Facile Preparation of Self-Assembled Hydrogel-like GdPO4·H2O

Langmuir 2008, 24, 8309-8313

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Facile Preparation of Self-Assembled Hydrogel-like GdPO4 · H2O Nanorods Chih-Chia Huang,† Yi-Wei Lo,† Wen-Shuo Kuo,† Jih-Ru Hwu,‡ Wu-Chou Su,§ Dar-Bin Shieh,| and Chen-Sheng Yeh*,† Department of Chemistry, National Cheng Kung UniVersity, No. 1 UniVersity Road, Tainan City, Taiwan, Department of Chemistry, National Tsing Hua UniVersity, Hsinchu, Taiwan 30013, Taiwan, Department of Internal Medicine, National Cheng Kung UniVersity, Tainan City, Taiwan, and Institute of Oral Medicine and Department of Stomatology, National Cheng Kung UniVersity, Tainan City, Taiwan ReceiVed March 18, 2008. ReVised Manuscript ReceiVed May 8, 2008 Of the methods employed in the preparation of one-dimensional lanthanide phosphate (LnPO4) nanorods/nanowires, such as GdPO4, the hydrothermal method has been mainly used as a synthetic route. In this study, we report a facile low-temperature solution approach to prepare GdPO4 · H2O nanorods by simply refluxing GdCl3 and KH2PO4 for only 15 min at 88 °C, an approach that can easily be scaled up by increasing the reagent amounts. We observed a highly viscous macroscopic hydrogel-like material when we mixed as-prepared GdPO4 · H2O nanomaterials with H2O. Hydrogels are an important class of biomaterials. Their building blocks, normally formed from protein-, peptide-, polymer-, and lipid-based materials, offer three-dimensional scaffolds for drug delivery, tissue engineering, and biosensors. Our preliminary results showed that GdPO4 · H2O hydrogels could be used for encapsulation and drug release, and that they were biocompatible, acting as scaffolds to foster cell proliferation. These findings suggested that they might have biomedical uses. Our findings may lead to the creation of other inorganic nanomaterial-based hydrogels apart from the organic and biomolecular protein-, peptide-, polymer-, and lipid-based building blocks.

Introduction Because of their unique electronic and optical characteristics arising from 4f electrons, lanthanide-based materials have attracted great interest in the fields of photoluminescence and catalyst applications. Among them, significant effort has been devoted to developing rare earth ion-doped lanthanide phosphate (LnPO4) nanoparticles, nanorods, and nanowires because of their high selectivity and sensitivity in luminescence signals.1–4 For example, Fang et al. prepared LnPO4 nanorods/nanowires and investigated the photoluminescent properties of LaPO4/Eu nanowires.2 The luminescence lines of the LaPO4/Eu nanowire emission were characterized to be similar with those bulk LaPO4/Eu materials. Haase’s groups studied nanocrystal shape-dependent emission by investigating the orientation of the unique crystal axis relative to the polarization vector of the incident light.1d They prepared core-shell structured CePO4:Tb/LaPO4 nanoparticles.4 The LaPO4 shell formation can reduce the quenching of quantum yield originating from undoped LaPO4 nanoparticles. For biological consideration, dextran-coated GdPO4 rod-like nanoparticles have been prepared to be treated as MR-positive * To whom correspondence should be addressed. E-mail: csyeh@ mail.ncku.edu.tw. † Department of Chemistry, National Cheng Kung University. ‡ National Tsing Hua University. § Department of Internal Medicine, National Cheng Kung University. | Institute of Oral Medicine and Department of Stomatology, National Cheng Kung University. (1) (a) Riwotzki, K.; Meyssamy, H.; Schnablegger, H.; Kornowski, A.; Haase, M. Angew. Chem., Int. Ed. 2001, 40, 573–576. (b) Schuetz, P.; Caruso, F. Chem. Mater. 2002, 14, 4509–4516. (c) Heer, S.; Lehmann, O.; Haase, M.; Gudel, H. U. Angew. Chem., Int. Ed. 2003, 42, 3179–3182. (d) Meyssamy, H.; Riwotzki, K.; Kornowski, A.; Naused, S.; Haase, M. AdV. Mater. 1999, 11, 840–844. (e) Yan, R.; Sun, X.; Wang, X.; Peng, Q.; Li, Y. Chem.;Eur. J. 2005, 11, 2183–2195. (f) Li, Q.; Yan, V. W. W. Angew. Chem., Int. Ed. 2007, 46, 3486–3489. (2) Fang, Y. P.; Xu, A. W.; Song, R. Q.; Zhang, H. X.; You, L. P.; Yu, J. C.; Liu, H. Q. J. Am. Chem. Soc. 2003, 125, 16025–16034. (3) Zhang, Y. W.; Yan, Z. G.; You, L. P.; Si, R.; Yan, C. H. Eur. J. Inorg. Chem. 2003, 4099–4104. (4) Hompe, K.; Borchert, H.; Storz, J.; Lobo, A.; Adam, S.; Moller, T.; Haase, M. Angew. Chem., Int. Ed. 2003, 42, 5513–5516.

