Stabilization of Superparamagnetic Iron Oxide Core− Gold Shell

Jul 31, 2009 - Steric repulsion is the main factor to prevent the particles from falling into a deep attractive well. The well is only about −0.4 kT...
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Stabilization of Superparamagnetic Iron Oxide Core-Gold Shell Nanoparticles in High Ionic Strength Media Jit Kang Lim,†, Sara A. Majetich,‡ and Robert D. Tilton*,†,§ Department of Chemical Engineering, ‡Department of Physics, and §Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, and School of Chemical Engineering, Universiti Sains Malaysia, 14300 Seberang Prai Selatan, Penang, Malaysia

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Received June 2, 2009. Revised Manuscript Received July 17, 2009 Nanoparticles with monodisperse, spherical magnetic iron oxide cores and contiguous gold shells (Fe/Au NPs) have been synthesized in order to combine magnetophoretic responsiveness and localized surface plasmon resonance in a single nanoparticle. Such particles are sufficiently charged to be stable against flocculation in low ionic strength media, but they require surface modification to be stably dispersed in elevated ionic strength media that are appropriate for biotechnological applications. Dynamic light scattering and ultraviolet-visible spectrophotometry are used to monitor the colloidal stability of Fe/Au NPs in pH 7.4 phosphate buffered saline containing 154 mM NaCl (PBS). While uncoated particles flocculate immediately upon introduction to PBS, Fe/Au NPs with adsorbed layers of bovine serum albumin or the amphiphilic triblock copolymers Pluronic F127 and Pluronic F68 resist flocculation after more than 5 days in PBS. Adsorbed dextran allowed flocculation that was limited to the formation of small clusters, while poly(ethylene glycol) homopolymers ranging in molecular weight from 6000 to 100 000 were ineffective steric stabilizers. The effectiveness of adsorbed Pluronic copolymers as steric stabilizers was interpreted in terms of the measured adsorbed layer thickness and extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory predictions of interparticle interactions.

Introduction Magnetic nanoparticles are of interest for a variety of biomedical applications, including magnetic resonance imaging contrast agents, hyperthermia therapies and targeted drug delivery.1,2 Magnetite (Fe3O4) and maghemite (γ-Fe2O3) nanoparticles have been the most intensely studied materials, because of their ease of synthesis across a range of controllable sizes and their biocompatibility.3 Particles with dimensions on the order of 10-100 nm approach the size of important biological targets, including large proteins or protein clusters (∼5-50 nm), genes (10100 nm), or organelles (∼25-2500 nm). Such nanoparticles therefore may provide the ability to directly probe the chemical and physical properties of individual copies of these important targets, perhaps under magnetophoretic guidance in externally imposed high-gradient magnetic fields. Successfully applying magnetic nanoparticles in biological environments generally requires good colloidal stability in biological media of moderate to high ionic strength. The current state of the art in stabilizing iron oxide nanoparticles for use in biological media involves grafting a layer of macromolecules onto the particles via strong specific linkages between carboxyl or other functional groups on the macromolecule and iron oxide surface sites.4,5 This often requires chemical *To whom correspondence should be addressed. Address: Department of Chemical Engineering, Carnegie Mellon University Pittsburgh, PA 15213. E-mail: [email protected]; tel: 1-412-268-1159; fax: 1-412-268-7139. (1) Pankhurst, Q. A.; Connolly, J.; Jones, S. K.; Dobson, J. J. Phys. D: Appl. Phys. 2003, 36, R167. (2) Tartaj, P; Morales, M. P.; Veintemillas-Verdaguer, S; Gonzalez-Carreno, T.; Serna, C. J. J. Phys. D:. Appl. Phys. 2003, 36, R182. (3) Torchilin, V. P. Nanoparticulates as Drug Carriers; Imperial College Press: London, 2006; pp 401-402. (4) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26, 3995. (5) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Elst, L. V.; Muller, R. N. Chem. Rev. 2008, 108, 2064.

