Controlled Magnetic Nanofiber Hydrogels by Clustering Ferritin

Oct 11, 2008 - Min Kyoon Shin, Sun I. Kim and Seon Jeong Kim*. Center for Bio-Artificial ... Dong-Pyo Jang, Young-Bo Kim and Zang-Hee Cho. Neuroscienc...
1 downloads 0 Views 1MB Size
Download PDF
Langmuir 2008, 24, 12107-12111

12107

Controlled Magnetic Nanofiber Hydrogels by Clustering Ferritin Min Kyoon Shin, Sun I. Kim, and Seon Jeong Kim* Center for Bio-Artificial Muscle and Department of Biomedical Engineering, Hanyang UniVersity, Seoul 133-791, Korea

Sang Yoon Park, Young Hoon Hyun, and YoungPak Lee q-Psi & Department of Physics, Hanyang UniVersity, Seoul 133-791, Korea

Kyung Eun Lee and Sung-Sik Han School of Life Sciences & Biotechnology, Korea UniVersity, Seoul 136-713, Korea

Dong-Pyo Jang, Young-Bo Kim, and Zang-Hee Cho Neuroscience Research Institute, Gachon UniVersity of Medicine and Science, Incheon 405-760, Korea

Insuk So Center for Bio-Artificial Muscle and Department of Physiology, Seoul National UniVersity, Seoul 110-744, Korea

Geoffrey M. Spinks ARC Center of Excellence in Electromaterials Science and Intelligent Polymer Research Institute, UniVersity of Wollongong, Wollongong NSW 2522, Australia ReceiVed July 8, 2008. ReVised Manuscript ReceiVed August 18, 2008 We have fabricated biocompatible nanofiber hydrogels with diverse sizes of ferritin clusters according to the mixing temperature of solutions employing electrospinning. Poly(vinyl alcohol) (PVA) was used as a polymeric matrix for fabricating nanocomposites. By thermal means we controlled the interaction between the host PVA hydrogel and the protein shell on ferritin bionanoparticles to vary the size and concentration of ferritin clusters. The clustering of ferritin was based on the partial unfolding of a protein shell of ferritin. By studying the magnetic properties of the PVA/ferritin nanofibers according to the mixing temperature of the PVA/ferritin solutions, we confirmed that the clustering process of the ferritin was related to changes in the superparamagnetic properties and magnetic resonance imaging (MRI) contrast of the PVA/ferritin nanofibers. PVA/ferritin nanofiber hydrogels with diverse spatial distributions of ferritin nanoparticles are applicable as MRI-based noninvasive detectable cell culture scaffolds and as artificial muscles because of their improved superparamagnetic properties.

Introduction Synthetic hydrogels have been developed for a number of in vivo applications. Drug encapsulants,1 stent coatings,2 tissue scaffolds,3 and even artificial muscles4 have been extensively researched and developed using a wide variety of hydrogel materials. Many of these applications would benefit from noninvasive monitoring of the hydrogel implant using commonplace body-scanning technology such as magnetic resonance imaging (MRI). The utilization of magnetic nanoparticles in this direction can provide contrast for MRI imaging to the hydrogel that requires the assessment of their function in real time and in vivo. Superparamagnetic iron oxide (SPIO) nanoparticles have been commonly used in molecular imaging as MRI contrast agents because of their localized shortening of spin-spin (T2) proton * Corresponding author. Tel: 82-2-2220-2321. Fax: 82-2-2291-2320. E-mail [email protected]. (1) Peppas, N. A.; Klier, J. J. Controlled Release 1991, 16, 203. (2) West, J. L.; Hubbell, J. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13188. (3) Lee, K. Y.; Mooney, D. J. Chem. ReV. 2001, 101, 1869. (4) Osada, Y.; Okuzaki, H.; Hori, H. Nature 1992, 355, 242.

