5′-Guanosine Monophosphate Mediated Biocompatible Porous

Article pubs.acs.org/JPCB

5′-Guanosine Monophosphate Mediated Biocompatible Porous Hydrogel of β‑FeOOHViscoelastic Behavior, Loading, and Release Capabilities of Freeze-Dried Gel Anil Kumar* and Sudhir Kumar Gupta Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India S Supporting Information *

ABSTRACT: The present manuscript reports the characterization, optimization of rheological properties, and loading and release capabilities of 5′-GMP mediated β-FeOOH hydrogel. Circular dichroism (CD) analysis indicates it to contain mainly the left-handed helix similar to that of Z-DNA. The highest viscosity (>300 cP) corresponds to the sample containing 2.5 × 10−3 mol dm−3 of 5′-GMP (SP2H). Field emission scanning electron microscope (FESEM) and transmission electron microscope (TEM) studies indicate the freeze-dried (FD) SP2H to be porous in nature, which is also supported by its high Brunauer−Emmett− Teller (BET) surface area of 226 m2/g as compared to that of SP3H (75 m2/g). Selected area electron diffraction (SAED) analysis and Raman spectroscopy show it to contain β-FeOOH phase. The FD SP2H exhibits the high swelling ratio (326%) and loading capacity for methylene blue (MB) dye. It displays a controlled and efficient release (>90%) for optimized [MB] (2.5 × 10−4 mol dm−3) in 48 h. The low toxicity of as synthesized FD SP2H nanostructures against MDA-MB-231 (breast cancer cells) up to 100 μg/mL suggests its biocompatible nature. The high porosity, surface area, % swelling, and loading and release performance of the hydrogel indicate its potential for drug delivery and other biological applications. Since β-FeOOH based nanoparticles have been observed to have low toxicity and biocompatibility,21,22 the biological applications of such materials could further be facilitated by developing magnetic hydrogel from these materials. Hydrogels have been found to have tremendous applications in biology and medicine because of increased functional capabilities like encapsulation and controlled release.23−25 In the literature, we have come across some papers on the formation of hydrogel employing 5′-GMP alone26−29 and one article on Ag+ mediated 5′-GMP30 hydrogel. In all of these reports, a fairly high concentration of 5′-GMP has been used for the formation of hydrogel. Moreover, we did not come across any article demonstrating loading and release capabilities. Recently, we have reported the synthesis of 5′-GMP-mediated porous hydrogel containing β-FeOOH nanostructures displaying superparamagnetic behavior with fairly high magnetization.31 In view of both 5′-GMP and β-FeOOH both being biocompatible and 5′-GMP mediated β-FeOOH hydrogel being superparamagnetic, we have explored the 5′-GMP templated β-FeOOH hydrogel for investigating its viscoelastic properties and loading and release capabilities in the context of their possible biomedical applications. The freeze-dried (FD)

1. INTRODUCTION In recent years, nanosized iron oxides (α-Fe2O3, β-Fe2O3, γFe2O3, Fe3O4) and iron oxyhydroxides (α-FeOOH, β-FeOOH, γ-FeOOH) have been investigated extensively for their multidisciplinary applications in the areas of environment,1 catalysis,2 data storage,3 magnetic devices,4 and biomedicine5,6 because of their characteristic structural features, magnetic properties, and biocompatibility.7,8 Among iron oxyhydroxide polymorphs, β-FeOOH has drawn considerable attention for its specific tunnel like structure with favorable adsorption behavior.9,10 These features could further be enhanced by synthesizing them in different shapes and morphologies. The colloidal approach provides a convenient method for the manipulation of the morphology of nanostructures by controlling their nucleation and growth.11,12 The surface modification may furnish them the additional characteristic features due to functionalization like increased solubility, specific sites to bind to the target, and enhanced optical and magnetic properties.13−16 In this regard, it will be interesting to modify the surface of the core inorganic nanostructures with biomolecules so as to enhance their physicochemical properties and also provide them the biocompatibility.17 Lately, a few reports have appeared to produce biocompatible iron oxide nanostructures for their biomedical applications such as drug delivery and as contrast agents for magnetic resonance imaging.18−20 © 2014 American Chemical Society