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modification of the macromolecules prior to grafting. Physisorption of macromolecules has also been attempted, but the stability of the stabilizing layers over more than several hours has been a source of difficulty.6 Recently there has been considerable interest in developing magnetic iron oxide nanoparticles with noble metal shells. Because of the high surface energy of gold,7 many stabilizing agents may be able to absorb strongly onto the gold surface and provide a robust steric barrier against flocculation. The prospects for combining magnetic responsiveness and localized surface plasmon resonance (LSPR) in a single composite nanoparticle have stimulated several efforts to synthesize iron oxide core/gold shell nanoparticles (Fe/Au NPs).8-14 The gold-shell LSPR provides for single nanoparticle sensing15 due to its extremely large molar extinction coefficient.16 Magnetically responsive particles can be (6) Woo, K.; Hong, J. IEEE Trans. Magn. 2005, 41, 4137. (7) Wu, S. Polymer Interface and Adhesion; Marcel Dekker, Inc: New York, 1982. (8) Lim, J. K.; Eggeman, A.; Lanni, F.; Tilton, R. D.; Majetich, S. M. Adv. Mater. 2008, 20, 1721. (9) Lyon, J. L.; Fleming, D. A.; Stone, M. B.; Schiffer, P.; Williams, M. E. Nano Lett. 2004, 4, 719. (10) Lu, Q. H.; Yao, K. L.; Xi, D.; Liu, Z. L.; Luo, X. P.; Ning, Q. J. Magn. Magn. Mater. 2006, 301, 44. (11) Lim, J. K.; Tilton, R. D.; Eggeman, A.; Majetich, S. M. J. Magn. Magn. Mater. 2007, 311, 78. (12) Zhang, J.; Post, M.; Veres, T.; Jakubek, Z. J.; Guan, J.; Wang D.; Normandin, F.; Deslandes, Y.; Simard, B. J. Phys. Chem. B. 2006, 110 7122. (13) Chen, M.; Yamamuro, S.; Farrell, D.; Majetich, S. A. J. Appl. Phys. 2003, 93, 7551. (14) Seino, S.; Kinoshita, T.; Otome, Y.; Nakagawa, T.; Okitsu, K.; Mizukoshi, Y.; Nakayama, T.; Sekino, T; Niihara, K.; Yamamoto, T. A. J. Magn. Magn. Mater. 2005, 293, 144. (15) Raschke, G.; Susha, B.; Rogach, A. L.; Klar, T. A.; Feldmann, J.; Fieres, B.; Petkov, N.; Bein, T.; Nichtl, A.; Kurzinger, K Nano Lett. 2004, 4, 1853. (16) Raschke, G.; Brogl, S.; Susha, A. S.; Rogach, A. L.; Klar, T. A.; Feldmann, J.; Fieres, B.; Petkov, N.; Bein, T.; Nichtl, A.; Kurzinger, K. Nano Lett. 2004, 4, 1853.

