the counterions previously neutralizing the polymer charges and, due to the subsequent lowering of the swelling pressure, causes the gel to collapse. As micelles form in the gel, the outer part will collapse first, causing a phase separation with a dense, outer, micelle-rich surface phase and a still swollen, micelle-lean core. As more surfactant is absorbed into the gel, the surface phase will grow at the expense of the core. The regular deswelling behavior is that the gel continuously deswells as the collapsed surface phase grows, exerting higher pressure on the core, while the core is gradually (32) Nilsson, P.; Hansson, P. J. Colloid Interface Sci. 2008, 325, 316–323.
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converted to surface phase, until the entire gel has collapsed. With increasing amount of bound surfactant, the particle evolves via a core/shell structure to a fully collapsed state.32 An indication of the structural characteristics of these biocompatible DNA particles can be deduced from the amount of DNA that can be released once the breakup of the particles was mechanically promoted. The percentages of DNA released in the supernatant are also summarized in Table 1. These values suggest that by using ALA or LAM most of the DNA is complexed during the particle formation. The formation of these fully collapsed particles differs from that obtained using surfactants with longer chain length. In our previous studies using CTAB,21 between 20% and 50% of the encapsulated DNA remains free to be released. It has been argued that longer surfactant chain length promotes irregular deswelling, where the gel turns into structures with a dense outer layer surrounding a liquid-filled core.32 The formation of these fully collapsed particles is also supported by visual inspection (see Figure 2). A similar correlation between DNA distribution and morphological aspects has been recently found for DNA particles formed with mixed protein systems.23 Particle Swelling and Deswelling Kinetics. Gels are considered to have great potential as drug reservoirs. Loaded drugs would be released by diffusion from the gels or by erosion of them. Hence, the release mechanism can be controlled by swelling or dissolution of the gels. Figure 3 shows the relative weight of the different gel particles after exposure to a buffer solution (TrisHCl pH 7.6). When the surfactant-DNA gel particles are inserted into a medium, different responses are encountered: swelling, deswelling, and dissolution. In all cases, particles placed in pH 7.6, 10 mM Tris HCl buffer show water uptake from the medium probably due to osmotic swelling. The extension of the swelling process depends on both the surfactant and the nucleic acid structures. Particles containing ALA exhibited the largest (relative weight ratio, RW 2-2.5) and the longest (more than 1300 h) swelling process. In the case of particles containing LAM, they swell (RW 1.5-2.0 using the maximum points as estimate) for up to 10 or 200 h, as a function of the secondary structure of the polyelectrolyte, and then start to shrink. The results suggest that the stability of the gel particles is given mainly by the electrostatic interaction between DNA and the oppositely charged surfactant. More stable particles are obtained for ALA than for LAM, probably due to its double charge. In addition, for the latter case, a higher stability is found when denatured DNA is used. In the context of DNA-surfactant interactions, we need to consider the amphiphilic nature of DNA. The segregation between hydrophilic and lipophilic parts is not pronounced, and the force opposing self-assembly is very strong due to the high charge density and large persistence length. However, from the binding isotherms and the phase diagrams we can infer a considerably stronger association of cationic surfactants for ss-DNA than for ds-DNA.33 DNA Release from Particles. Particles were suspended in pH 7.6, 10 mM Tris HCl buffer to determine the kinetics of DNA release (see Figure 4). Generally, the release pattern seems congruent with that observed in the swelling/dissolution profiles. Thus, LAM-DNA particles exhibited a faster release than ALA-DNA particles. The DNA release profiles from LAMdsDNA particles display the complete release after 400 h; (33) Dias, R. S.; Miguel, M. G.; Lindman, B. DNA as an Amphiphilic Polymer, in Dias, R. S., Lindman, B., Eds. DNA Interactions with Polymers and Surfactants; Wiley Interscience: New York, 2008.
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Figure 3. Relative weight ratio measurements performed on the different DNA gel particles after exposure to pH 7.6, 10 mM Tris HCl buffer solutions.
