DNA Hybrid Hydrogels

Article pubs.acs.org/Biomac

Preparation and Characterization of Imogolite/DNA Hybrid Hydrogels Nattha Jiravanichanun,† Kazuya Yamamoto,† Kenichi Kato,‡ Jungeun Kim,‡ Shin Horiuchi,§ Weng-On Yah,∥ Hideyuki Otsuka,†,∥ and Atsushi Takahara*,†,∥ †

Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan § Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan ∥ Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan ‡

ABSTRACT: Imogolite is one of the clay minerals contained in volcanic ash soils. The novel hybrid hydrogels were prepared from imogolite nanofibers and DNA by utilizing strong interaction between the aluminol groups on imogolite surface and phosphate groups of DNA. The hybrid hydrogels of imogolite and DNA were prepared in various feed ratios, and their physicochemical properties and molecular aggregation states were investigated in both dispersion and gel states. The maximum DNA content in the hybrid gels was shown in equivalent molar ratio of imogolite and DNA. The physical properties of the hybrid gels were changed by varying DNA blend ratios. In the dispersion state, the hybrid gels showed a fibrous structure of imogolite, whereas a continuous network structure was observed in pure imogolite, indicating that the hybrid with DNA enhanced the dispersion of imogolite. In the gel state, DNA and imogolite nanofibers formed a 3D network structure.

1. INTRODUCTION Aluminol (AlOH) groups have been known to have great affinity for phosphonic acid groups. The adsorption and binding of nucleic acid and clay minerals has attracted great interest because the complexes provide protection against the degradation by nucleases but maintain their biological activity such as gene delivery.1−4 The interaction of clays such as montmorillonite with important biological polymers such as DNA and RNA has been investigated.5−7 However, the study on the interaction between imogolite and DNA has not yet been reported. Imogolite is one of the important clay minerals contained in volcanic ash soils.8,9 Imogolite is a hydrous aluminosilicate nanofiber having outer surface of AlOH groups. The diameter of the nanofiber is ∼2 nm. The AlOH groups on the outer surface of imogolite are pH-dependent, possessing positive charge at low pH and negative charge at high pH. Point of zero charge (pzc) for imogolite was reported to be about pH 6−9.10 Additionally, imogolite can also be synthesized chemically from tetraethoxysilane and aluminum chloride.11−13 Because of these unique physicochemical properties, imogolite is categorized as an interesting nanomaterial. The self-assembly of polyelectrolyte homopolymers with imogolite by solution mixing was reported.14 Hydrogen bonding and ionic interactions between imogolite and polyelectrolyte contribute to the self-assembly. Double-helix DNA molecule has diameter of ∼2 nm similar to imogolite nanofiber. Both nanomaterials can be expected to exhibit good compatibility in aqueous media. Importantly, © 2011 American Chemical Society

phosphate groups on the outside of DNA double helix have strong interaction with aluminol groups on the imogolite surface. However, DNA could not be inserted into the inner pore of imogolite because the inner diameter of imogolite is ∼1 nm, which is smaller than that of DNA. These reasons stimulated our interest in the combination of DNA and imogolite. Furthermore, this hybridization can be performed without surface modification; therefore, the preparation process is very simple. In the present paper, we report preparation of the imogolite/DNA hybrid gels and investigation of their characteristics. The optimum adsorption of DNA on imogolite was determined, and the mass ratio of imogolite-to-DNA in the hybrid gels was examined. To reveal the structure−property relationships of the hybrid gels, dynamic viscoelastic measurement, transmission electron microscopy (TEM) observation, and wide-angle X-ray diffraction (WAXD) measurement were performed.

2. EXPERIMENTAL SECTION Materials. DNA (double strand) from herring testes in sodium salt form was purchased from Sigma Aldrich. The molecular weight was polydisperse between 400 and 1000 base pairs (bp) with center of distribution at ca. 700 bp. Imogolite was synthesized according to a previously reported method.13 HCl, NaHCO3, acetate buffer, tris buffer, and NaHCO3/NaOH buffer were prepared as buffer solutions Received: November 16, 2011 Revised: December 13, 2011 Published: December 13, 2011 276