contrast agents.5 EuPO4 and TbPO4 nanorods were examined for their nontoxic nature in in Vitro assays and were applied for novel fluorescent biolabel agents.6 In this study, we report that an inorganic nanomaterial, GdPO4 · H2O nanorod, exhibiting a hydrogel-like property, could be used for encapsulation and drug release, and was biocompatible. Hydrogels are an important class of biomaterials with building blocks normally formed from protein-, peptide-, polymer-, and lipid-based materials. They offer three-dimensional (3D) scaffolds for drug delivery, tissue engineering, and biosensors.7 Hydrothermal methodology has been used primarily for synthesizing one-dimensional LnPO4 nanomaterials. Hydrothermal treatment usually requires a relatively high temperature, in the range of 150-240 °C, and a reaction period from 2 h to 2 days, depending on the experimental circumstances.1d,e,2,3,5 A room-temperature route has also been used to synthesize Tb3+doped CePO4 nanowires.1f Herein, we developed a facile lowtemperature solution approach to prepare GdPO4 · H2O nanorods by simply refluxing GdCl3 and KH2PO4 for only 15 min at 88 °C. The number of GdPO4 · H2O nanorods produced could easily (5) Hifumi, H.; Yamaoka, S.; Tanimoto, A.; Citterio, D.; Suzuki, K. J. Am. Chem. Soc. 2006, 128, 15090–15091. (6) Patra, C. R.; Bhattacharya, R.; Patra, S.; Basu, S.; Mukherjee, P.; Mukhopadhyay, D. J. Nanobiotechnol. 2006, 4, 11. (7) (a) Kim, S. W.; Bae, Y. H.; Okano, T. Pharm. Res. 1992, 9, 283–290. (b) Kisiday, J.; Jin, M.; Kurz, B.; Huang, H.; Semino, C.; Zhang, S.; Grodzinsky, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9996–10001. (c) Kohler, K.; Forster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A. Angew. Chem., Int. Ed. 2004, 43, 245–247. (d) Kretsinger, J. K.; Haines, L. A.; Ozbas, B.; Pochan, D. J.; Schneider, J. P. Biomaterials 2005, 26, 5177–5186. (e) Ishihara, M.; Obara, K.; Nakamura, S.; Fujita, M.; Masuoka, K.; Kanatani, Y.; Takase, B.; Hattori, H.; Morimoto, Y.; Ishihara, M.; Maehara, T.; Kikuchi, M. J. Artif. Organs 2006, 9, 8–16. (f) Gutierrez, M. C.; Garcia-Carvajal, Z. Y.; Jobbagy, M.; Yuste, L.; Rojo, F.; Abrusci, C.; Catalina, F.; del Monte, F.; Ferrer, M. L. Chem. Mater. 2007, 19, 1968–1973. (g) Murphy, W. L.; Dillmore, S.; Modica, J.; Mrksich, M. Angew. Chem., Int. Ed. 2007, 46, 3066–3069. (h) Thornton, R.; Mart, R. J.; Ulijn, R. V. AdV. Mater. 2007, 19, 1252–1256. (i) Oh, J. K.; Siegwart, D. J.; Lee, H.-i.; Sherwood, G.; Peteanu, L.; Hollinger, J. O.; Kataoka, K.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 5939–5945.