Received: April 19, 2014 Revised: August 5, 2014 Published: August 13, 2014 10543

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sized by adding 0.49 g of FeCl3 in 100 mL (3.0 × 10−2 mol dm−3) of aqueous solution containing varied amounts of 5′-GMP (1.0 × 10−3 to 5.0 × 10−3 mol dm−3) following the previously developed method.31 The solutions were refluxed in an oil bath for 6 h at 100 °C under continuous stirring and were allowed to cool up to the room temperature. Thereafter, the resulting solutions were dialyzed (12−15 h) for the removal of excess Fe3+ and 5′-GMP. The colloidal samples thus obtained were stored at room temperature, and within a few weeks, the colloidal samples underwent spontaneous transformation to hydrogel. The gelified samples were used for the rheological measurements. The hydrogel samples were freeze-dried and then lyophilized at −55 °C and used for the swelling and MB release experiments. In a control experiment, β-iron oxyhydroxide sample was also synthesized in the absence of 5′-GMP under similar experimental conditions (SB). Herein, we obtained a turbid suspension which did not result in any gelification. The resulting turbid suspension was dried in a rotatory evaporator at 30 °C and then dried at 30− 35 °C in a vacuum oven to obtain it in the powder form. The βiron oxyhydroxide samples containing varied amounts of 5′GMP (mol dm−3) have been labeled as SB (0.000), SP1 (0.001), SP2 (0.0025), and SP3 (0.005) and the respective aged/hydrogel samples as SP1H, SP2H, and SP3H, respectively. The gelation for SP1H began in 1 month, and in the case of SP2H and SP3H, it takes about 3 weeks. Therefore, in the present work, mainly SP2H and SP3H have been investigated. 2.4. Preparation of Samples for CD Spectroscopy, Rheology, Raman Spectroscopy, Surface Area Analysis, Zetasizer, FESEM, TEM, and Swelling Experiments. The CD spectra of the samples were recorded as such without any dilution. The concentrations of 5′-GMP and Fe3+ taken in blank experiments were the same as was determined in SP2. The ellipticities of the as synthesized samples have been recorded by using a cuvette with a 1 mm path length and subtracting the baseline using water as the solvent in the range 180−350 nm. In order to measure the rheological properties of the samples, the hydrogel portion of the sample was used as such. The FD hydrogel samples of SP2H and SP3H were used as such for recording the Raman spectra, surface area, and loading and release measurements. The zeta potentials of SP2H and SP3H were measured by taking the hydrogel samples directly in the capillary cuvette fitted with Cu electrodes. For FESEM analysis, sample preparation was carried out by applying the sample on a conducting substrate using both sided tape. The surface of these samples was coated with gold in order to make them conducting, and their FESEM images were then recorded by applying an acceleration voltage of 20 kV. For TEM analysis, a small amount of FD hydrogel sample was sonicated in ethanol for 10 min. A drop of this solution was applied on the carbon coated copper grid and was dried at room temperature in the dark. For the swelling study, 0.1 g portions of as synthesized FD samples of SP2H and SP3H were taken. To these samples, about 5 mL of phosphate buffer saline (PBS) of pH 7.2 was added at room temperature. In these experiments, swelling was monitored by recording its weight at various time intervals. The digital images of the hydrogel samples, SP2H and SP3H, in inverted position were captured after removing the excess water by centrifugation. 2.5. Swelling Experiments. % Swelling ratio was worked out by using the following equation:

hydrogel has been used for analyzing its loading and release capabilities by employing methylene blue (MB) dye as a probe molecule. Biocompatibility of the hydrogel has been examined by performing cytotoxicity tests against MDA-MB-231 (breast cancer cells). The supramolecular interactions of β-FeOOH with the functionalities of 5′-GMP control its porosity, viscoelastic properties, and loading and release capacities, and to the best of our knowledge, this is the first report investigating these properties of β-FeOOH hydrogel.

