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Chem. Mater. 2006, 18, 956-959
Surface Modification Using Cubic Silsesquioxane Ligands. Facile Synthesis of Water-Soluble Metal Oxide Nanoparticles Benjamin L. Frankamp,† Nicholas O. Fischer,†,‡ Rui Hong,† Sudhanshu Srivastava,† and Vincent M. Rotello*,†,‡ Department of Chemistry and Molecular and Cellular Biology Program, UniVersity of Massachusettss Amherst, Amherst, Massachusetts 01003 ReceiVed October 4, 2005. ReVised Manuscript ReceiVed December 23, 2005
The use of magnetic nanoparticles in biological applications is hampered by the lack of versatile methods to transfer them into aqueous solutions. Monolayer exchange using anionic octa(tetramethylammonium)polyhedral oligomeric silsesquioxane provides individual particles that are soluble in aqueous environments and possess excellent stability in biologically relevant pH ranges and salt concentrations. In addition, this surface exchange reaction proved to be general in nature, allowing facile functionalization of a variety of magnetic nanoparticles.
Introduction Iron oxide nanoparticles have many biological applications, including MRI contrast agents,1 magnetic separation2 or localization,3 thermal ablation,4 and hyperthermia.5 A recent suite of topical reviews outlines the current state-of-the-art in terms of application,6 synthesis,7 and functionalization8 of such particles. The effective use of magnetic nanoparticles in a given application is based primarily on two factors: the physical properties of the particle (size, composition, etc.) and the ability to tailor its surface chemistry to promote specific interactions with target biomolecules. Several reports have demonstrated that high temperature decomposition of iron precursors in the presence of capping ligands leads to high quality magnetic particles featuring dramatically improved shape, size control, crystallinity, and size monodispersity as compared to more traditional coprecipitation/ dextran methods.9-11 Despite the ability to synthesize discrete * To whom correspondence should be addressed. E-mail: chem.umass.edu. † Department of Chemistry. ‡ Molecular and Cellular Biology Program.
rotello@
(1) Brigger, D. C.; Dubernet, C.; Couvreur, P. AdV. Drug DeliVery ReV. 2002, 54, 631-651. (2) (a) Rheinlander, T.; Kotitz, R.; Weitzchies, W.; Semmlar, W. J. Magn. Magn. Mater. 2000, 219, 219-228. (b) Todd, P.; Cooper, R.; Doyle, J.; Dunn, S.; Vellinger, J.; Deuser, M. J. Magn. Magn. Mater. 2001, 225, 294-300. (3) Tibbe, A.; de Grooth, B.; Greve, J.; Liberti, P.; Dolan, G.; Terstappen, L. Nat. Biotechnol. 1999, 17, 1210-1213. (4) (a) Jordan, A.; Wust, P.; Scholz, R.; Tesche, B.; Fahling, H.; Mitrovics, T.; Vogl, T.; Cervos-Navarro, J.; Felix, R. Int. J. Hyperthermia 1996, 12, 705-722. (b) Hilger, I.; Andra, W.; Hergt, R.; Hiergeist, R.; Schubert, H.; Kaiser, W. A. Radiology 2001, 218, 570-575. (5) Neilsen, O. S.; Horsman, M.; Overgaard, J. E. J. Cancer 2001, 37, 1587-1589. (6) Pankhurst, Q. A.; Connolly, J.; Jones, S. K.; Dobson, J. J. Phys. D: Appl. Phys. 2003, 36, 167-181. (7) Tartaj, P.; del Puerto Morales, M.; Veintemilas-Verdaguer, S.; Gonzalex-Carreno, T.; Serna, C. J. J. Phys. D: Appl. Phys. 2003, 36, 182-197. (8) Berry, C. C.; Curtis, A. S. G. J. Phys. D: Appl. Phys. 2003, 36, 198206. (9) Rockenberger, J.; Scher, E. C.; Alivisatos, A. P. J. Am. Chem. Soc. 1999, 121, 11595-11596.