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used for magnetophoretically guided gene delivery,17 intracellular tracking18 and guidance,19 dynamic mechanical and rheological property measurement in20 or on21 cells, sorting of cells22 or biomolecules,23 and nanosensors.24 Progress has been made in each of these areas using magnetically responsive nanoparticles or micrometer-scale particles. Incorporation of plasmonic behavior in magnetically responsive core-shell nanoparticles will make it possible to combine magnetophoresis and optical sensing as well as optical tracking of single nanoparticles with sizes that are comparable to molecular and subcellular structures. The initial development of Fe/Au NPs has been devoted to synthesis and characterization of their optical and magnetic properties.8-14 The Fe/Au NPs are generally electrostatically stabilized in deionized water.8-11 Screening of the electrostatic repulsion between nanoparticles in biological media of elevated ionic strength necessitates the use of steric stabilization schemes to prevent flocculation. Whereas the magnetophoretic mobility and LSPR absorbance spectrum of discrete Fe/Au NPs are welldefined and can be estimated quite well, these properties change dramatically upon flocculation. Estimating the collective magnetophoretic mobility of a floc of such nanoparticles is difficult and depends on the uncertain, and polydisperse, total amount of magnetic material in the flocs25 and their shape.26 Maintaining colloidal stability at elevated ionic strengths is therefore essential for most of the intended biotechnological applications of such nanoparticles. Amal and co-workers recently stabilized iron oxide core/gold shell nanoparticles at ionic strengths up to 10 mM using poly(ethyleneimine),27 but there are not yet any reports of longterm colloidal stability at the 150 mM ionic strength that is typical of physiological fluids. A variety of macromolecules have been used to stabilize magnetic iron oxide nanoparticles at elevated ionic strengths.4,5 The most commonly used macromolecules are derivatives of dextran5 or polyethylene glycol28,29 (PEG), amphiphilic polyether triblock copolymers such as Pluronics,30 and proteins.31 For example, covalent grafting of dopamine-modified PEG maintained the stability of 9 nm magnetite nanoparticles in phosphate buffered saline (PBS) for up to 24 h. PEG-silane is another popular candidate for covalent grafting of a stabilizer to iron oxide nanoparticles.32 For most of the above-mentioned stabilizers, covalent bonds or strong specific interactions between the stabilizer and the iron oxide surface were required. The simplest mechanism to provide steric stabilization is to physically adsorb water-soluble macromolecules to the particle.33 Intermediate layers sometimes play a role in this process. For example, iron oxide nanoparticles (17) Salem, A. K.; Searson, P. C.; Leong, K. W. Nat. Mater. 2003, 2, 668. (18) Jesephson, L.; Tung, C. H.; Moore, A.; Weissleder, R Bioconjugate Chem. 1999, 10, 186. (19) Gao, J.; Zhang, W.; Huang, P.; Zhang, B.; Zhang, X.; Xu, B. J. Am. Chem. Soc. 2008, 130, 3710. (20) Bausch, A. R.; Moller, W.; Sackmann, E. Biophys. J. 1999, 76, 573. (21) Hu, S.; Eberhard, L.; Chen, J.; Love, C.; Bulter, J. P.; Fredberg, J. J.; Whitesides, G. M.; Wang, N. Am. J. Physiol. Cell Physiol. 2004, 287, C1184. (22) McCloskey, K. E.; Chalmers, J. J.; Zborowski, M. Cytometry 2000, 40, 307. (23) Kraus, R. H.; Zhou, F.; Nolan, J. P. U.S. Patent 7232691, Nov. 27, 2001. (24) Perez, J. M.; Josephson, L.; Weissleder, R. ChemBioChem 2004, 5, 261. (25) Ditsch, A.; Lindenmann, S.; Laibinis, P. E.; Wang, D. I. C.; Hatton, T. A. Ind. Eng. Chem. Res. 2005, 44, 6824. (26) Moller, W.; Nemoto, I.; Heyder, J. Eur. Cells Mater. 2002, 3, 30. (27) Goon, L. Y.; Leo, M. H.; Lai, M. L.; Munroe, P.; Gooding, J. J.; Amal, R. Chem. Mater. 2009, 21, 673. (28) Xie, J.; Xu, C.; Kohler, N.; Hou, Y.; Sun, S. Adv. Mater. 2007, 19, 3136. (29) Zhang, Y.; Kohler, N.; Zhang, M. Biomaterials 2002, 23, 1553. (30) Jain, T. K.; Morales, M. A.; Sahoo, S. K.; Leslie-Pelecky, D. L.; Labhasetwar, V. Mol. Pharmacol. 2005, 2, 194. (31) Soenen, S. J. H.; Hodenius, M.; Schmitz-Rode, T.; De Cuyper, M. J Magn. Magn. Mater. 2008, 320, 634. (32) Kohler, N; Fryxell, G. E.; Zhang, M. J. Am. Chem. Soc. 2004, 126, 7206. (33) Gonzales, M.; Krishnan, K. M. J. Magn. Magn. Mater. 2007, 311, 59.

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coated by oleic acid to promote their dispersibility in organic solvents subsequently can be dispersed into water by adsorbing a layer of Pluronic F127 triblock copolymer onto the modified nanoparticle surface.30 Most of the prior literature on iron oxide nanoparticle stabilization in high ionic strength media takes a pragmatic approach, emphasizing short-term stability for immediate usage of the particles. The long-term stability (days and beyond) is not typically reported. The main challenge is to overcome the long-range magnetic and van der Waals attractions by relying entirely on steric repulsions at ionic strengths where electrostatic double layer repulsions are insignificant. Figure 1 sketches the interaction of the core-shell nanoparticles with adsorbed polymer layers that serve as steric stabilizers. Here we compare the ability of macromolecules similar to those noted above to stabilize magnetic iron oxide nanoparticles, namely PEG homopolymers (but lacking any specific functionalization), Pluronic F127 and F68, dextran (nonfunctionalized), and bovine serum albumin (BSA), to stabilize Fe/Au NPs in pH 7.4 150 mM PBS over the course of several days (to weeks in some cases). PBS represents the typical ionic strength and pH of physiological media and is a common biological buffer. Flocculation is monitored by dynamic light scattering (DLS) and ultraviolet-visible (UV-vis) spectrophotometry. The latter exploits the strong sensitivity of the LSPR spectrum to aggregation of gold shell particles. These macromolecules are approved by the U.S. Food and Drug Administration for medical applications.34 Of the systems tested, Pluronic F127 and F68 provided the most effective stabilization. Dextran allowed arrested flocculation to the point of creating small clusters, while polyethylene oxide (PEO) homopolymers were ineffective stabilizers.