10.1021/la800847d CCC: $40.75  2008 American Chemical Society Published on Web 06/21/2008

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be scaled up by increasing the reagent amounts. For example, if the amount of reagents was increased 16 times, then the product yield increased approximately 15 times. The as-prepared GdPO4 nanorods were only 3-5 nm in diameter as opposed to the GdPO4 nanorods/nanowires usually produced with diameters of at least 10 nm.

Experimental Section Preparing GdPO4 · H2O Nanorods. All chemicals used in this work were analytical-grade reagents obtained without further purification. The GdCl3 (99.9%; Alfa Aesar, Ward Hill, MA) and KH2PO4 (99.6%; J. T. Backer, Inc., Phillsburg, NJ) were dissolved in aqueous solution to concentrations of 0.2 and 0.0165 M, respectively, as the stock solutions. In a typical preparation, 1.25 mL of KH2PO4 was mixed with 2.5 mL of ethanol (99.9%; J. T. Backer) and was preheated for 5 min of refluxing at 88 °C, after which 0.125 mL of GdCl3 aqueous solution was added to react for 15 min. White precipitates were obtained and were collected using centrifugation (8000-11000 rpm). The precipitates were washed several times with ethanol/water for further characterization. This synthetic approach can be readily scaled up. For example, if the amount of the reagent were increased 16 times, the product yield increased approximately 15 times. The GdPO4 · H2O hydrogels were prepared by suspending GdPO4 · H2O nanorods in distilled water with a final GdPO4 · H2O concentration of 0.7-2.5 wt % and aged for 30 min to form gel structures. Release Studies of the GdPO4 · H2O Hydrogels. First, the preformed GdPO4 · H2O hydrogels were loaded with fluorescent molecules, such as Rhodamine 6G (Rh6G; Exiton, Dayton, OH) and fluorescein isothiocyanate (FITC; Sigma-Aldrich Co., St. Louis, MO), EuNO3 (99.9%; Aldrich) and CuCl2 (99%; RiedeldeHaen). Preformed GdPO4 · H2O hydrogel (0.07 mL) was mixed with an aqueous solution (0.03 mL) containing concentrations of 0.5 mM for fluorescent molecules, 0.1 M for Eu3+ ions, and 30 mM for Cu2+ ions, respectively. The resulting moleculeloaded GdPO4 · H2O hydrogels (2.5 wt%) were then aged for 30 min at room temperature (approximately 25 °C). Next, distilled water (1.3 mL; pH ∼ 5.61) was added on top of the hydrogel to study the release of the entrapped molecules. At different time points (0.5, 1.5, 4, 8, 12, 18, and 24 h), the supernatants were collected and subjected to signal quantification. The fluorescent signals of FITC (excitation at 457 nm and emission at 514 nm), Rh 6G (excitation at 348 nm and emission at 550 nm), and Eu3+ ions (excitation at 380 nm and emission at 591 nm) were recorded using a fluorescence spectrophotometer (F-2500; Hitachi Koki Co., Ltd., Tokyo, Japan). The release of Cu2+ ions from GdPO4 · H2O hydrogels exhibited a broad d-d transition absorption at 800 nm and was monitored by a UV-visible spectrophotometer (Hewlett-Packard 8452A). The Biocompatibility of GdPO4 · H2O Hydrogels. To evaluate the biocompatibility of GdPO4 · H2O hydrogels, both WST-1 and MTT assays were used on the Vero cell line. WST-1 Assay. This assay measured mitochondrial dehydrogenase activity known to be associated with cell viability. This assay is based on the formation of dark-red formazan by the metabolically active cells after their exposure to WST-1 (tetrazolium salt). The amount of formazan is related directly to the number of metabolically active cells in the medium and can be quantified by measuring its absorbance using an ELISA reader. The absorbance of the formazan dye solution is in direct proportion to the number of viable cells. The Vero cells were cultured in a 96-well microplate with modified Eagle’s medium (MEM) containing 10% fetal bovine serum (FBS), 1% L-glutamine, 1%