particles by other means, dextran-coated particles continue to dominate the aforementioned applications as a result of their inherent water solubility and relative ease of functionalization.12 To take advantage of the higher quality particles available using high-temperature syntheses, several groups have published surface modification techniques including the surface exchange of cyclodextran13 or dimercaptosuccinic acid,14 intercalation of surfactants,15 and covalent attachment of poly(ethylene glycol) using trimethoxysilane.16 Here we report the direct surface ligand exchange of high quality particles with commercially available anionic octa(tetramethylammonium)-polyhedral oligomeric silsesquioxane (TMA-POSS), resulting in particles that are discrete and water-soluble. In addition, we have shown that the particles are stable over wide ranges of pH and salt concentrations and can interact with proteins via surface charge complementarity.17 Finally, we have demonstrated that TMA-POSS exchange is a general strategy that can be applied to different monolayer protected magnetic nanoparticles. Results and Discussion Surface Exchange with TMA-POSS. TMA-POSS (Figure 1a) is an octahedral molecule featuring eight siloxy groups. It is readily water-soluble, making it a potentially (10) (a) Guo, Q.; Teng, X.; Rahman, S.; Yang, H. J. Am. Chem. Soc. 2003, 125, 630-631. (b) Also see, Hyeon, T.; Lee, S. S.; Park, J.; Chung, Y.; Na, H. B. J. Am. Chem. Soc. 2001, 123, 12798-12801. (11) Sun, S.; Murray, C.; Weller, D.; Folks, L.; Moser, A. Science 2000, 287, 1989-1992. (12) Pardoe, H.; Chua-anusorn, W.; St. Pierre, T. G.; Dobson, J. J. Magn. Magn. Mater. 2001, 225, 41-46. (13) Wang, Y.; Wong, J. F.; Teng, X.; Zhang Lin, X.; Yang, H. Nano Lett. 2003, 3, 1555-1559. (14) Fauconnier, N.; Pons, J. N.; Roger, J.; Bee, A. J. Colloid Interface Sci. 1997, 194, 427-433. (15) Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktyshu, D.; Rogach, A. L.; Keller, S.; Radler, J.; Natile, G.; Parak, W. J. Nano Lett. 2004, 4, 703-707. (16) Kohler, N.; Fryxell, G. E.; Zhang, M. J. Am. Chem. Soc. 2004, 126, 7206-7211.
10.1021/cm052205i CCC: $33.50 © 2006 American Chemical Society Published on Web 01/28/2006
TMA-POSS Functionalized Iron Oxide Nanoparticles
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Figure 2. IR spectra of (a) MPN-1, (b) TMA-POSS, and (c) MPN-2. The spectra of MPN-2 exhibit characteristics from both MPN-1 (peaks at ca. 2900 cm-1 from residual alkane ligands) and TMA-POSS (1488 and 949 cm-1, with broadening at 1190 cm-1). The broad peak at 3450 cm-1 indicates that some siloxyl groups are protonated.
Figure 1. (a) Schematic of passive ligand exchange between hydrophobic MPN-1 and TMA-POSS, resulting in water-soluble MPN-2. (b) TEM images of magnetic nanoparticles exchanged with TMA-POSS. Images represent MPN-1 (in hexanes) and MPN-2 (in water) with the inset clearly showing particles transferred from nonpolar to polar solvent after exchange (scale bar ) 20 nm).
interesting candidate for the surface modification of iron oxide nanoparticles. Previously, we found that suitable ligands for surface modification exhibited two properties: multidentate surface interactions and bulkiness or branching.18 Both of these criteria are satisfied as the cubic shape of the TMA-POSS molecule lends itself to multipodal binding and its rigid three-dimensional structure gives it inherent bulkiness. To test the ability of TMA-POSS to impart water solubility to monolayer protected nanoparticles (MPNs), iron oxide nanoparticles were prepared by thermal decomposition of the iron cupferron precursor in the presence of octylamine and trioctylamine,9 resulting in the toluene/hexane soluble MPN-1. To generate water-soluble MPN-2, a solution of MPN-1 in toluene was stirred with an aqueous TMA-POSS solution. Over the course of 12 h, the particles transfer completely to the aqueous phase (Figure 1b, insets). Transmission electron microscopy (TEM) images of the particles were obtained before and after exchange (Figure 1b), demonstrating that the TMA-POSS-coated nanoparticles remain discrete and that the core size is unchanged. IR analysis was used to characterize the surface of MPN-2 upon TMA-POSS exchange. Iron oxide nanoparticles alone (MPN-1) are characterized by a grouping of peaks below 3000 cm-1, arising from the octylamine and trioctylamine ligands (Figure 2). The most prominent features of TMAPOSS arise from the silica cage, with a broad peak at 1070 (17) Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5018-5023. Hong, R.; Fischer, N.; Verma, A.; Goodman, C.; Emrick, T.; Rotello, V. J. Am. Chem. Soc. 2004, 126, 739-743. (18) Boal, A. K.; Das, K.; Gray, M.; Rotello, V. M. Chem. Mater. 2002, 14, 2628-2636.