dx.doi.org/10.1021/bm201616m | Biomacromolecules 2012, 13, 276−281

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according to the equation. Q = (Wwet − Wdry)/Wdry, where Wwet and Wdry are the weights of the wet gel and dried gel, respectively. Desorption of DNA from Hybrid Gels. The protection of DNA by the hybrid gel was tested by incubated the hybrid gel under various conditions such as different pH, salt concentration, and temperature. Acetate buffer, tris buffer, and NaHCO3/NaOH buffer were applied for pH 4, 7−9, and 10−11, respectively. The freeze-dried hybrid gels having DNA content of 70 wt % were incubated under given buffer solution, salt concentration, and temperature. The amounts of DNA release from the hybrid gels were monitored by UV−vis spectroscopy. Morphology and Structure of Hybrid Gels. Transmission electron microscopic (TEM) images were observed with H-7500 (Hitachi High-Technologies Corporation, Tokyo, Japan) at 100 kV of acceleration voltage and 10 μA of beam current. Wide-angle X-ray diffraction (WAXD) measurements were carried out at room temperature on a large Debye−Scherrer camera at the BL02B2 beamline of SPring-8. The incident beam from the bending magnet was monochromatized to λ = 0.1 nm. The dried powder samples were packed into glass capillary tubes with an outer diameter of 0.5 mm, and the tubes were rotated during the measurements. The data were collected in a 2θ range from 1 to 75° with a step interval of 0.01°. The rheological properties of hybrid gels were measured with MCR101 rheometer. A plate with diameter 12 mm was applied, and measured temperature was controlled at 25 °C. Each sample was measured three times to get an average value.

of pH 4−11. All other reagents were chemical grade and used as received. Preparation of Hybrid Gels in Varying Imogolite/DNA Blend Ratio. Hybrid gels of imogolite and DNA were prepared by mixing 1 mg/mL solutions of imogolite and DNA, which were prepared and adjusted to pH 4 by diluted HCl (Scheme 1). The several hybrid gels

Scheme 1. Schematic Representation for the Preparation of Hybrid Gel from Imogolite and DNA

Table 1. Concentration of Imogolite and DNA Feeds for the Hybrid Gel Preparation gel no.

1

2

3

4

5

6

7

8

DNA content (wt %) Imogolite content (wt %)

9 91

17 83

29 71

38 62

50 50

63 37

71 29

83 17

were prepared by varying DNA to imogolite feed ratio, as listed in Table 1, which shows the concentration of imogolite and DNA in wt %. Solvent contents were the same for all samples. Adsorption of DNA on imogolite was rapidly observed visually as gel formation. The mixture was further shaken at 37 °C overnight. Then, all mixtures were centrifuged to separate the hybrid gels and supernatants. The collected hybrid gels were washed with water by centrifugation at 3000 rpm and repeated three times until no more DNA was desorbed. Characterization of Adsorbed DNA in Hybrid Gels. The DNA concentration in supernatants was measured by a UV−vis spectrometer, Lamda 35 (Perkin-Elmer Japan) at a wavelength region of 190 to 450 nm. DNA absorbs UV-light at λmax 260 nm. The remained DNA in supernatant was calculated based on the initial amount of DNA. The collected hybrid gels were freeze-dried and weighed to calculate % yield based on the initial weight of imogolite and DNA. FT-IR measurement of the hybrid gels was carried out as KBr pellet by a Spectrum 100 spectrometer (PerkinElmer Japan). The amount of bound DNA on imogolite was estimated by the following experiment. The known-weight gels were dissolved in concentrated HCl for 1 week, neutralized with saturated NaHCO3, integrated volume, and measured DNA amount by UV−vis spectroscopy. The DNA content (wt %) in the hybrid gel was calculated by dividing the DNA amount in HCl solution by the initial weight of DNA. The measurement for each sample was repeated three times for confirmation. Water Content and Swelling Degree. Water content in the hybrid gel was estimated by drying the collected gel and was calculating by subtracting the weight of dried gel from the initial weight of wet gel. A swelling experiment was done by equilibrating the freezedried gel in water for 24 h. Then, swelling degrees (Q) were estimated

Figure 1. Amount of DNA remained in supernatant (blue plots) and yield of the obtained hybrid gels (green plots) as a function of DNA feed content.

Figure 2. IR spectra of imogolite, DNA, and the hybrid gels varied DNA/imogolite feed ratios. 277