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pyruvate, 1% nonessential amino acid, and 1% penicillin/ streptomycin/neomycin (PSN). The initial density was 4 × 103 cells/well. The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. After 24 h, serial dilutions of the GdPO4 · H2O hydrogel, at concentrations of 200, 100, 10, 1, and 0.1 µg mL-1, were added to the culture wells to replace the original culture medium with a final volume of 100 µL. The cells were incubated with the particles for 24 h. The culture medium was then removed and replaced with 100 µL of the new culture medium containing 10% WST-1 reagent. The cells were then incubated for 1.5 h at 37 °C to allow formazan dye to form. Next, the culture medium in each well was centrifuged (to prevent the nanoparticles from interfering with the spectrophotometric measurement), and then transferred to an ELISA plate. The quantification determining cell viability was done using optical absorbance (450/690 nm) and an ELISA plate reader. MTT Assay. This method is based on the formation of darkred formazan by the metabolically active cells after their exposure to MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Once again, serial dilutions of the GdPO4 · H2O hydrogel at concentrations of 200, 100, 10, 1, and 0.1 µg mL-1 were added to the culture wells to replace the original culture medium with a final volume of 100 µL. The cells were incubated with the particles for 24 h. The culture medium was then removed and replaced with 100 µL of the new culture medium containing 10% MTT reagent. The cells were then incubated for 4 h at 37 °C to allow formazan dye to form. Next, the culture medium in each well was removed, and dimethyl sulfoxide (DMSO) (200 µL/well) was added for an additional 10 min incubation. After the cells had been centrifuged, the resulting formazan in each well was transferred to an ELISA plate. The quantification determining cell viability was done using optical absorbance (540/650 nm) and an ELISA plate reader. The Cell Proliferation. A 1 wt % portion of GdPO4 · H2O gel was prepared as a film-like scaffold to foster cell proliferation. The film was prepared by deposition of 50 µL of GdPO4 · H2O (1 wt %) on a 96-well cell culture plate. Thereafter, those gels were slowly dried into the solid scaffold in an incubator at 37 °C with a humidified atmosphere of 5% CO2 for 7 days. The resulting film was inspected by the optical microscopy to ensure full coverage of the GdPO4 · H2O over the plate. The films were then left overnight with 200 µL of MEM containing 10% FBS, 1% L-glutamine, 1% pyruvate, 1% nonessential amino acid, and 1% PSN. Next, the MEM was removed, and 100 µL of MEM with Vero cells (2500 cells) was added over the film. Cells were cultured for 4 h, and 1, 2, and 3 days, and the viability of the cells was determined using MTT assay as described above. Each data point was recorded after 5 repetitions. Characterization. The crystalline structures were identified using an X-ray diffractometer (XRD-7000S; Shimadzu Corporation, Tokyo, Japan) with Cu Ka radiation (λ ) 1.54060 Å) at 30 kV and 30 mA. An Al template was used as a sample holder. Electron micrographs using transmission electron microscopes (FE-2000; Hitachi Koki; and Zeiss 10c; Carl Zeiss MicroImaging, Inc., Thornwood, NY) were attained by placing a drop of the sample on a copper mesh coated with an amorphous carbon film, followed by evaporation of the solvent in a vacuum desiccator. Field-emission scanning electron microscopic (FE-SEM) images of the GdPO4 nanorods on a Si wafer substrate were taken using a field-emission scanning electron microscope (XL-40FEG; Philips Research Europe, Eindhoven, The Netherlands). IR spectra were measured using a Fourier transformation infrared (FT-IR) spectrometer (200E; JASCO International Co., Ltd., Tokyo, Japan) by KBr plate. Fluorescence spectrophotometers (F-2500; Hitachi Koki) were used as controlled-

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Figure 2. TGA profile for GdPO4 · H2O nanorods.