2. EXPERIMENTAL SECTION 2.1. Reagents. Iron(III) chloride (Merck), 5′-guanosine monophosphate disodium salt (SRL), methylene blue (Thomas Baker), HClO4 (Qualigens), NaOH, HCl (s-d Fine chemicals), phosphate buffer saline (PBS) buffer pH 7.2 (HIMEDIA), carbon coated copper grids for the transmission electron microscope (TEM) (Polaron), seamless dialysis tubing/dialysis tubing closures (Sigma), and MDA-MB-231 breast cancer cells (ATCC) were used. All the reagents used were of analytical grade and used without any further purification. Different solutions were prepared freshly in Millipore water (Bedford, MA, USA). 2.2. Characterization Techniques. Raman spectra of the samples were recorded on an inVia Raman spectrometer (serial no. 021R88) equipped with a 514 nm Ar ion laser and a confocal microscope. The viscosity and rheological measurements of the hydrogel samples were carried out on an MCR 102 rheometer from Anton Paar using a cone−plate geometry with a diameter of 40 mm and a 1° cone angle. UV−visible (200−800 nm) spectra were recorded on a Shimadzu 2100S spectrophotometer. Circular dichroism (CD) spectra of the various samples were recorded on a Chirascan spectropolarimeter procured from Applied Photophysics, U.K. The surface morphology and the elemental analysis of the nanostructures were performed on a FEI-QUANTA 200F field emission scanning electron microscope (FESEM) coupled with an energy dispersive X-ray analysis (EDAX) accessory by applying a 20 kV acceleration voltage. The electron micrographs and selected area electron diffraction (SAED) measurements of the samples were carried out on a FEI, TECNAI G2 20 S-TWIN transmission electron microscope operating at an accelerating voltage of 200 kV equipped with a CCD camera having a point resolution of 0.24 nm. The stability of the hydrogel sample containing 5′-GMP mediated β-FeOOH nanostructures was analyzed on a Malvern Instruments Zetasizer ZS90 using a 632 nm He−Ne laser as the light source. The surface area analysis of the FD gel sample was carried out on a NOVA 2200e High Speed Automated Surface Area Analyzer using Nova Win software. The reaction was carried out in a round silicon oil bath equipped with a PID controller, operating at 500 W, 230 V, and 50 Hz procured from Medica Instruments MFG., Co. The water was removed from the colloidal samples on a Buchi Rotavapor R-114. These samples were dried in a vacuum oven, model no. LVP 500, from Labtech, Daihan Labtech Co. Ltd. The hydrogel samples were freeze-dried and then lyophilized in a freeze drier (Lyophilyzer) equipped with a Pirani gauge and a digital temperature controller from Biosync Teknology, Delhi. For the MB loading and release experiments, the FD hydrogel samples were shaken using a water bath shaker NSW-133, NSW India, at room temperature. The pH of the solutions was measured on a pH510 pH meter from Eutech instruments. 2.3. Synthesis of 5′-GMP-Templated β-Iron Oxyhydroxide Hydrogel. 5′-Guanosine monophosphate disodium salt (5′-GMP) mediated β-iron oxyhydroxide hydrogel was synthe10544

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Figure 1. (a) Structure of 5′-GMP. (b) Digital images of the vials containing SB and SP2H and SP3H hydrogels before (vertical) and after (inverted) removing water.