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3. RESULTS AND DISCUSSION 3.1. Preparation of DNA/Imogolite Hybrid Gels. The residual in supernatant represents the fraction of unbound DNA. The fraction of remained DNA in supernatants with varying DNA/imogolite feed ratio is shown in Figure 1 (blue plots). When DNA feed content was lower than 50 wt %, DNA molecules were completely immobilized on imogolite to form the hybrid gels. In contrast, when DNA feed content was higher than 50 wt %, the amount of unbound DNA started to increase sharply. The yield of the obtained imogolite/DNA hybrid gels is shown in Figure 1 (green plots). The amount of the obtained hybrid gels increased as increasing DNA feed content. The maximum amount of the hybrid gel was obtained at DNA to imogolite feed ratio 63/37 (gel 6). Above this point, the amount of obtained gels decreased because the amount of imogolite decreased. Figure 2 shows FT-IR spectra of the hybrid gels with varied DNA/imogolite feed ratio. The characteristic peaks of imogolite were observed at 930 and 960 cm−1 corresponding to the stretching of Al−O−Si groups. The prominent bands at 1090, 1236, and 1700 cm−1 correspond to PO2− and P−O groups in DNA. These results confirmed the existence of imogolite and DNA in the hybrid gels. When DNA feed content increased, there is an increase in the intensity band of P−O and PO2− stretching. The FT-IR spectra provided an average quantitative DNA amount in the hybrid gels. The bound DNA into imogolite in the hybrid gel was estimated as shown in Figure 3a. UV−vis spectra of DNA adsorption for each hybrid gel are shown in Figure 3b. Increase in the DNA feed content led to an increase of DNA content in the hybrid gels. Gel 7 showed the highest DNA content in the hybrid gel (75%), which was made from DNA/imogolite feed ratio of 71/29. DNA/ Imogolite weight ratio at 75/25 could calculate to be 1:1 molar ratio. Above this point, DNA content decreased, which may be caused by electrostatic repulsion generated by the excessive negative charges of DNA hindered the formation of hybrid gels.

Figure 3. (a) DNA content in the hybrid gels and (b) normalized UV−vis absorption spectra showing an increase in DNA content in the hybrid gel against DNA feed content.

Figure 4. Photographs of the hybrid gels 1, 3, 5, and 7; (a) front view and (b) up view and their model structures. DNA/imogolite feed ratio for each gels is shown in Table 1. 278


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Figure 5. (a) Storage, (b) loss moduli, and (c) complex viscosity (G′, G″, and η*, respectively) as a function of frequency for the hybrid gels 1 (black), 3 (red), 5 (blue), and 7 (green). Figure 6. Releasing of DNA from the hybrid gels; (a) varying pH 7−11 (at 37 °C), (b) varying NaCl concentration (at pH 9, 37 °C), and (c) varying temperature (at pH 9).

3.2. Physical Properties of DNA/Imogolite Hybrid Gels. The aggregation occurred when imogolite and DNA solutions were mixed. The initial state of the obtained gels after collection by centrifugation exhibited a difference in physical properties depending on DNA feed content, as shown in Figure 4. Gels 1, 3, 5, and 7 were prepared by varied DNA to imogolite feed ratio as shown in Table 1. The frequency dependence of dynamic viscoelasticity of the hybrid gels is shown in Figure 5. Gels 1 and 3 were gel-like and high viscosity, which did not fall down even upturn the vials. Lower modulus in gel 1 than gel 3 is probably due to the lower density of cross-linking point in hybrid network. Gels 5 and 7 were fluid-like and lower viscosity than gels 1 and 3. This is because of the incompleteness of network structure. Figure 4b shows photographs of the gel surface (up side view), and the

smoothness of gels was ordered from roughness to smoothness; gels 3 > 1 > 5 > 7. The different amount of DNA in the hybrid gels influenced the gel viscosity and physical properties. The aluminol groups of imogolite can be both positively and negatively charged depending on the pH in the aqueous environment. In the cases of gels 1 and 3, which have higher imogolite content, the entanglement of imogolite themselves and imogolite with DNA molecules like a spontaneous aggregation might lead to highly viscous gels. In gels 5 and 7, which have higher DNA content, coverage of imogolite−DNA network surface by DNA molecules induced repulsion between small aggregations and led to low viscous liquid-like gels. 279