Experimental Section Materials. Potassium hydroxide (greater than 85% purity), sodium hydroxide (97% purity), tetramethylammonium hydroxide pentahydrate (TMAOH, 97% purity), PBS solution (containing 150 mM NaCl, ionic strength equivalent to 154 mM NaCl), oleylamine (70% purity), oleic acid (90% purity), iron(III) acetylacetonate (greater than 99.9% purity), iron(III) oxide hydrated (30-50 mesh), benzyl ether (99% purity), 1-octadecene (90% purity), anhydrous ethanol, anhydrous toluene (99.8% purity), 1,2hexadecanediol (90% purity), tetrakis (hydroxymethyl) phosphonium chloride solution (THPC, 80% in water), 11-mercaptoundecanoic acid (MUA, 5 mM in ethanol), Pluronic F127, Pluronic F68, dextran (MW 43,200), L-ascorbic acid, BSA (min 99%, essentially fatty acid and globulin free), and hydrogen tetrachloroaurate (III) (99.9þ%) were obtained from Sigma-Aldrich, Inc., and used as received. Gold(I) sodium thiosulfate (99.9%) was purchased from Alfa Aesar. PEG with a molecular weight of 6000 was purchased from Fluka, and 10 000 and 100 000 molecular weight PEG samples were purchased from Polysciences and used as received. All water was deionized by reverse osmosis and further treated by the Milli-Q Plus system (Millipore) to 18 MΩ cm resistivity. The hydrodynamic radius measured by DLS is listed in Table 1 for each of the macromolecules used in this study in PBS and compared with literature data for aqueous solutions.35-39 Synthesis of Iron Oxide Nanoparticles. We used the twostage, high-temperature decomposition process described in detail (34) FDA Center for Drug Evaluation and Research, Inactive Ingredient Search for Approved Drug Products, Data through July 2, 2009; http://www. accessdata.fda.gov/scripts/cder/iig/index.cfm (accessed July 17, 2009). (35) Zhang, Y.; Lam, Y. M.; Tan, W. S. J. Colloid Interface Sci. 2005, 285, 74. (36) Borbely, S.; Pedersen, J. S. Phys. B 2000, 276-278, 363. (37) Armstrong, J. K.; Wenby, R. B.; Meiselman, H. J.; Fisher, T. C. Biophys. J. 2004, 87, 4259. (38) Ioan, C. E.; Aberle, T.; Burchard, W. Macromolecules 2000, 33, 5730. (39) Yoon, J. Y.; Park, H. Y.; Kim, J. H.; Kim, W. S. J. Colloid Interface Sci. 1996, 177, 613.

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Figure 1. Schematic of two core-shell particles (dark core = iron oxide, lighter shell = gold) coated with adsorbed polymer. r1 ≈ 10 nm is the iron oxide core radius, and r2 ≈ 25 nm is the radius of the gold-coated particle (15 nm gold shell thickness). r is the particle center-to-center separation distance, and h is the separation distance between the surfaces of the gold shells (h = r - 2r2). L is the thickness of the adsorbed polymer layer. Table 1. Hydrodynamic Radii for Dissolved Macromolecules in Solution as Measured by DLS, and Adsorbed Layer Thickness of Pluronic F127 and F68 on Iron Oxide Core-Gold Shell Nanoparticles Determined by DLS and Electrokinetic Measurements coating thickness from molecular weight (g/mol)

hydrodynamic radius from DLS (nm)

hydrodynamic radius from literature (nm)

Zeta potential of particlea (mV)

DLS (nm)

electrokinetics (nm)

bare particle 25.2 -12.6 12 600 10.4 9.935 -3.5 13.4 9.3 Pluronic F127b 8400 2.7 3.336 -4.7 9.4 7.1 Pluronic F68b 37 PEG 6k 6000 3.4 2.29-2.48 PEG 10k 10 000 4.2 2.92-3.4937 PEG 100k 100 000 15.9 11.9537 dextran 43 200 6.6 5-1038 BSA 67 000 4.6 3.839 b a Measured in 1 mM NaCl solution. Pluronics are amphiphilic triblock copolymers composed of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO). Pluronic F127 is 70 wt % PEO, and F68 is 80 wt % PEO.40 Critical micelle concentrations (CMCs) at room temperature for Pluronic F127 and Pluronic 68 are 0.007 g/cm3 and 2.69 g/cm3, respectively.40

previously8 to synthesize superparamagnetic iron oxide nanoparticles. The resulting iron oxide nanoparticles were dispersed in toluene. After gently washing three times with 10 v/v% tetramethylammonium hydroxide11 and transferring into deonized water, the final product is a suspension of 18.2 ( 1.7 nm diameter iron oxide nanoparticles. The suspension had a particle concentration of 4.36  1012 cm-3. The particles had a specific magnetization of 54 emu/g and a zeta potential, ζ, of -41.9 mV in the absence of added electrolyte.8