cm-1 and two sharp peaks at 1488 and 949 cm-1. After passive modification with TMA-POSS and subsequent purification, the IR spectrum of the MPN-2 nanoparticle reflects the key characteristics of both TMA-POSS and MPN1. The two TMA-POSS-derived peaks at 1488 and 949 cm-1 are present, and a broad peak emerges at 1190 cm-1. In addition, a very broad Si-OH peak was visible at 3453 cm-1, signifying that some siloxyl groups have been protonated. Interestingly, the peaks around 2900 cm-1 indicate that some of the initial ligands remain on the MPN-2 surface. MPN-2 Stability and Biomolecule Interaction. In addition to intrinsic magnetic properties, the utility of watersoluble magnetic nanoparticles is dictated by numerous factors, including stability in physiological environments and interactions with biomolecules. Many applications require magnetic nanoparticles to exhibit stability to biologically relevant pH ranges and salt concentrations. MPN-2 shows excellent long-term solution stability, remaining stable in deionized water for more than 6 months under normal laboratory conditions. In addition, Figure 3 illustrates the stability of MPN-2 to a wide range of salt concentrations and pH. No aggregation was observed at 0.5 M NaCl, suggesting that the particles are stable above physiological salt concentrations (Figure 3a). Similarly, MPN-2 is stable in a pH range of 2-12 (Figure 3b). In addition to stability at the physiological condition, the ability to interact with biomolecules is important, having implications in cell labeling and particle uptake.19 Nanoparticles with anionic surface charge can be internalized by cells and have been successfully used as intracellular contrast agents.20 To demonstrate the negative surface charge of MPN-2 at neutral pH, agarose gel electrophoresis was conducted in the presence of two oppositely charged proteins, anionic bovine serum albumin (BSA) and cationic lysozyme (pI ≈ 5 and 11, respectively). The mobility of BSA and lysozyme alone are shown for comparison in lanes 1 and 2. As a result of an overall anionic surface charge contributed by TMA-POSS, MPN-2 exhibited significant mobility (lane 3). While BSA incubation had no effect on MPN-2 migration toward the anode (lane 4), lysozyme incubation decreased the MPN-2 mobility (lane 5). These data indicate that MPN-2 readily interacts with the cationic lysozyme by electrostatic comple(19) Nichol, C.; Kim, E. E.; J. Nucl. Med. 2001, 42, 1368-1374. (20) Wilhelm, C.; Billotey, C.; Roger, J.; Pons, J. N.; Bacri, J.-C.; Gazeau, F. Biomaterials 2003, 24, 1001-1011.
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Figure 4. TEM images of oleic acid iron oxide (a, b) and FePt (c, d) nanoparticles in hexanes (a, c) and water (b, d), respectively (scale bars ) 20 nm). Figure 3. Stability of MPN-2 (0.2 mg/mL) to increasing (a) concentrations of NaCl and (b) pH. (c) Gel electrophoresis demonstrates anionic surface charge and selective protein interaction of MPN-2 (lanes 1-5: BSA, lysozyme, MPN-2, MPN-2 + BSA, MPN-2 + lysozyme; [MPN-2] ) 10 mg/mL, [protein] ) 0.2 mg/mL).
mentarity, resulting in decreased mobility due to increased particle size and charge attenuation. Surface Modification of Diverse Magnetic Cores. Given the ease at which MPN-1 was transferred into water and our understanding of the ligand binding process to metal oxide nanoparticles, we expect that TMA-POSS would prove a general ligand to surface exchange. To determine this, we repeated the TMA-POSS surface modification with two additional MPNs featuring different starting ligand layers and cores: iron oxide nanoparticles stabilized with oleic acid10 and FePt nanoparticles11 stabilized with oleic acid, oleylamine, and hexadecanediol. Given the combination of different core materials and capping ligands, we anticipated that these two particles would provide useful insight into the versatility of our system. Both particles were effectively transferred into water upon exchange, and TEM images again illustrate that these particles are discrete (Figure 4). These studies demonstrate the versatility of TMA-POSS surface modification of three oxide nanoparticles featuring diverse sizes and ligand coatings. We anticipate that this approach is applicable to a wide range of oxide nanoparticles, allowing one to choose a specific magnetic candidate for a given application. In conclusion, we have demonstrated that passive exchange of TMA-POSS onto the surface of iron oxide nanoparticles results in water-soluble scaffolds for biologically relevant applications. Two additional nanoparticle cores were also exchanged with TMA-POSS and characterized using TEM, outlining the versatility of the method. Modification of the surface through functionalization of the TMA-POSS surface allows tailoring of the nanoparticle surface with moieties capable of imparting specificity against a given target. The TMA-POSS-modified nanoparticle was shown to be stable over a wide range of pH and salt concentrations. This method