Figure 4. Release of DNA from the different DNA gel particles in pH 7.6, 10 mM Tris HCl buffer solutions.
LAM-ssDNA shows complete DNA release after 800 h. When the formulation contains ALA, the DNA release was slower. Complete DNA release is only achieved after 1800 h. With respect to the observed differences between ssDNA and dsDNA release, the results agree with our previous studies on both surfactant and protein systems, which have shown a stronger interaction with ssDNA compared to dsDNA.20,21 The rate and the final cumulative DNA release depends on its secondary structure. This strongly indicates the important role of hydrophobic interaction in DNA when the bases are more exposed, as in the case of ssDNA. Secondary Structure of DNA in the Particles. Fluorescence microscopy (FM) using the fluorescence dye acridine orange (AO) was used to confirm the presence of DNA, as well as to assess the secondary structure of the nucleic acid in the particles. AO (excitation 500 nm/emission 526 nm) intercalates into doublestranded DNA as a monomer, whereas it binds to single-stranded DNA as an aggregate. Upon excitation, the monomeric acridine orange bound to double-stranded DNA fluoresces green, with an emission maximum at 530 nm. The aggregated acridine orange on single-stranded DNA fluoresces red, with an emission at about 10610 DOI: 10.1021/la100818p
640 nm.34,35 On the basis of the observation of green or red fluorescence, acridine orange has been used to differentiate native, double-stranded DNA from denatured, single-stranded DNA in the surfactant-DNA particles. FM images of freshly prepared particles showed green emission (Figure 5), independently of the initial secondary structure of the DNA. The absence of red emission in the particles containing denatured DNA suggests that the accessibility of free DNA to the dye is hindered. This observation is consistent with our data on DNA distribution for the present systems and can be confirmed from our previous observations for CTAB-DNA particles.21 The percentages of ssDNA released is close to 0.1% (see Table 1), confirming the total complexation of the DNA in the presence of ALA and LAM surfactants. However, using CTAB, the ssDNA release achieved 20%, making possible its detection in the FM studies. Studies of the accessibility of AO in ALA-DNA particles were carried out after 1300 h. On the basis of the green and red emission (34) Ichimura, S.; Zama, M.; Fujita, H. Biochim. Biophys. Acta 197l, 240, 485–495. (35) Peacocke, A. R. The interaction of acridines with nucleic acids, in Acridines, Acheson, R. M., Ed.; Interscience Publishers: New York, 1973; pp 723-754.
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Figure 5. Fluorescence micrographs of the different DNA gel particles in the presence of the fluorescent dye acridine orange.
in the case of ALA-dsDNA and ALA-ssDNA, respectively, the formation of particles with conserved secondary DNA structure is confirmed (Figure 5). Microstructure of the DNA-surfactant Complexes. The qualitative phase behavior of the different formulations was initially studied by means of polarized light microscopy. All complexes obtained are found to be birefringent under a polarizing microscope. Figure 6 reports some examples of optical micrographs at 25 °C, where very different textures can be inferred. ALA-dsDNA and LAM-ssDNA revealed the formation of a hexagonal texture from the apperarance of the smokey optical texture and the well-defined spherulites that exhibit the classical maltese-cross extinction pattern, respectively. The textures formed by ALA-ssDNA could be attributed to a lamellar structure because of the presence of some maltese crosses. LAM-dsDNA did not show a recognizable texture. Further insight into the structural properties of the constituent phases of the different formulations was obtained by examination of small-angle X-ray scattering data. The SAXS profiles obtained from transmission measurements of these same samples show a relatively broad peak with maxima in the Langmuir 2010, 26(13), 10606–10613
range of q = 1.3-1.6 nm-1 (Figure 7). This corresponds to repeat distances from 4.0 to 4.7 nm. In the case of samples containing LAM, a second peak is apparent at q values twice as large as those of the first peak. This could indicate a lamellar structure of the complexes prepared with LAM. There are, however, several arguments against this assignation of structure. If we calculate the repeating distance for a lamellar structure, it is difficult to accommodate in this distance for a LAM bilayer plus DNA rods. In a lamellar arrangement, we should consider the repeat distance as formed by twice the surfactant hydrophobic length and two hydrophilic heads (hydrated bilayer thickness of 5.1 nm36). Moreover, we should allow some room to accommodate the DNA rods, which have a diameter of about 2 to 2.5 nm.37,38 If hydrophobic interdigitation occurred, this value of 5.1 would be reduced to 3.5 nm to which still we should add the space for the (36) Perez, L.; Pinazo, A.; Infante, M. R.; Pons, R. J. Phys. Chem. B 2007, 111, 11379–11387. (37) Krishnaswamy, R.; Pabst, G.; Rappolt, M.; Raghunathan, V. A.; Sood, A. K. Phys. Rev. E 2006, 73, 031904–031908. (38) Leal, C.; Wads€o, L.; Olofsson, G.; Miguel, M.; Wennerstr€om, H. J. Phys. Chem. B 2004, 108, 3044–3050.