Figure 1. (a) X-ray diffraction patterns obtained from ethanol/H2O and H2O systems. (b) A TEM image of GdPO4 · H2O (0.02 wt%) in dried form and (c) an HRTEM image of a single GdPO4 · H2O nanorod.

release monitors. N2 adsorption measurements were done at 77 K using an accelerated surface area and porosimetry analyzer (ASAP 2010; Micromeritics Instrument Corporation, Norcross, GA) using Brunauer-Emmett-Teller (BET) calculations for the surface area and Barrett-Joyner-Halenda (BJH) calculations for the pore-size distribution of dry-form powders from the adsorption branch of the isotherm.

Figure 3. FT-IR spectra of GdPO4 · H2O nanorods. The absorption bands in the range of 475-1300 cm-1 and at around 3400 cm-1 are attributable to the active vibrations of tetrahedral PO43- and the O-H stretching in molecular H2O, respectively.

Results and Discussion In a typical reaction, 0.125 mL of GdCl3 (0.2 M) aqueous solution was added to 3.75 mL of a preheated water (1.25 mL) and ethanol (2.5 mL) mixture containing KH2PO4, and then allowed to reflux at 88 °C for 15 min, which resulted in 4.4 mg of white precipitates. The precipitates were then washed and collected for further characterization. The resulting precipitates were indexed to the hexagonal GdPO4 · nH2O (JPCDS card: 00039-0232) (Figure 1a). We also synthesized GdPO4 · nH2O nanorods using pure water without ethanol. However, the product yield was only 25% of that obtained using water plus ethanol. Figure 1b shows a low-resolution transmission electron microscope (TEM) image of GdPO4 · H2O (0.02 wt %) in dried form. The resulting GdPO4 · H2O nanorods have an average length of 200-300 nm. Figure 1c displays a high-resolution TEM (HRTEM) image of a single GdPO4 · H2O nanorod (diameter ) ∼4.8 nm), showing a crystalline structure with lattice fringes of 0.29, 0.21, and 0.19 nm, corresponding to the (200), (003), and (301) planes, respectively. GdPO4 · H2O nanorods are highly crystalline enclosed with {100} planes and prefer growth along the 〈001〉 direction. Thermogravimetric analysis (TGA) showed a weight loss in the range of 130-500 °C, which indicated that this phosphate hydrates as GdPO4 · 1H2O (Figure 2). FT-IR measurements indicated that the absorption bands, in the range of 475-1200 cm-1 and at around 3400 cm-1, were attributable to the active vibrations of tetrahedral PO43- and the O-H stretching in molecular H2O, respectively (Figure 3). There are no absorption bands originating from P2O7 (1265-1267 cm-1) and HPO42- (880 cm-1). The formation of the GdPO4 · H2O was carefully conducted using time-dependent morphology evolution (Figure 4). We found that the urchin-like structures, consisting of needle-like structures ∼120 nm long radiating from the center, appeared as early as 10 s from the initial reaction (Figure 4a). After 7 min of reaction time, the urchin-like particles began to

Figure 4. TEM images showing morphology evolution for GdPO4 · H2O nanorods as a function of reaction time: (a) 10 s, (b) 50 s, (c) 2 min, and (d) 7 min. The arrow in (d) indicates urchin-like structures that collapsed to form nanorod domains.