Preparation of Iron Oxide Core/Gold Shell Nanoparticles. Following our previously described method,8 the iron oxide 13386 DOI: 10.1021/la9019734

nanoparticles were coated by MUA to promote the covalent attachment of 1.5-3 nm gold nanoparticles that had been made by Duff’s method.41 These gold-seeded nanoparticles were then subjected to an electroless gold deposition method42 to produce contiguous gold shells on the nanoparticles. The method mixes a suspension of the gold-seeded iron oxide nanoparticles with a (40) Sakai, T.; Alexandridis, P. J. Phys. Chem. B 2005, 109, 7766. (41) Duff, D. G.; Baiker, A. Langmuir 1993, 9, 2301. (42) Lam, P.; Kumar, K.; Wnek, G. E.; Przybycien, T. M. J. Electrochem. Soc. 1999, 146, 2517.

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Figure 2. Intensity weighted average hydrodynamic diameter for core-shell nanoparticles with different adsorbed macromolecules in PBS. Extensive aggregation is evident with PEG 6k, PEG10k, and PEG100k, while BSA, Pluronic F127, and Pluronic F68 provided stable hydrodynamic diameters over the course of 5 days. Although far slower than the PEG-coated nanoparticles, dextran-coated particles did aggregate, as seen in the figure on the right that reproduces the Pluronic, BSA, and dextran data on a finer scale. “Day 0” corresponds to the start of the overnight adsorption of polymers to the particles. plating solution of gold sodium thiosulfate and ascorbic acid in deionized water, with the pH adjusted to 6.4 by titration with 1 M NaOH. Once the formation of a sky blue suspension was observed, an excess of deionized water is added to quench the gold reduction. At this stage, the nanoparticles are electrostatically stabilized in deionized water with a negatively charged surface (ζ=-12.6 mV in 1 mM NaCl). The intensity-weighted mean hydrodynamic diameter of the Fe/Au NPs measured by DLS is 50 ( 25 nm (see Supporting Information, Figure S1).

Steric Stabilization of Iron Oxide Core/Gold Shell Nanoparticles. To form a sterically stabilized nanoparticle suspension, the final quenching step described above for the electroless gold deposition procedure is performed in the presence of the macromolecule of interest (BSA, Dextran 43200, PEG 6k, PEG 10k, PEG 100k, Pluronic F127 or F68) at a concentration of 0.0025 g/cm3. This macromolecule concentration is chosen to ensure that there is at least 5 orders of magnitude more polymer than would be needed to saturate the nanoparticle surfaces in the final suspension of 5.27  1012 particles/cm3 (see Supporting Information, Table S1). Immediately after adding the macromolecule solution, the suspension was ultrasonicated (Fisher Scientific ultrasonic probe FS-14) for 10 min and then incubated overnight with continuous gentle agitation by an end-over-end rotating mixer at 22 rpm to allow the macromolecules to adsorb to the nanoparticles. The macromolecule-coated nanoparticles were collected by a permanent magnet while the supernatant was decanted, and the retentate was resuspended into 5 mL of a macromolecule-free, 154 mM ionic strength, pH 7.4 PBS solution under ultrasonication for 10 min. Nanoparticle Characterization. The size distributions of the nanoparticles before and after macromolecule adsorption were determined by DLS (Malvern Instruments Nanosizer ZS). The light scattering intensity autocorrelation function was fitted by the CONTIN algorithm to produce an intensity-weighted distribution of hydrodynamic radii. DLS also provides a means to monitor nanoparticle flocculation by recording shifts in the particle size distribution to higher values, and a broadening of the size distribution (as quantified by full width at half-maximum, fwhm) as flocs nucleate and grow. The electrophoretic mobility of the Fe/Au NPs was measured (Malvern Instruments Nanosizer ZS) before and after adsorption, and was converted to ζ using the Helmholtz-Smoluchowski (high κa) limit for high ionic strength conditions or the H€ uckel (low κa) limit for low ionic strength conditions. Here κ is the reciprocal of the Debye screening length, and a is the particle hydrodynamic radius. Langmuir 2009, 25(23), 13384–13393

UV-vis absorbance spectra of nanoparticle suspensions were recorded (Cary 300 Scan, Varian, Inc.) to monitor flocculation via the shift in the LSPR peak.