% Swelling =

(Wt − W0) × 100 W0

where W0 stands for the weight of the sample before being soaked in the PBS buffer and Wt stands for the weight of the sample after being soaked in PBS buffer for time, t. The percentage swelling refers to the % increase in the weight of the FD hydrogel due to the uptake of water from the buffer. 2.6. Dye Loading and Release Efficiency. In order to perform the loading and release experiments and their optimization, the FD hydrogel samples (0.5−10.0 mg/mL) were shaken with varied amounts of MB (5 × 10−6 to 5 × 10−4 mol dm−3) keeping the volume of solution at 10 mL at room temperature for 4 h. The release of dye was then monitored spectrophotometrically in PBS buffer at a pH of 7.2. These results were reproducible within ±5%. 2.7. Cytotoxicity Assay. For the cell viability test, MDAMB-231 breast cancer cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 accordingly to the supplier. The cell viability was measured in triplicate for each sample after incubating them for 48 h according to the supplier (Promega, G7571) with a Tecan F200 instrument.

3. RESULTS AND DISCUSSION Synthesis and characterization of 5′-GMP (Figure 1a) mediated hydrogel of β-FeOOH has been reported in our previous paper31 and will not be described fully here. The digital images of the SP2H and SP3H along with SB are shown in Figure 1b. Both of the hydrogel samples were negatively charged and had an average zeta potential of ∼ −40 mV (Figure S1, Supporting Information). The formation of hydrogel has earlier been reported to take place in the process of self-assembly involving supramolecular interactions among different moieties of 5′-GMP, viz., pyrimidine/imidazole, sugar, PO32−, and β-FeOOH. This aspect has been further analyzed using CD spectroscopy. 3.1. Circular Dichroism Spectroscopic Analysis. The CD spectra of the 5′-GMP, Fe3+ and 5′-GMP mixture, SP2, and SP2H are shown in Figure 2. The CD spectrum of 5′-GMP was found to be very similar to that observed earlier for the lowest concentration of 5′-GMP employed by them.29 In the Fe3+ and 5′-GMP mixture, all peaks due to 5′-GMP are now red-shifted with fairly high negative ellipticity. The negative peaks are observed at (−) 211, (−) 240, and (−) 300 (Figure 2a). Similar changes in ellipticity have earlier been observed upon binding of alkali metal ions with 5′-GMP.29

Figure 2. CD spectra of (a) 5′-GMP and Fe3+−5′-GMP mixture and (b) SP2 and SP2H at 22 °C.

The 5′-GMP mediated β-FeOOH nanoparticles (SP2) show a blue-shifted CD spectrum containing bands at (−) 203, (−) 220, (−) 240, and (−) 260 nm with reduced ellipticity (Figure 2b) as compared to the Fe3+−5′-GMP mixture. It may be understood by the presence of different associated structures of 5′-GMP. In SP2H, all of these peaks exhibit further high negative ellipticity, indicating an increase in the left-handed helix of 5′GMP in the process of gelation. A careful examination of this spectrum shows an unusual strong negative band in the range 200−220 nm and a small negative band at ∼300 nm. These features are similar to Z-DNA species with left-handed helix formation.32 Thus, the CD spectrum due to SP2H appears to contain a mixture of conformations existing simultaneously in different proportions. All of the observed changes in CD spectra can be understood by the presence of different associated structures of 5′-GMP in the self-assembly. 10545

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Figure 3. (a) Viscosity of the various samples at a fixed shear rate of 100 s−1. (b) Viscosity of SP2 upon aging at a fixed shear rate of 100 s−1. (c) Viscosity profile for the shear sweep of the hydrogel samples.

periodic oscillation of strain at a fixed angular frequency (10 rad/ s) (Figure 4a). These data need to be understood in terms of the following correlations of stress (σ) and strain (γ) with G′ and G″:

The samples SP2H and SP3H employed for the analysis of viscoelastic behavior were optimized with respect to the time of gelation, morphology, and magnetic properties.31 3.2. Rheological Studies. In the process of gelation, one of the important criteria to be followed is the change in viscosity as a function of the concentration of matrix (5′-GMP) and time. This matrix having multifunctional sites could be involved in the supramolecular interactions by coordination to inorganic nanoparticles as well as involving other 5′-GMP molecules. The variation of viscosity as a function of [5′-GMP] follows the trend SP1H < SP2H > SP3H, showing that the viscosity initially increases with an increase in [5′-GMP] and then reduces drastically at its higher concentrations (Figure 3a). The highest viscosity corresponds to SP2H containing 2.5 × 10−3 mol dm−3 of 5′-GMP aged for 21 days. To further optimize the time of gelation for SP2H, the viscosity of SP2 was monitored with days of aging (Figure 3b). It reveals that the viscosity increases regularly with time up to 21 days and did not show any appreciable change thereafter. Therefore, all the viscoelastic experiments were designed with the sample aged for 3 weeks unless mentioned otherwise. Interestingly, the optimum concentration of 5′-GMP found for the hydrogel formation in the present system is more than an order of magnitude lower as compared to the previous reports30 on 5′-GMP based hydrogel. In order to further investigate the stability of these hydrogels, the viscosities of SP2H and SP3H were followed as a function of shear rate (Figure 3c). In both cases, the shear thinning is observed to increase with shear rate, which suggests them to be the hydrogel. The higher value of the viscosity for SP2H indicates it to be more stable as compared to SP3H. An analysis of the viscoelastic nature of hydrogel was further carried out by recording their dynamic mechanical behavior. In these experiments, the viscoelastic properties of SP2H and SP3H hydrogel samples were monitored by recording the response of the elastic modulus (G′) and viscous modulus (G″) under

σ = G′(ω)γ /cos(δ)

Figure 4. Viscoelastic behavior of gel: (a) Amplitude sweep curves for SP2H and SP3H samples at an angular frequency fixed at 10 rad s−1. (b) Frequency sweep curve for SP2H and SP3H samples at a strain fixed at 0.5% recorded at 20 °C. 10546

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where G′ is the elastic modulus which denotes the energy stored, when stress and strain are in phase, and δ is the phase angle, and σ = G″(ω)γ /sin(δ)

where G″ describes the viscous modulus and denotes the energy dissipated as heat, when stress and strain are out of phase. An examination of curves initially depicts a linear variation of G′ and G″ with strain for both samples (Figure 4a), as has been observed earlier for hydrogels.33 G′ and G″ cross each other at a percentage strain of 15.4 ± 0.1 and 11.4 ± 1.9, indicating the yield strain(s) for SP2H and SP3H, respectively. It suggests that SP2H can withstand higher strain as compared to SP3H before flow. The lower values of G″ and G′ of SP3H with respect to those of SP2H suggest it to have relatively poor viscoelastic properties. Increased 5′-GMP in SP3H possibly causes the reduction in interaction between 5′-GMP present in the shell with the core β-FeOOH of other building blocks. The difference in elastic modulus can be attributed to the difference in stoichiometry of 5′-GMP and β-FeOOH in the hydrogel samples (SP2H and SP3H). SP3H having a higher concentration of 5′GMP with respect to the concentration of β-FeOOH would thus have a lower degree of cross-linking between them. This explains the lower mechanical properties of SP3H. The value of strain for SP2H and SP3H was optimized from the linear viscosity range observed in the amplitude sweep plot (Figure 4a). From this curve, it is apparent that G′ and G″ remain well separated and are constant up to 4% for both samples, reflecting the stability of the gel in this range. Therefore, for further oscillatory (frequency sweep) measurements, the % strain was fixed at 0.5. The mechanical nature of the gel was examined by recording the mechanical spectrum of gel over a frequency range of 10−1 to 102 rad/s at a fixed strain of 0.5% (Figure 4b). From this experiment, it can be noted that G′ is always >G″, indicating the solid-like viscoelastic networking in hydrogel.30 A close examination of curves for G′ and G″, however, reveals that the observed spectrum depicts a slight frequency dependence in lower and higher angular frequency range. At lower frequency, the ratio of G′ to G″ is more than an order of magnitude higher. It may be noted that the prolonged aging of SP2H beyond 3 weeks to 3 months results in the enhancement of its viscoelastic parameters G′ and G″ (Figure S2a and b, Supporting Information). This hydrogel remains quite stable up to 3 months, as was revealed by an increase in its yield strain from 15.4 ± 0.1 (observed for the 3 weeks aged sample) to 16.3 ± 0.1%. The increase in yield strain was associated with an increase in elastic modulus from ∼250 to 1100 Pa, as was observed from their frequency sweep plots (Figures 4b and S2b, Supporting Information). Very similar viscoelastic properties have been reported for Ag−5′-GMP hydrogel.30 These changes in the properties are understood by the increased interactions and reorganization of the various functionalities of building blocks containing 5′-GMP and β-FeOOH in the process of selfassembly upon prolonged aging. In order to investigate the loading and release capabilities of as synthesized SP2H gel, the gel was lyophilized at −55 °C. The freeze-dried gel has been characterized earlier by XPS and IR spectroscopy.31 In the present work, it has been further characterized by employing Raman, FESEM, TEM, and Brunauer−Emmett−Teller (BET) surface area analyzer. 3.3. Raman Spectra. The presence of the β-FeOOH phase in FD SP2H was verified by recording its Raman spectrum (Figure 5). This spectrum clearly shows the presence of eight