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Figure 6. Representative optical polarizing micrographs displayed by the different samples at 25 °C. Table 2. Repeat Distance (d ) and Lattice Spacing (a) Parameteres Derived from the Diffraction Patterns at 25 °C for the Suggested Hexagonal Model
Figure 7. Scattered X-ray intensity of DNA particles as a function of scattering vector at 25 °C. The curves have been multiplied by factors of 4 to avoid overlapping.
DNA rods. Hexagonal packing of rod-like √ particles should produce a scattering pattern with peaks in a 1: 3:2 order. The absence of a second and/or third reflection from a hexagonal structure can be due to the occurrence of minima of the scattering form factor close to the expected reflections as was shown by Krishnaswamy et al.37 Also, a preferred orientation of the rods perpendicular to the skin normal would hinder the observation of √ the 3 peak; some indication of this has been obtained from the GISAXS patterns of ordered samples of these complexes formed by deposition on a surface.39 However, the exact structure of the (39) Moran, M. C.; Pons, R. To be published.
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system
d (nm)
a (nm)
ALA-dsDNA ALA-ssDNA LAM-dsDNA LAM-ssDNA
4.22 ( 0.05 3.95 ( 0.05 4.69 ( 0.05 4.52 ( 0.05
4.87 ( 0.05 4.56 ( 0.05 5.41 ( 0.05 5.22 ( 0.05
particle skin is still an open question that deserves further research. Considering an hexagonal packing as the most plausible structure, regardless of being a direct or an inverse hexagonal (see ref 37 for further details on the direct or reverse structure of DNA-CTAB complexes), we can calculate the lattice parameter of these samples. Those, together with the repeat distances, are shown in Table 2. We can establish a correlation between the DNA-amphiphile interaction and the lattice parameters obtained from these SAXS experiments. For the strongest interaction, an overall neutrality on the complexes can be achieved. Consequently, at charge compensation, water solvation can be minimal and the shorter lattice parameters are expected. Electroneutral complexes dissociate with more difficulty, leading to slower release kinetics. There exists a clear correlation between the DNA release rates and the lattice parameters obtained from these SAXS experiments: the slower the release, the shorter the lattice parameter. The lattice parameter is consistently smaller for the singlestranded DNA than for the double-stranded DNA. We also consistently have a lower lattice parameter for ALA than for the LAM surfactant. This leads to the conclusion that the shorter the lattice parameter (i.e., the more compact the structure), the stronger the surfactant-DNA interaction. These observed characteristic distances correlate quite well with the observed release half-life as shown in Figure 8 in which Langmuir 2010, 26(13), 10606–10613
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Figure 8. Half-life release values plotted as a function of the SAXS repeat distance d.