relaxation times.5-7 Specifically, the control of the spatial distribution of SPIO nanoparticles provides the optimization for detailed imaging applications because T2 relaxation times can be modified according to the size of nanoparticle aggregates.8-11 Although T2 contrast enhancement from SPIO nanoparticle aggregation is a well-established phenomenon, all examples have used inorganic SPIO nanoparticles with surface modification, and any direct method to control SPIO nanoparticle aggregation in a hydrogel has not been reported. (5) Sosnovik, D. E.; Nahrendorf, M.; Weissleder, R. Circulation 2007, 115, 2076. (6) Wu, E. X.; Tang, H. Y.; Jensen, J. H. NMR Biomed. 2004, 17, 478. (7) Duguet, E.; Vasseur, S.; Mornet, S.; Devoisselle, J. M. Nanomedicine 2006, 1, 157. (8) Tsourkas, A.; Hofstetter, O.; Hofstetter, H.; Weissleder, R.; Josephson, L. Angew. Chem., Int. Ed. 2004, 43, 2395. (9) Larsen, B. A.; Haag, M. A. B.; Stowell, M. H.; Walther, D. C.; Pisano, A. P.; Stoldt, C. R. Proc. SPIE 2007, 652519. (10) Moffat, B. A.; Reddy, G. R.; McConville, P.; Hall, D. E.; Chenevert, T. L.; Kopelman, R. R.; Philbert, M.; Weissleder, R.; Rehemtulla, A.; Ross, B. D. Mol. Imaging 2003, 2, 324. (11) Berret, J. F.; Schonbeck, N.; Gazeau, F.; El Kharrat, D.; Sandre, O.; Vacher, A.; Airiau, M. J. Am. Chem. Soc. 2006, 128, 1755.

10.1021/la802155a CCC: $40.75  2008 American Chemical Society Published on Web 10/11/2008

12108 Langmuir, Vol. 24, No. 21, 2008

Ferritin bionanoparticles are good candidates for controlling the T2 relaxation time because they contain a superparamagnetic ferrihydrite (5Fe2O3 · 9H2O) crystal12 that accelerates the transverse nuclear magnetic resonance relaxation of water. The distribution of ferritin in the liver and in the brain has been studied using MRI.13,14 The transverse relaxation rate induced by ferritin was different according to the kind of tissue containing ferritin.15 The phenomenon is related to the clustering of ferritin, and the ferritin clusters make body organs easily visible in MRI. Ferritin is also a uniform magnetic nanoparticle consisting of a protein shell with dimensions of ∼2 nm and an iron core with a diameter of about 10 nm.16 This unique structural property of ferritin makes it possible for it to play a significant role as a biocontrast agent at room temperature. In addition, unlike inorganic magnetic nanoparticles that are unlikely to be biocompatible over long time periods (polymer coatings on metal nanoparticles are biodegradable17,18 and thus synthetic magnetic nanoparticles are not suitable for implanting materials), ferritin that naturally occurs in the body does not pose toxicity risks. In this work, we produced nanofiber hydrogels containing different sizes of ferritin clusters and demonstrated both in vitro and in vivo that the ferritin clusters improve the magnetic properties of the nanofiber hydrogel.