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temperature. Upon temperature decrease, thermal fluctuations continuously reduced; therefore, DNA motion decreased, leading to lower amount of DNA release. Under severe conditions such as strong alkaline solutions or NaCl solutions, the hybridization with imogolite provided enough protection to DNA molecules. This is probably due to the very strong chemical interactions such as electrostatic interaction between aluminol groups of imogolite and phosphate groups of DNA. The other reason is that an entanglement of DNA molecules and imogolite nanofibers provided physical protection from the added salt or alkaline solution. 3.3. Molecular Aggregation States of DNA/Imogolite Hybrid Gels. The morphology of freeze-dried imogolite and the hybrid gel was observed by TEM, as shown in Figure 7. Figure 7a shows imogolite bundles forming continuous network structure. Interestingly, imogolite presented different morphology in the hybrid gel (Figure 7b). TEM image of the hybrid gel showed two characteristic of imogolite bundles. One is the fibrous morphology of imogolite bundles and another is an entanglement between imogolite fibers. First, a good dispersion of imogolite fibers is suggested so that DNA molecules wrap around a single imogolite nanofiber; this leads to a better fiber dispersion than that of pure imogolite. When DNA molecules bridged between imogolite fibers, imogolite fibers were curled and entangled forming web-like structures. Good affinity of AlOH groups of imogolite and phosphate functional groups of DNA is a driving force for DNA adsorption on imogolite. There was no significant difference in morphology for all hybrid gels. WAXD measurement of freeze-dried imogolite, DNA, and the hybrid gels was carried out by SR-powder diffractometer at BL02B2 in SPring-8. Figure 8 shows WAXD profiles of freezedried hybrid hydrogels. The WAXD pattern of DNA showed one broad range at d spacing 5−30 nm. Imogolite exhibited peaks at d spacing 1.53, 0.92, and 0.66 nm assigned to the diffraction from individual tube16 and a peak at 2.2 nm associated with center-tocenter tube distance of imogolite bundles.17 Two hybrid gels showed the WAXD pattern similar to imogolite, but there are some differences at q = 2−5 and 11−18 nm−1. The shift of peak at q = 2−5 nm−1 region might be suggestive of distortion of imogolite packing upon binding by DNA. This result agreed with observation on TEM images. The higher intensity of the peak at q = 11−18 nm−1 indicated the difference of DNA content in the hybrid gels. The gel 7 with DNA feed content of 70 wt % clearly showed a higher intensity compared with other patterns.

Figure 7. TEM images of (a) imogolite and (b) the hybrid gel.

Figure 8. WAXD profiles of freeze-dried imogolite, DNA, and the hybrid gels.

Water content of the hybrid gels was higher than 99%. However, once the hybrid gels were dried, their swelling degree was not so high. The swelling degrees (Q) for gels 1, 3, 5, and 7 with increasing DNA feed ratio were 14.7, 35.8, 15.2, and 12.3, respectively. The dried hybrid gels could adsorb water only 10 to 40 times by weight. The drying process led to the strong aggregation, which made it difficult to redisperse the imogolite nanofibers. Gel 3 showed the highest swelling degree probably due to the presence of effective cross-linking points. The charge of imogolite surface is dependent on pH and has point of zero charge (pzc) about pH 6−8.2 Theoretically, DNA is expected to release from imogolite at pH higher than pzc because AlOH2+ groups are deprotonated. From the experimental results, DNA was not released from the hybrid gel at pH 4 because the surface of imogolite was positively charged. Figure 6a shows DNA release in the buffer with pH of 7, 7.5, 8, 9, 10, and 11 monitored for 24 h. DNA release amount increased as pH increased. However, the DNA release amount was only 12% even at pH 11. This suggests that DNA exhibits a stronger interaction with imogolite. Figure 6b shows that the DNA release amount decreased as NaCl concentration increased to 0.1 M. The results are opposed to the phenomena of salt-induced desorption of DNA complexes with polycations.15 Probably, the added salt would dissociate surface charge around the hybrid gel instead of attacking the gel. These results indicate that the adsorption of DNA on imogolite protected DNA from attack of salt ions. Figure 6c shows that DNA release amount decreased at lower

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CONCLUSIONS The imogolite/DNA hybrid gels were successfully prepared by a simple mixing method, utilizing aluminol functionality of imogolite to bind to phosphate ester of DNA. Varying imogolite and DNA feed ratio could control morphology and physical properties of the hybrid gels. Additionally, DNA could be protected even under severe conditions by the hybridization with imogolite. The structures of the imogolite/DNA hybrid gels were investigated using methods such as WAXD and TEM. In a dried state, TEM images of the hybrid gels showed fibrous imogolite morphology, which differed from a continuous network structure in pure imogolite. Also, WAXD results were consistent with TEM observation. In the present Article, we have demonstrated the specific interaction between imogolite and DNA for the first time. The results obtained here are important to be the stage for further works to investigate the detailed properties such as rheology and to develop the imogolite/DNA hybrid gels for various biomaterial applications. 280


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +81-92-802-2518. Tel: +81-92-802-2517.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support of a Grant-inAid for Scientific Research (No. 19205031) and a Grant-in-Aid for the Global COE Program, “Science for Future Molecular System” from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. The synchrotron radiation experiments were performed at BL02B2 in SPring-8 with the approval of the JASRI (Proposal No. 2010A1454).

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DNA Hybrid Hydrogels

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