Results and Discussion Dynamic Light Scattering. DLS was employed to measure the size distribution of Fe/Au NPs over the course of 5 days in suspension in 154 mM ionic strength PBS. Uncoated Fe/Au NPs flocculated immediately and extensively after their introduction to PBS. The intensity weighted average hydrodynamic diameter DH is plotted over 5 days in PBS for each of the macromoleculecoated Fe/Au NPs in Figure 2. Both PEG 6k and PEG 10k tentatively stabilized the particles in PBS for the first 24 to 48 h, consistent with previous observations on PEG-coated iron oxide nanoparticles.6 After this period of relative stability, aggregation accelerated to produce large aggregates by day 3. Since these PEG samples have rather low degrees of polymerization, the loss of stability over a day or two could have been due to slow PEG desorption that would not be expected of larger polymers. Nevertheless, PEG100k-coated Fe/Au NPs were not as well stabilized as the PEG6k- or PEG10k-coated ones, despite the higher degree of polymerization that one might expect to produce greater adsorbed layer thicknesses and therefore longer ranged steric forces. Submicrometer aggregates were detected by DLS already on day 1. With a hydrodynamic diameter of 32 nm, PEG100k is comparable in size to the Fe/Au NPs themselves. For this case where the polymer and nanoparticle have similar sizes, one expects the formation of less dense adsorbed layers and fewer adsorbed chains per particle compared to cases with a larger particle-to-polymer size ratio. We hypothesize that, in such a case, bridging flocculation is more probable. This is consistent with prior observations that larger poly(styrene sulfonate) (PSS) chains were less effective steric stabilizers than smaller PSS chains for quasi-single domain magnetic nanoparticles with Fe0 cores and magnetite shells, when the polymers and particles were of comparable size. This correlated with the adsorption of far less dense layers for the larger polymer chains.43 (43) Phenrat, T.; Saleh, N.; Sirk, K.; Kim, H. J.; Tilton, R. D.; Lowry, G. V. J. Nanopart. Res. 2008, 10, 795.

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Figure 3. Intensity weighted DLS size distributions for Pluronic F127-, F68-, BSA- and dextran-coated Fe/Au NPs in PBS.

Compared to the PEG homopolymer results, the mean hydrodynamic diameter for Fe/Au NPs coated with Pluronic F68, Pluronic F127, BSA or dextran was considerably more stable in PBS for 5 days. Close inspection of the data in Figure 2 shows that the average DH of BSA-coated and Pluronic-coated Fe/Au NPs was unchanged for 5 days, but dextran-coated particles experienced a small increase in DH from 77 nm at day 1 to 105 nm at day 5. The intensity weighted size distributions are plotted in Figure 3 for each of the Fe/Au NPs that showed better stability over 5 days. Although the dextran-coated nanoparticles showed the most significant increase in DH over 5 days, the size distribution remained relatively sharp. The fwhm on day 5 was fairly similar for dextran-coated particles (269 nm) and for F68-coated particles (274 nm). The sharpest distribution, with fwhm = 103 nm, is for Pluronic F127-coated particles. The broadest size distribution, with fwhm = 448 nm, is for BSA-coated nanoparticles. For comparison, the fwhm for the uncoated Fe/Au NPs dispersed in deionized water is 92 nm (see Supporting Information, Figure S1). The significant broadening of the size distribution of BSAcoated particles at day 5 would suggest the formation of small clusters, but DLS alone is not conclusive here. A small peak is evident in the day 1 size distribution that matches the size of unadsorbed BSA in solution. The distribution on day 5 shows a shoulder below 10 nm. This shoulder contributes to the broadening of the distribution. Deconvolution of a bimodal size distribution sometimes fails to resolve neighboring peaks when they are not sufficiently separated in size. The large fwhm on day 13388 DOI: 10.1021/la9019734

5 could merely be an artifact of the inability of the CONTIN algorithm to successfully deconvolute the nanoparticle and unadsorbed BSA contributions to the autocorrelation function. The LSPR absorbance data, shown below, do support the conclusion that BSA-coated particles in fact are colloidally stable. On the basis of the stability of DH and fwhm, Pluronic F127 and F68 appear to be the most effective stabilizers. Figure 4 shows a typical TEM image of Pluronic F127-coated Fe/Au NPs that had been dispersed in PBS for 5 days before microscopy observation. The samples for TEM were prepared by drying suspensions in PBS directly onto the carbon-coated TEM grid. The absence of particle aggregates, even after drying for TEM, is evident. These observations indicate that steric stabilization by Pluronic F127 maintains stable suspensions in a high ionic strength environment. It is a known problem that due to its high surface energy, gold is easily contaminated by impurities from the surrounding fluid, often leading to colloidal instability. Hence, besides preventing the particles from aggregation, the stabilizing agents should also play a role in preventing nonspecific and irreversible adsorption of foreign protein onto the particles. For example, Fe/Au NPs might be employed in protein-rich biological fluids, such as blood plasma. Then, one should be concerned about the displacement of the adsorbed stabilizer by blood proteins, the most abundant of which is serum albumin. Thus, we suspended the F127-coated Fe/Au NPs into PBS that also contained 0.03 g/cm3 BSA. The typical plasma protein concentration in blood is around 0.03-0.05 g/cm3. Langmuir 2009, 25(23), 13384–13393

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Figure 4. Transmission electron micrograph of Fe/Au NPs after adsorption of Pluronic F127, transfer into PBS, and deposition onto a TEM grid.