Article
Gel Retardation Assay. When placed in semipermeable media, like an agarose gel, DNA will follow the electric field and will replicate throughout the gel. Larger fragments migrate more slowly, at a rate approximately proportional to its charge to mass ratio, toward the positive electrode. Complete complexation and neutralization of DNA is indicated by complete retention of the DNA in the wells of the gel.45 The gel retardation assay has been used here to find out if there are cosolutes binding a fragment of DNA (in a DNA-cosolute complex) by watching how fast the DNA fragment moves through an electric field and seeing whether it moves more slowly when a particular cosolute is also present. Agarose gel electrophoresis of DNA gel particles confirmed that DNA on these DNA gel particles forms significantly stable complexes, with a decreasing amount of DNA migration in the gel compared to free DNA (Figure 9). In addition, DNA on ALA-DNA gel particles do not migrate in the electric field. The signal UV active in the wells might indicate that the nonmigrating DNA is accessible to Gelstar intercalation.
Conclusion
Figure 9. Gel retardation assay for monitoring DNA binding in DNA/amino acid-based surfactants particles, compared with naked DNA.
the half-life release values have been plotted as a function of the SAXS repeat distance d. With respect to the observed differences between ssDNA and dsDNA in the release, the results agree with previous studies, both experimental20,21,40 and theoretical,41-44 which have shown a stronger interaction with amphiphiles for ssDNA compared to those with dsDNA. This fact strongly indicates the important role of hydrophobic interaction in DNA when the bases are more exposed, as in the case of ssDNA. (40) Rosa, M.; Dias, R.; Miguel, M. G.; Lindman, B. Biomacromolecules 2005, 6, 2164–2171. (41) Wallin, T.; Linse, P. J. Phys. Chem. B 1997, 101, 5506–5513. (42) Wallin, T.; Linse, P. J. Phys. Chem. 1996, 100, 17873–17880. (43) Wallin, T.; Linse, P. Langmuir 1996, 12, 305–314. (44) Kwak, J. C. T. Polymer-Surfactant Systems; Marcel Dekker: New York, 1998.
Langmuir 2010, 26(13), 10606–10613
Novel DNA gel particles, in which surfactants with the cationic functionality based on an amino acid structure can interact with nucleic acids to form biocompatible devices for the controlled encapsulation and release of DNA, have been prepared. We report here on the formation of DNA gel particles mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with two different single-chain amino acid-based surfactants: arginineN-lauroyl amide dihydrochloride (ALA) and NR-lauroyl-arginine-methyl ester hydrochloride (LAM). It was shown that DNA was effectively entrapped in the surfactant solutions, protecting its secondary structure. Changes in the magnitude of the swelling behavior and the DNA release kinetics by differences in the structure of the surfactant are demonstrated. The stronger interaction with amphiphiles for ssDNA compared with dsDNA suggest the important role of hydrophobic interactions in DNA. Looking at the microstructure of the complexes using small-angle X-ray scattering (SAXS) strongly suggests they have a hexagonal packing. There exists a clear correlation between the DNA release rates and the lattice parameters obtained from these SAXS experiments: the slower the release, the shorter the lattice parameter. These results can be explained by the complexation and neutralization of DNA on the DNA gel particles and they were confirmed also by agarose gel electrophoresis measurements. This new generation of amino acid-based surfactant complexes with DNA contributes to the increasing demand for biocompatible vehicles for pharmaceutical applications. Acknowledgment. This work was supported by projects from the Fundac-~ao para a Ci^encia e Tecnologia (FCT, POCTI/QUI/ 45344/02 and POCTI/QUI/58689/2004) and postdoctoral grant SFRH/BPD/43838/2008. Also, was supported by the CICYT under Project CTQ2006-01582. We thank the bilateral project FCT/CSIC, references 27/CSIC/08 and 2007PT0050). We are thankful to Jaume Caelles of the SAXS-WAXS Service of IQAC for the SAXS measurements. (45) Rickwood, D.; Hames, D. H. Gel electrophoresis of nucleic acids: a practical approach; IRL Press: Washington, DC, 1982.
DOI: 10.1021/la100818p
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