Figure 5. Raman spectra of FD SP2H.

prominent peaks at 249, 316, 381, 681, 721, 1066, 1335, and 1591 cm−1. The peaks (cm−1) at 249, 681, and 1066 match those of pure 5′-GMP, and all the other peaks correspond to the βFeOOH phase, as was confirmed by recording the blank sample of pure 5′-GMP and SB (Figure S3, Supporting Information) and with the literature data.34−36 To further analyze the nature of interactions between 5′-GMP and β-FeOOH in SP3H, the Raman spectrum of SP3H was also recorded (Figure S4, Supporting Information). This spectrum shows several additional peaks due to 5′-GMP and peaks due to β-FeOOH become less intense and poorly resolved. These changes are understood to have arisen due to increased coverage of core β-FeOOH by 5′-GMP shell as compared to the Raman spectrum of SP2H. Increased interactions among 5′-GMP molecules in the shells of different building blocks of SP3H and decreased interactions between the 5′-GMP shell and core βFeOOH of the neighboring building blocks might have contributed to the observed change (Scheme 1). This aspect has been further explored by IR spectroscopy. 3.4. FESEM Analysis. The FESEM image of the FD SP2H is shown in Figure 6a. This shows it to contain a porous structure. EDAX analysis of this image at the marked (+) location shows the presence of Fe, O, and Cl contributed by the inorganic nanostructure and the elements pertaining to the matrix, 5′-GMP (C, N, O, and P) (Figure 6b and c). The elemental mapping of this sample shows a homogeneous distribution of C, N, O, P, Cl, and Fe all along the nanohybrid (figure not shown). 3.5. TEM Analysis. TEM micrographs of FD SP2H and FD SP3H along with their SAED patterns have been shown in Figure 7. The SAED pattern of SP2H depicts diffused rings masked with some spots. Indexing of the SAED pattern shows different rings corresponding to (103), (112), (114), and (512) planes matching the β-FeOOH phase (JCPDS file 80-1770) with monoclinic structure. The SAED pattern of SP3H exhibited highly diffused rings without any spots, indicating this sample to be highly amorphous. The presence of the β-FeOOH phase in both SP2H and SP3H was observed by Raman analysis (vide inf ra). The TEM image of FD SP2H was further analyzed by recording a 3D view of its TEM image using ImageJ software. Its top and lateral views are presented in Figure S5 (Supporting Information). 3.6. Swelling StudiesAnalysis of Porosity. The porosity of the gel was further examined by determining % 10547

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Scheme 1. Difference in Supramolecular Interactions between SP2H and SP3H: Involving Core β-FeOOH and 5′-GMP of Neighboring Building Blocks (Red in SP2H) and 5′-GMP Molecules of Two Neighboring Building Blocks (Green in SP3H) upon Increasing the Concentration of 5′-GMP