DLS measurements made after 72 h in BSA-PBS solution, shown in Figure 5, revealed no significant change in DH or fwhm when challenged by BSA in solution. Thus, Pluronic F127-coated Fe/Au NPs were not destabilized by these serum proteins. While DLS confirms that these particles retain their colloidal stability during a BSA challenge, it does not directly prove that BSA could not displace preadsorbed Pluronic F127. Nevertheless, this finding is consistent with a previous finding that preadsorbed Pluronics impart protein adsorption resistance to surfaces.44 Spectrophotometry. Unmodified Fe/Au NPs form blue suspensions in deionized water as a result of the optical properties and geometry of the core-shell constituents.8 As Fe/Au NPs flocculate to form clusters of varying size and shape, a rapid color change is observed (see Supporting Information, Figure S2) due to the plasmon-plasmon interaction among the closely spaced particles.45 The plasmon coupling broadens and shifts the LSPR peak to higher wavelengths.46 Formation of flocs with different shapes furthermore produces higher order multipole terms that also contribute to the LSPR spectral broadening.47 Figure 6 compares UV-vis absorbance spectra of Fe/Au NPs with and without adsorbed macromolecules in both deionized water and in PBS. The spectrum of bare Fe/Au NPs broadens dramatically after being transferred into PBS as a result of the extensive flocculation that occurs when the electrostatic double layer repulsion between the bare particles is screened at high ionic strength. The absorbance spectrum of flocculated samples contains two broad resonance peaks with maxima at 526 and 685 nm. The development of the longer wavelength peak is consistent with the plasmon-plasmon interaction between clustered particles as (44) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. J. Biomed. Mater. Res. 1998, 42, 165. (45) Oldenburg, S. J.; Averitt, R. D.; Westcott, S. L.; Halas, N. J. Chem. Phys. Lett. 1998, 228, 243. (46) Haes, A. J.; Haynes, C. L.; McFarland, A. D.; Schatz, G. C.; Van Duyne, R. P.; Zou, S. MRS Bull. 2005, 30, 368. (47) Link, S.; El-Sayed, M. A. Int. Rev. Phys. Chem. 2000, 19, 409. (48) Westcott, S. L.; Oldenburg, S. J.; Lee, T. R.; Halas, N. J. Chem. Phys. Lett. 1999, 300, 651.

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Figure 5. Size distribution obtained by DLS for Pluronic F127coated Fe/Au NPs in deionized water, in 154 mM PBS, and in a 0.03 g/cm3 BSA solution in 154 mM PBS.

predicted and observed by others.48 Thus, the LSPR spectrum provides a convenient and sensitive indication of Fe/Au NP flocculation. There is a small deviation of less than ∼5 nm in the resonance peak for Fe/Au NPs with different coatings, in deionized water or in PBS. Adsorption of macromolecules or transfer into PBS perturbs the local dielectric environment and shifts the LSPR resonance, as expected from Mie theory.49 Figure 6a-c shows that the absorbance spectra of Fe/Au NPs coated with Pluronic F127, F68, or BSA are extremely similar before and after being transferred into PBS. In contrast, dextrancoated particles (Figure 6d) show a dramatic spectral broadening within 30 min of transfer into PBS, although the spectrum is still quite different from that of the extensively flocculated bare Fe/Au NPs in PBS. No visible sedimentation of flocs was observed. The spectra before and after dextran adsorption on the particles in deionized water were only slightly shifted relative to one another, consistent with the altered local dielectric environment but without flocculation. Together with the small increase in DH measured by DLS, the spectrophotometry results suggest that dextrancoated Fe/Au NPs do indeed flocculate, but the process is limited to the formation of relatively small clusters. The spectrophotometry results are revealing, in light of the somewhat uncertain conclusions that could be drawn from the DLS size distribution for BSA-coated Fe/Au NPs. Figure 6c shows very little shift in the LSPR peak upon transfer into PBS. The results are similar to those obtained for Pluronic F127 and F68. Thus, the spectrophotometry data indicate that BSA should also be considered to be an effective stabilizer in PBS. Importance of the Gold Shell for Colloidal Stability. A batch of the core iron oxide nanoparticles was held aside without depositing the gold shell. These particles were electrostatically stabilized in deionized water. We attempted to stabilize these particles in 150 mM PBS by adsorbing each of the polymers tested above in deionized water, then transferring into PBS. None was able to stabilize the iron oxide particles, as each sample flocculated soon after the transfer. This is consistent with previous (49) McFarland, A. D.; Van Duyne, R. P. Nano Lett. 2003, 3, 1057.