collapse and form nanorods (Figure 4d). It is known that the anistropic nanostructure of LnPO4 · H2O nanorods/nanowires exhibit a hexagon structure growing along the c chain.1e,2,3 Although the detailed mechanisms remain to be resolved, the formation of GdPO4 · H2O nanorods was believed to occur on the basis of its intrinsic structure. We observed a highly viscous macroscopic hydrogel-like material when as-prepared GdPO4 · H2O nanomaterials were mixed with H2O (Figure 5a). The SEM image in Figure 5b shows that the GdPO4 · H2O gel consisted of a dense 3D interconnected network visible even in dilute aqueous solution (0.7 wt %). Although as-prepared GdPO4 · H2O nanorods were 200-300 nm long, the self-assembled nanorods aggregated and self-assembled into nanowires extending over micrometers. The aggregation process resulting in gel formation was driven by polar interactions, e.g., hydrogen bonds. Water could be placed on top of the resulting

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Figure 5. (a) Formation of GdPO4 · H2O hydrogels exhibiting a mechanically rigid conformation. (b) SEM image of the self-assembled GdPO4 · H2O (0.7 wt %) in dried form.

Figure 6. N2 adsorption-desorption isotherm of as-obtained GdPO4 · H2O hydrogels resulting in a surface area of 58.8 m2 g-1.

hydrogels. On the other hand, the self-assembled GdPO4 · H2O lost its gel character at temperatures above 55 °C and appeared as a fluid. Hydrated hydrogels have been increasingly studied for encapsulation and controlled-release drug delivery systems. Figure 5b shows that the GdPO4 · H2O gel consisted of a self-assembled network with interstitial spaces, although this SEM image was taken of the dried form of the gel sample. The BET analysis of the N2 adsorption-desorption measurements indicated that the porous cavity size had a distribution of 3.5-80 nm for the dried gel (Figure 6). Since the hydrogels swell in water or aqueous solution, an expansive network conformation is expected in humid conditions. Herein, the preformed GdPO4 · H2O hydrogel (2.5 wt %) was loaded with Rh6G dye solution. Figure 7a illustrates the encapsulation of the dye molecules by GdPO4 · H2O hydrogel and also shows that the gel was manipulated into a desired pattern, the letters “NCKU”. The Rh6G-loaded hydrogel was illuminated by white light (left panel) and UV excitation (right panel) (Figure 7a). Figure 7b shows the results of release experiments done using Rh6G-loaded, FITC-loaded, Cu2+ ion-loaded, and Eu3+ ion-loaded hydrogels. The gel containing releasing molecules was covered with distilled water, and the diffusion of the releasing molecules out of the hydrogel and into the surrounding water was monitored as a function of time. The entrapped fluorescent molecules were released, and a release profile was recorded by measuring the fluorescence intensity of the released fluorescent molecules. Rh6G was promptly released, and the release speed increased over time. Rh6G was 100% released after 18 h. On

Huang et al.

Figure 7. (a) The letters NCKU were written using Rh6G-doped GdPO4 · H2O hydrogels and were illuminated by white light (left panel) and UV light (right panel). (b) The release profiles of the entrapped Rh6G, FITC, Cu2+, and Eu3+ species were monitored as a function of time.