Experimental Section Materials. Ferritin samples (type I from horse spleen) were purchased from Sigma Chemicals and were dissolved to a concentration of 76 mg/mL in a 0.15 M NaCl solution. The poly(vinyl alcohol) (PVA, Mw ≈ 124 000-186 000, 99+% hydrolyzed, degree of polymerization ≈ 3300) and methyl alcohol used were obtained from Aldrich Chemicals. Solution Preparation. To prepare a 7.5 wt % PVA/ferritin solution in water (the mass ratio of ferritin to PVA was about 0.01.), PVA was dissolved in water and then stirred for 1.5 h at 90 °C before being allowed to cool to 30 °C. Then, the PVA/water solution was mixed with a ferritin/water solution using a magnetic stirrer for 1 h at room temperature. To prepare 7.5 wt % PVA/ferritin solutions with ferritin clusters, PVA/ferritin/water solutions forming the homogeneous mixture at 30 °C were reheated to 60 and 80 °C. These solutions were stirred for 10 min at their respective temperatures before being allowed to cool to 30 °C. Fabrication of Nanofiber Hydrogels. To fabricate PVA/ferritin nanofibers by electrospinning, a PVA/ferritin solution was loaded into a plastic syringe equipped with a stainless steel needle. The polymer solution was fed at a flow rate of 10 µL/min using a syringe pump (KD Scientific) located in a horizontal mount. A voltage of 10 kV was applied between the syringe needle and the grounded electrodes using a high-voltage power supply (Nano Technics, Korea). The syringe needle acted as an anode, and an aluminum electrode acted as a cathode. The distance between the syringe needle and the aluminum electrode was 20 cm. To fabricate water-insoluble nanofiber hydrogels, PVA/ferritin nanofibers deposited on the aluminum electrode after electrospinning were immersed in methanol for 12 h, and then the methanol-treated nanofibers were separated from the aluminum electrode. The separated nanofibers were dried on a Teflon sheet at room temperature. Characterization. To prepare samples for cross-sectional TEM images, the PVA/ferritin nanofiber hydrogels were placed on a hat(12) Harrison, P. M.; Arosio, P. Biochim. Biophys. Acta 1996, 1275, 161. (13) Bonkovsky, H. L.; Rubin, R. B.; Cable, E. E.; Davidoff, A.; Rijcken, T. H. P.; Stark, D. D. Radiology 1999, 212, 227. (14) Schenck, J. F. J. Neurol. Sci. 1995, 134S, 10. (15) Gossuin, Y.; Burtea, C.; Monseux, A.; Toubeau, G.; Roch, A.; Muller, R. N.; Gillis, P. J. Magn. Reson. Imaging 2004, 20, 690. (16) Theil, C. E. Annu. ReV. Biochem. 1987, 56, 289. (17) Jordan, A.; Wust, P.; Scholz, R.; Tesche, B.; Fahling, H.; Mitrovics, T.; Vogl, T.; Cervos-Navarro, J.; Felix, R. Int. J. Hyperthermia 1996, 12, 705. (18) Yeh, T.-C.; Zhang, W.; Ildstad, S. T.; Ho, C. Magn. Reson. Med. 1993, 30, 617.