represents a viable platform for the preparation of high quality magnetic nanoparticles for multiple biologically relevant applications. Experimental Section Materials. All the chemicals were purchased from Aldrich or Acros and were used without further purification. Solvents were purchased from Fisher Scientific or VWR and were used as received unless otherwise specified. Synthesis of MPN-1. The synthesis follows that outlined in ref 9. Briefly, 5 mL of a 0.3 M octylamine solution of iron cupferron was degassed and then slowly added to a hot, 200 °C solution of trioctylamine under an inert atmosphere. After a short, vigorous reaction the temperature was maintained for 30 min, and then the mixture was cooled and precipitated by the addition of excess ethanol. Synthesis of Oleic Acid Fe Oxide. The synthesis follows that outlined in ref 10. Briefly, 6 mL of octyl ether, 120 µL of iron pentacarbonyl, and 880 µL of oleic acid were added to a dry roundbottom flask and slowly heated to reflux. Reflux was maintained for 1 h, and then the mixture was cooled and precipitated by the addition of excess ethanol. Synthesis of FePt Nanoparticles. The synthesis follows that outlined ref 11. Briefly, 100 mg of Pt(acac)2 and 200 mg of hexadecanediol were heated in an octyl ether solution (10 mL) at 100 °C and purged with argon for 10 min. Oleic acid (80 µL), oleylamine (tech grade, 85 µL), and Fe(CO)5 (65 µL) were then added. The mixture was refluxed under argon for 0.5 h before being cooled down to room temperature. The nanoparticles were collected by precipitation in ethanol and centrifugation. Preparation of MPN-2. We observed that this reaction proceeded to completion over a wide range of TMA-POSS/nanoparticle ratios. In general, a 10:1 ratio by weight was used with an initial concentration of 10 mg/mL MPN-1 (in nonpolar solvent) and 100 mg/mL of TMA-POSS (in water). This bilayer system was sparged with argon for 2-3 min, capped, and stirred rapidly for 24 h during which time the particles transfer to the aqueous phase. The layers were carefully separated, and water-soluble MPN-2 was run through a 0.22 µM filter, dialyzed against deionized/distilled water (4-6 h) to remove free TMA-POSS, and concentrated using a spin filter
TMA-POSS Functionalized Iron Oxide Nanoparticles to the desired final concentration. The oleic acid Fe oxide and FePt particles were exchanged using the method outlined above. Infrared Spectroscopy. IR spectra were taken of KBr pellets formed from powder samples of TMA-POSS, MPN-1 and MPN-2 using a MIDAC M1200-SP3 spectrophotometer. Transmission Electron Microscopy. TEM images were acquired on a JEOL 2000FX operating at 200 keV. Samples were dropcast, either from hexanes or water, onto a copper-coated grid, dried, and imaged. Electrophoresis. Stock solutions of MPN-2 nanoparticles (20 mg/mL), lysozyme (2 mg/mL), and BSA (2 mg/mL) were prepared in distilled/deionized water. MPN-2 nanoparticles (10 µL) were incubated with proteins (2 µL) in a final volume of 20 µL for 5 min. Prior to loading, 3 µL of 80% glycerol were added to each sample. Samples were run on a 1% agarose gel in 5 mM sodium phosphate buffer (pH 7.4) at 100 V for 20 min. Proteins were stained (0.2% Brilliant Blue, 50% methanol, 10% acetic acid, aqueous solution) for 1 h, followed by extensive destaining. Stability Assays. To investigate salt stability, 10 µL of MPN-2 nanoparticles (20 mg/mL stock in distilled/deionized water) was added to 990 µL of an aqueous sodium chloride solution (0-1 M
Chem. Mater., Vol. 18, No. 4, 2006 959 range). UV-visible absorbance spectra (HP 8452A spectrophotometer) were obtained after 1 h of incubation at ambient temperature. pH stability was determined in phosphate-buffered saline adjusted with either HCl or NaOH (pH range 2-12). Again, spectra were obtained after 1 h of incubation at ambient temperature
Acknowledgment. This research was funded by the Army Breast Cancer Research Program (DAMD17-03-1-0419). B.L.F. also recognizes the ACS division of Organic Chemistry and Proctor and Gamble for a graduate student fellowship and the University of Massachusetts for a University Fellowship. N.O.F. acknowledges the U.S. DOD fellowship No. DAMD17-03-10257. Supporting Information Available: Additional characterizations, including thermogravimetric analysis, IR, UV, and gel electrophoresis (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. CM052205I