the contrary, the release of the fluorochrome (FITC) into the supernatant was slow and showed no further release after reaching a plateau of ∼57% after 18 h. Forty-three percent of the FITC molecules were locked inside the hydrogel. Such a distinct release behavior was related to the interaction between GdPO4 · H2O hydrogel and the entrapped molecules. A ζ-potential measurement indicated a positive surface charge of +32.9 mV for GdPO4 · H2O hydrogel. Different from the neutral Rh6G molecules, FITC is a proteolytic acid with three pKa values of 2.2, 4.4, and 6.7. It has been reported that FITC presents as a neutral and monoanion species with a pH between 2.8 and 5.5.8 Distilled water with a pH of ∼5.6 was measured for our release studies. The interaction between the -COO- groups of the FITC molecules and the positive charge of the hydrogel might have trapped some anionic FITC molecules inside the hydrogel. We did a parallel experiment in which the highly negative charge of DNA-Cy5 (Cy5-5′AAAAAAAAAAAAAAAAAAAAA-3′) was loaded into hydrogel, but it showed no release of DNA-bearing Cy5 fluorescent molecules. However, the larger molecule (7.3 kDa) originating from DNA-Cy5 giving rise to the inhibition of diffusion release cannot be excluded. Further studies on the molecular weight effect are necessary. In additional, the metal ion release studies were also investigated for Eu3+ ion-loaded and Cu2+ ion-loaded GdPO4 · H2O hydrogels (Figure 7b). The release of the positively charged Cu2+ ions from GdPO4 · H2O hydrogel was rapidly increased, reaching 100% release after 18 h. On the contrary, a slow release was observed for the Eu3+ ions. It only releases 57% after a prolonged 24 h. This could be stemmed from the possible formation of highly insoluble Eu- containing phosphate hydrates on the phosphate surfaces of GdPO4 · H2O nanorods.1e,2,4,6 In addition to providing a platform for drug delivery, successful hydrogel materials ought to serve as scaffolds to foster cell proliferation for tissue regeneration and engineering. Therefore, cell viability experiments were conducted on a Vero cell line (monkey kidney cell line) using two well-established WST-1 and MTT assays.9,10 The GdPO4 · H2O hydrogels were delivered over a range of dosages (0-200 µg/mL). Both assays showed satisfactory results that supported the biocompatibility of the (8) Fuh, M.-R. S.; Burgess, L. W.; Hirschfeld, T.; Christian, G. D.; Wang, F. Analyst 1987, 112, 1159–1163. (9) (a) Su, C. H.; Sheu, H. S.; Lin, C. Y.; Lo, Y. W.; Pu, Y. C.; Weng, J. C.; Shieh, D. B.; Chen, J. H.; Yeh, C. S. J. Am. Chem. Soc. 2007, 129, 2139–2146. (b) Cook, J. A.; Mitchell, J. B. Anal. Biochem. 1989, 179, 1–7. (10) Mosmann, T. Immunol. Methods 1983, 65, 55–63.

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positively charged surface of the resultant GdPO4 · H2O gel could be favorable to attach the negative charge of the surface of cell membranes. GdPO4 · H2O gel was examined for the ability to support proliferation. Vero cells were suspended on the GdPO4 · H2O film-like substrate on consecutive days. A 1 wt % portion of GdPO4 · H2O gel was prepared as a film-like scaffold to foster cell proliferation. The film was prepared by deposition of 50 µL of GdPO4 · H2O (1 wt %) on a 96-well cell culture plate. Cells were cultured for 4 h, and 1, 2, and 3 days, and cell proliferation was determined by MTT assay (Figure 8b). The experiments revealed that the cell growth profile increased with increasing culture period. These results indicate that the GdPO4 · H2O gel is nontoxic and supportive of cell attachment for cell proliferation.

Conclusions

Figure 8. (a) The biocompatibility of the GdPO4 · H2O hydrogels was analyzed using WST-1 and MTT assays. Vero cells were incubated with hydrogels for 24 h. (b) Cell proliferation based on MTT assays by culturing Vero cells (2500 cells per well) on the GdPO4 · H2O film-like scaffold.

hydrogels in all dosages and, interestingly, had some slightly higher values (Figure 8a), which might imply that the hydrogel could serve as a substrate for cell attachment and growth. The

In summary, we report the development of a facile preparation route synthesizing GdPO4 · H2O nanorods, a route that can be easily scaled up to produce nanorods in quantity. The selfassembled GdPO4 · H2O nanorods yielded a highly dense 3D network and resulted in a gel state. Our preliminary results showed that the GdPO4 · H2O hydrogels could be used for encapsulation and drug release, and that they were biocompatible as a scaffold for cell proliferation. These findings suggested that they might have biomedical uses. Although further characterization is required and is currently being investigated in our laboratory, our findings may lead to the creation of inorganic nanomaterialbased hydrogels other than those using organic and biomolecular protein-, peptide-, polymer-, and lipid-based building blocks. Acknowledgment. We thank the National Science Council of Taiwan for financially supporting this work. LA800847D