Letters shaped copper disk and sandwiched by placing another hat-shaped copper disk over them prior to freezing. The copper sandwich structures were frozen using an RMC MF-7200 propane jet freezer. The frozen samples were freeze substituted with 2% OsO4 in acetone at -80 °C for 3 days, -20 °C for 3 h, and then at 4 °C for 1 h. The samples were rinsed three times in pure acetone at 4 °C for 30 min each time. After being rinsed, the samples were infiltrated with Spurr’s medium for 1 day. After infiltration, the samples were embedded and polymerized at 60 °C for 1 day. Sections of plasticembedded samples were cut to a thickness of approximately 70 nm. Tilt series were acquired using field-emission transmission electron microscopy (FE-TEM, FEI TECNAI G2, JEOL (Japan), model JEM 2100F, accelerating voltage ) 200 kV). The internal structure and energy-disperse X-ray spectroscopy (EDXS) of the nanofibers were characterized using high-resolution transmission electron microscopy (HRTEM, JEOL (Japan), model JEM 2100F, accelerating voltage ) 200 kV). The magnetic properties of the PVA/ferritin nanofibrous mats were measured using a superconducting quantum interference device (SQUID) magnetometer. To confirm the chemical state of the ferritin iron core, X-ray photoelectron spectroscopy (XPS) measurements were performed using a standard Al KR (λ ) 1486.7 eV) excitation source in an electron spectrometer (model ESCA 5700, PHI Ltd.) at a residual gas pressure of about 2 × 10-10 Torr. The T2 relaxivity of the PVA/ferritin/water, ferritin/water, and PVA/water solutions was determined using a conventional spin-echo sequence employing a 1.5 T MRI scanner (Avanto, Siemens). The following parameters were used: TR ) 3000 ms, TE ) 20, 40, 80, 150, 300, 500, and 1000 ms, field of view ) 20 × 20 cm2, and matrix size ) 256 × 256 points. The in vitro MRI study of the PVA/ferritin nanofiber hydrogel and the PVA nanofiber hydrogel was performed using a research prototype scanner equipped with an in-house high-sensitivity 5-cmdiameter coil. To compare the magnetic properties of PVA/ferritin and PVA nanofiber hydrogels, the two materials were contained in tubes containing DI water, and then the samples were scanned using a conventional 2-D gradient echo sequence (TR/TE ) 500 ms/7.0 ms, field of view ) 30 × 30 mm2, matrix size 256 × 256 points, in-plane resolution ) 117 µm, slice thickness ) 1.5 mm). The in vivo animal experiments were carried out with an ∼12week-old Sprague-Dawley (SD) rat at Gachon University. All procedures were performed in accordance with the National Institutes of Health Guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals) and were approved by the Institutional Animal Care and Use Committee of Gachon University. Animals were housed in a vivarium with a 12 h light/dark cycle (lights on at 8:00 a.m.), 50-60% humidity, and free access to food and water. The animals were acclimatized to the laboratory 4 days before the beginning of the experiments. We implanted the PVA/ferritin nanofiber hydrogel in the leg of the rat, and the T2-weighted images were acquired before implantation and after 3 days of implantation. For T2-weighted imaging, we used a 3-D gradient echo sequence (TR/TE ) 100 ms/5.8 ms, field of view ) 50 × 60 mm2, matrix size ) 208 × 256 points, slice thickness ) 1.0 mm). The weight of the PVA/ferritin samples used was in the range of 4 to 5 mg in the dry state.

Results and Discussion We used commercial ferritin biomolecules that generally have about 2000 Fe atoms19 and the mass ratio of the iron core to ferritin (iron core + protein shell) is about 10.7%20 to fabricate biocompatible nanofiber hydrogels with superparamagnetic properties. Figure 1a shows the typical shape of the horse spleen ferritin used in the nanocomposites and the lattice structure of a ferritin core. From Figure 1a, we confirmed that the ferritin (19) Galvez, N.; Fernandez, B.; Sanchez, P.; Cuesta, R.; Ceolin, M.; ClementeLeon, M.; Trasobares, S.; Lopez-Haro, M.; Calvino, J. J.; Stephan, O.; DominguezVera, J. M. J. Am. Chem. Soc. 2008, 130, 8062. (20) St. Pierre, T. G.; Gorham, N. T.; Allen, P. D.; Costa-Kramer, J. L.; Rao, K. V. Phys. ReV. B 2001, 65, 024436.

Letters

Langmuir, Vol. 24, No. 21, 2008 12109

Figure 1. (a) TEM images showing the iron core and protein shell of ferritin. (b) TEM image showing the ferritin clusters formed by thermal energy at 80 °C. (c) EDXS analysis showing typical peaks from Fe and P in a ferritin iron core after thermal treatment at 30 and 80 °C for 10 min. (d) XPS analysis showing the Fe3+ state of a ferritin core after thermal treatment at 30 and 80 °C for 10 min.