Figure 7. TEM image of FD SP2H (a) and its SAED pattern (b); TEM image of FD SP3H (c) and its SAED pattern (d).

swelling for 5 h in PBS at pH 7.2 in an aqueous medium. From these experiments, SP2H and SP3H demonstrate 326 and 93% swelling, respectively (Figure 8). The reduced swelling in the

Figure 6. FESEM images of FD SP2H (a), EDX analysis of the marked region (b), and EDX spectra of the marked region (c). 10548

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these experiments, loading of MB was carried out by immersing FD gel (2 mg/mL) for an optimized time of 4 h in its solution (2.0 × 10−5 mol dm−3). This exercise was carried out for both samples SP2H and SP3H. From these experiments, it is observed that the loading capacity of SP2H is more than 3-fold higher (71%) compared to that of SP3H (22%). That is very similar to that arrived by swelling experiments (vide ut supra). The amount of SP2H was optimized by designing % loading and % release experiments for different concentrations of SP2H (Figure 9). From these experiments, the best concentration of

Figure 8. % Swelling for various hydrogel samples in PBS buffer pH 7.2 at room temperature.

case of SP3H is demonstrated by its reduced surface area. This finding is also supported by the lower degree of cross-linking in SP3H, resulting in poor viscoelastic behavior. The BET surface area of FD SP2H was determined by degassing it at 100 °C for 3 h under a vacuum followed by adsorption of N2 gas. From the five-point method, the surface area was determined to be 226 m2/g, which is about 3 times higher than that of SP3H (75 m2/g). Thus, BET analysis supports that SP2H consists of a more porous structure as compared to that of SP3H (Figure 7a and c). The increased porosity is also evidenced by the enhanced rheological properties of SP2H, as has been observed earlier for polysaccharide hydrogels.37 3.7. FTIR Spectra. A difference in the interactions between SP2H and SP3H is shown in Scheme 1. This scheme is based on the interactions of 5′-GMP with β-FeOOH as observed by IR spectroscopy (Table S1 and Figure S6, Supporting Information). In SP2H, the main interactions of β-FeOOH were through PO32−, NH2, >CO, and nitrogen of pyrimidine and imidazole rings of 5′-GMP,31 whereas, in the case of SP3H, the vibrational bands due to CN ring skeletal vibrations, CO sugar ring, PO 5′-sugar, and PO stretching completely vanished. The peaks (cm−1) observed earlier in SP2H31 due to N(7)C(8) pyrimidine (1482), imidazole (1402 and 1359), and PO32− (989) shifted to lower energy in SP3H at 1467(w), 1379(w), 1327(w), and 982 (w), respectively. It also resulted in the broadening and masking of FeOFe stretching at 687(br), 631(w), and 471(br). The observed changes in the IR spectrum of SP3H evidently indicate increased interactions between β-FeOOH and 5′-GMP within a building block and decreased cross interactions with the neighboring building blocks (Scheme 1), which result in the masking of the Fe−O−Fe stretching peaks in SP3H. Both IR and Raman spectroscopy suggest that increased intermolecular interactions among different moieties of 5′-GMP involving weak supramolecular bonding is possibly responsible for the broadening of the peaks, leading to the change in morphologies in SP3H. The change in morphology also supports the enhanced viscoelastic properties of SP2H hydrogel as compared to those of SP3H. 3.8. Dye Loading−Release Study. The porous nature of FD gel indicates its utility for possible encapsulation and controlled release applications. In view of the negative charge on the hydrogel with a ζ-potential of −40 mV, the cationic dye MB was employed to examine its loading and release capabilities. In

Figure 9. % Loading and % release for FD SP2H concentration variation against 2 × 10−5 mol dm−3 MB solution.

SP2H was selected to be 2 mg/mL for which the maximum % release for minimum % loading was found to be 49 and 71%, respectively. For the optimized concentration of SP2H, the amount of dye was varied from 5 × 10−6 to 5 × 10−4 mol dm−3, and for each concentration, the loading and release were monitored (Figure 10). The maximum amount of release was obtained for about 2.5

Figure 10. MB concentration variation profile for loading and release of MB against 2 mg/mL SP2H.