consisted of an iron core with lattice dimensions of ∼0.2 nm and a protein shell. Figure 1b shows ferritin clusters formed by thermally induced phase separation at 80 °C. The protein shell of ferritin can be partially unfolded when the temperature of the solution containing the ferritin increases. Although the ferritin forms a uniform monodispersion in the polymer solution without agglomeration, ferritin with an unfolded protein shell due to the thermal energy forms small clusters in solution.21 The clustering of ferritin is promoted as the temperature of the solution is increased, and thus the size of the ferritin clusters can be controlled in solution. In Figure 1b,c, we confirmed that the original lattice structure of the ferritin iron core was not changed and that Fe and P coexisted in the ferritin core observed in the 30 and 80 °C samples. (The coexistence of Fe and P means that the internal structure of the ferritin core was not changed.) Figure 1d shows the X-ray photoelectron spectroscopy (XPS) analysis of a ferritin core for samples fabricated at 30 and 80 °C. From Figure 1d, we confirmed that the chemical state of the ferritin iron cores was maintained as Fe3+, regardless of the mixing temperature. On the basis of the clustering process of ferritin, we controlled the dispersion of the ferritin nanoparticles incorporated into a biocompatible poly(vinyl alcohol) (PVA) solution by changing the mixing temperature of the PVA/water and ferritin/water solutions. Figure 2a shows the typical morphology of electrospun nanofiber hydrogels in the dry state. Electrospinning22 was carried out using a solution cooled to room temperature after maintaining the blend solutions at a mixing temperature of 30, 60, and 80 °C for 10 min. When the PVA/water solution was mixed with (21) Kim, S.-W.; Kim, Y.-H.; Lee, J. Biochem. Biophys. Res. Commun. 2001, 289, 125. (22) Shin, M. K.; Kim, S. I.; Kim, S. J.; Kim, S.-K.; Lee, H. Appl. Phys. Lett. 2006, 88, 193901.

Figure 2. PVA/ferritin nanofibers with the various spatial distributions of ferritin. (a) SEM image showing the PVA/ferritin nanofiber hydrogel in the dry state. (b-d) TEM images showing the spacing difference of the ferritin nanoparticles in the PVA/ferritin nanofibers fabricated at different mixing temperatures: (b) 30, (c) 60, and (d) 80 °C.

the ferritin/water solution at 30 °C, they formed a homogeneous mixture, and ferritin nanoparticles were monodispersed in the PVA/ferritin nanofibers electrospun from the blend solution without any aggregation (Figure 2b). However, the average size of the ferritin clusters was larger in the PVA nanofibers when the mixing temperature was increased (Figure 2c,d). The average sizes of the ferritin clusters calculated from the cross-sectional

12110 Langmuir, Vol. 24, No. 21, 2008

TEM images of the 60 and 80 °C samples were 7.3 × 104 and 74.3 × 104 nm2, respectively. In particular, nanofibers fabricated from a solution mixed at 60 °C showed a mixture of both welldispersed and clustered ferritin nanoparticles. Surprisingly, in the sample prepared at 80 °C, all of the ferritin nanoparticles participated in the formation of clusters, such as those shown in Figure 2d, and the interparticle spacing between the ferritin cores in the clusters was on the order of several nanometers, corresponding to a thickness of two ferritin protein shells. This shows that the ferritin shell plays a crucial role as a spacer separating the ferritin cores, even though the ferritin nanoparticles are clustered. From Figure 2, we confirmed that a mixing temperature of ∼60 °C was the transition point for forming highly aggregated nanoparticles. A close inspection of 3-D electron tomograms showed the densities of ferritin nanoparticles distributed within nanofiber hydrogels. Whereas these clusters were evenly dispersed and closely spaced in the 80 °C sample, very few clusters were observed in the 30 °C sample. The 60 °C showed distinct regions of both high and low cluster concentrations (movies S1-S3 in the Supporting Information). These results suggest that the close proximity of the ferritin cores within the clusters can result in magnetic ordering and increased magnetization. The typical parameter influencing the dispersion of nanoparticles in a polymer solution is the ratio of the polymer radius of gyration (Rg) to the particle radius (r).23 The value of Rg of PVA used was about 10 nm when calculated using Rg (Å) ) 4.09(DP/ 6)1/2 (where DP is the degree of polymerization, and the DP value of PVA used was >3300).24 The diameter of the ferritin cores was confirmed from TEM images and was 6 to 7 nm. Thus, when considering the protein shell of ferritin, the Rg/r ratio was about 2. Because the value of Rg/r was >1, it is possible for polymer chains to surround and disperse the ferritin. However, when the mixing temperature is increased, the generation of ferritin clusters means that the attractive forces between the ferritin nanoparticles may be more influential in the dispersion of ferritin in the PVA solution than the geometric parameters of the polymer chains and nanoparticles. The protein shell of ferritin has both hydrophilic and hydrophobic residues and ion channels,25 and ferritin is a water-soluble nanoparticle. Because the protein shell of ferritin partially unfolds on increasing the mixing temperature, the hydrophobicity in the ferritin shell increases as a result of the conformational change of the protein subunits in the ferritin shell. This can result in a strong attraction between ferritin nanoparticles in a hydrophilic PVA solution, thus ferritin clusters can form. In addition, Kim and co-workers suggested that at 50 °C the temperature-induced aggregation of human ferritin in water is related to the partial denaturing of the amino terminus of ferritin, even though ferritin is stable up to 85 °C.21 Therefore, the formation of ferritin clusters in PVA nanofibers according to the mixing temperature of the solutions can be attributed to the partial unfolding of the protein subunits because the geometric parameters of the ferritin and PVA chains and the pH of the PVA/ferritin solutions were almost constant. (The pH of the PVA/ferritin solutions was 6.8, independent of the mixing temperature.) It has been shown that MRI contrast can be improved by reducing the T2 relaxation times of water molecules.26 As shown in Figure 3a, the composition and mixing temperature (30, 60, (23) Mackay, M. E.; Tuteja, A.; Duxbury, P. M.; Hawker, C. J.; Van Horn, B.; Guan, Z.; Chen, G.; Krishnan, R. S. Science 2006, 311, 1740. (24) Lankveld, J. M. G.; Lyklema, J. J. Colloid Interface Sci. 1972, 41, 475. (25) Ford, G. C.; Harrison, P. M.; Rice, D. W.; Smith, J. M. A.; Treffry, A.; White, J. L.; Yariv, J. Philos. Trans. R. Soc. London, Sect. B 1984, 304, 551. (26) Perez, J. M.; Josephson, L.; Weissleder, R. ChemBioChem 2004, 5, 261.

Letters

Figure 3. Magnetic properties of the PVA/ferritin/water solutions and PVA/ferritin nanofibers fabricated at different mixing temperatures. (a) A graph showing T2 values of the PVA/ferritin/water solutions with increasing mass fraction of ferritin at 1.5 T. The inset of graph a shows T2 values of the PVA/ferritin/water and ferritin/water solutions. (b) Graph showing the magnetization induced by an external magnetic field on PVA/ferritin nanofibers fabricated at different mixing temperatures. The inset of graph b shows a plot of the magnetization shown in graph b at low magnetic fields. The magnetic properties of all the samples were measured at 300 K.

or 80 °C) significantly affected the T2 times of the PVA/ferritin/ water solutions. As the mixing temperature of the PVA/ferritin/ water solutions increased, the T2 times significantly decreased. It was observed that higher ferritin concentrations also decreased the T2 times. Moreover, the T2 values of the PVA/ferritin/water solutions were much smaller than those of the ferritin/water solution (inset of Figure 3a) and the PVA/water solution. Clearly, a synergistic effect occurs between the ferritin and the PVA chains in solution, leading to shorter relaxation times. It is known that the MRI relaxation can be enhanced using methods that increase the effective molecular weight of a contrast agent using a polymer or protein.27 The clustering of magnetic nanoparticles has been shown to reduce the T2 times of water molecules close to the clusters because of their larger effective cross-sectional area.26 The electrospun nanofibers prepared at the different mixing temperatures were tested for their magnetic behavior and MRI contrast. As shown in the inset of Figure 3b, the coercive field of all of the samples was about 18 Oe, which implies that the dipole-dipole interactions can be ignored. Therefore, all of the samples exhibited a superparamagnetic phase, which was almost independent of the solution mixing temperature. However, (27) Nicolle, G. M.; Toth, E.; Eisenwiener, K.-P.; Macke, H. R.; Merbach, A. E. J. Biol. Inorg. Chem. 2002, 7, 757.