× 10−4 mol dm−3 MB. The entire process involving the formation of hydrogel, morphology of the freeze-dried gel, loading of dye, and its release has been presented in Scheme 2. The time dependent release profile of the dye is also shown pictorially in Figure 11. The release increased linearly up to 10 h, and thereafter, it starts taking a plateau value after about 40 h. 3.9. Cell Toxicity Test. The cytotoxicity of the FD SP2H was examined with MDA231 (breast cancer cells) by using the Luciferase activity assay method. 500 cells were plated in 96-well 10549

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Scheme 2. Schematic Presentation of the Formation and Processing of Hydrogel with Dye

Figure 12. Cell viability test for FD SP2H for an incubation period of 48 h.

employed earlier for the formation of 5′-GMP based hydrogels. The FD SP2H has been characterized to contain a β-FeOOH phase with a porous structure. The porosity is also revealed by the fairly high surface area, swelling, and loading capabilities of these hydrogels. The controlled high % release, low toxicity, and biocompatibility of the investigated gel suggest its potential for biomedical applications.

culture plates. The day after seeding, compounds were added at different concentrations to the medium. FD SP2H was added to the cell culture at different concentrations ranging from 5 to 100 μg/mL along with a control experiment designed in its absence. Figure 12 shows the cell viability as a function of the concentration of FD SP2H. It shows the nontoxic behavior of as synthesized FD SP2H up to 100 μg/mL, which suggests its biocompatible nature.

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ASSOCIATED CONTENT

S Supporting Information *

Zeta potential distribution plot for SP2H and SP3H (Figure S1), viscoelastic behavior of 3 months aged SP2H: (a) amplitude sweep curves and (b) frequency sweep curves (Figure S2), Raman spectra of pure 5′-GMP (a) and SB (b) (Figure S3), Raman spectrum of SP3H and pure 5′-GMP altogether (Figure S4), 3D view of the TEM image of the FD SP2H: (a) top view and (b) lateral view (Figure S5), FTIR spectrum of SP3H (Figure S6), and FTIR data of 5′-GMP, β-FeOOH, FD SP2H, and FD SP3H. This material is available free of charge via the Internet at http://pubs.acs.org.

4. CONCLUSIONS The present manuscript reports the optimized viscoelastic properties of 5′-GMP mediated porous superparamagnetic βFeOOH hydrogels. CD analysis indicates the hydrogel to contain helical structures similar to that of Z-DNA. In hydrogel (SP2H), the viscosity is improved by more than 2 orders of magnitude as compared to that of its colloidal solution (SP2). Gelation is observed to take place for both higher (5.0 × 10−3 mol dm−3) and lower (1.0 × 10−3 mol dm−3) 5′-GMP, but SP2H was found to be the most appropriate in regard to the porosity and viscoelastic properties of hydrogel. The concentration of 5′-GMP in SP2H is more than an order of magnitude lower as compared to those

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Phone: +911332-285799. Fax: +91-1332-273560.

Figure 11. Time variation for the release profile of MB up to 48 h for 2 × 10−3 g mL−1 SP2H against 2.5 × 10−4 mol dm−3 MB at physiological pH in PBS buffer (a); the corresponding UV spectra up to 6 h (b). 10550

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of CSIR, New Delhi (grant no. 01(2758/13/ EMR-II)), to undertake this work is gratefully acknowledged. S.K.G. is thankful to MHRD, New Delhi, for the award of SRF. Thanks are also due to the Head IIC, IITR, Roorkee, for providing the facilities of FESEM and TEM. Thanks are also due to Dr. Flavio Rizzolio and Dr. Vinit Kumar, National Cancer Institute, Aviano, Italy, for performing the cell viability test.

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dx.doi.org/10.1021/jp5038427 | J. Phys. Chem. B 2014, 118, 10543−10551