Letters

Langmuir, Vol. 24, No. 21, 2008 12111

more contrast for the 80 °C sample than for the 30 °C sample and PVA nanofibers swollen in DI water in vitro (Figure 4a,b). The PVA/ferritin nanofiber hydrogel (80 °C sample) was clearly visible by MRI imaging in vivo when implanted in a mouse (Figure 4d). Therefore, the improved MRI contrast is derived from the enhanced superparamagnetic properties of the PVA/ ferritin nanofibers. Because the chemical state of the iron and the original lattice structure in the ferritin cores were unchanged with mixing temperature (Figure 1), the increased magnetization is most probably due to a change in magnetic ordering inside the ferritin core.

Conclusions Varying the magnetic properties of materials by controlling the spatial distribution of nanoparticles is also seen in nature.29,30 Inspired by nature, we have fabricated PVA/ferritin nanofiber hydrogels in which the size of ferritin clusters has been controlled by partial unfolding of the ferritin protein shell by changing the mixing temperature. The enhanced magnetic signal of the waterinsoluble PVA/ferritin nanofibers containing the ferritin clusters provides a breakthrough for the realization of excellent performance of biocompatible superparamagnetic nanofiber hydrogels. Moreover, because PVA is biocompatible and has good mechanical properties,31,32 the PVA/ferritin nanofiber hydrogels with improved superparamagnetic properties could be applied as noninvasive monitorable artificial muscles, materials for drug delivery, cartilage, and cell culture scaffolds in vivo. Nanofiber hydrogels may also be important in elucidating the relationship between the basic mechanical and thermal properties and nanoparticle clustering.

Figure 4. (a-c) In vitro MRI images showing cross-sections of the nanofiber hydrogels contained in DI water: (a) PVA/ferritin (80 °C sample), (b) PVA/ferritin (30 °C sample), and (c) PVA. (d) In vivo MRI image showing the pelvic limb of a mouse after implantation of the PVA/ferritin nanofiber hydrogel prepared at the mixing temperature of 80 °C.

samples prepared at the higher mixing temperatures had enhanced saturation magnetization per weight of ferritin compared with that of the 30 °C sample. Because the MRI contrast is closely related to superparamagnetism,28 the enhanced superparamagnetic properties of the PVA/ferritin nanofibers should improve their visibility in MRI. Indeed, MRI images taken of the PVA/ferritin nanofiber hydrogels swollen in DI water showed considerably (28) Wu, E. X.; Tang, H.; Jensen, J. H. NMR Biomed. 2004, 17, 478.

Acknowledgment. This work was supported by the Creative Research Initiative Center for Bio-Artificial Muscle of the Ministry of Education, Science and Technology (MEST)/the Korea Science and Engineering Foundation (KOSEF) in Korea. Supporting Information Available: Electron tomogram showing the 3-D structures of ferritin clusters incorporated into nanofiber hydrogels. This material is available free of charge via the Internet at http://pubs.acs.org. LA802155A (29) Doyle, F. H.; Pennock, J. M.; Banks, L. M.; McDonnell, M. J.; Bydder, G. M.; Steiner, R. E.; Young, I. R.; Clarke, G. J.; Pasmore, T.; Gilderdale, D. J. AJR Am. J. Roentgenol 1982, 138, 193. (30) Gossuin, Y.; Burtea, C.; Monseux, A.; Toubeau, G.; Roch, A.; Muller, R; Gillis, P. J. Magn. Reson. Imaging 2004, 20, 690. (31) Oka, M.; Noguchi, T.; Kumar, P.; Ikeuchi, K.; Yamamuro, T.; Hyon, S. H.; Ikada, Y. Clin. Mater. 1990, 6, 361. (32) Stammen, J. A.; Williams, S.; Ku, D. N.; Guldberg, R. E. Biomaterials 2001, 22, 799.