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Figure 6. Absorbance spectra of Fe/Au NPs with various adsorbed macromolecules, normalized to the maximum absorbance. Absorbances are normalized to account for variations in concentration. Gray continuous curves represent Fe/Au NPs with the indicated macromolecule coating in deionized water; black dashed curves represent Fe/Au NPs with the indicated macromolecule coating in PBS. Each plot also reproduces data for uncoated Fe/Au NPs in deionized water (black continuous curves) and uncoated Fe/Au NPs in PBS (gray dotted curves). (a) Pluronic F127 (spectra for coated and uncoated particles in deionized water perfectly overlap one another), (b) Pluronic F68, (c) BSA, and (d) Dextran 42 300.

observations discussed in the Introduction regarding the difficulty of stabilizing iron oxide nanoparticles by polymer physisorption from solution. The gold shell is apparently highly advantageous for sterically stabilizing these particles by physisorption of macromolecules. A notable feature of the gold shells is their roughness (Figure 4). Suresh and Walz50 have shown that increased roughness tends to increase the strength of van der Waals attractions at small separations and tends to lower the energy barrier against flocculation of electrostatically stabilized particles. This detrimental feature of the gold shell is evidently overcome by the strong adsorption of stabilizing macromolecules. Estimation of Adsorbed Pluronic F127 and F68 Layer Thicknesses. The thickness of the absorbed layers dictates the minimum interparticle spacing in suspension. The stability of the suspensions is directly related to this layer thickness. To help interpret the colloidal stability results, we used two independent methods to estimate adsorbed layer thicknesses for Pluronic F127 and F68, two of the successful stabilizers. The first method is to (50) Suresh, L; Walz, J. Y. J. Colloid Interface Sci. 1996, 183, 199.

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simply measure the mean hydrodynamic radius of the particle before and after adsorption via DLS (Figure 7). The difference provides a measure of the hydrodynamic layer thickness. These results are reported in Table 1. The layer thickness is comparable to (F127) or several-fold larger than (F68) the hydrodynamic diameter of the free polymer in solution, indicating a moderately stretched chain conformation on the surface. For verification, we also used a previously described method to estimate adsorbed nonionic polymer layer thicknesses based on the measurement of ζ-potential before and after polymer adsorption. Initially applied to polymer adsorption within porous media51 and on flat surfaces,52,53 here we make a simple modification to apply the method to spherical particles. The ζ-potential, defined as the electrical potential at the hydrodynamic plane of shear adjacent to a charged surface, (51) Pagac, E. S.; Prieve, D. C.; Y. Solomentsev, Y.; Tilton, R. D. Langmuir 1997, 13, 2993. (52) Braem, A. D.; Biggs, S.; Prieve, D. C.; Tilton, R. D. Langmuir 2003, 19, 2736. (53) Berglund, K. D.; Timko, A.; Przybycien, T. M.; Tilton, R. D. Prog. Colloid Polym. Sci. 2003, 122, 56.

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Article

Interaction Energies between Core/Shell Nanoparticles. The effect of the gold shell and adsorbed polymer layer on nanoparticle stability can be interpreted in terms of an extended Derjaguin-Landau-Verwey-Overbeek (DLVO) analysis of the interparticle interactions. The four major interaction energies at work here are van der Waals (UvdW) and magnetic (Umag) attractions, and electrostatic double layer (Uelec) and steric (Usteric) repulsions. These are assumed to be additive, so the total interparticle interaction energy becomes55 Utotal ¼ UvdW þ Umag þ Uelec þ Usteric

ð3Þ

We calculated interaction energies for a spherical iron oxide core surrounded by a concentric, contiguous gold shell, on which is adsorbed a polymer layer as shown in Figure 1. The van der Waals attraction between two homogeneous spherical particles of radii a1 and a2 at a center-to center separation distance r is Figure 7. Size distribution of uncoated and Pluronic F127-coated core-shell nanoparticles measured by DLS in deionized water.

dictates the electrophoretic mobility of a colloidal particle. In the case of neutral polymers, the primary effect of adsorption on the electrophoretic mobility is to shift the plane of shear further out from the charged surface, within the electrostatic double layer. The plane of shear moves to a distance determined mainly by the extension of polymer tail segments from the surface. Since electrical potential decays with increasing distance from the surface in the double layer, the ζ-potential measured after polymer adsorption is smaller than that measured before adsorption. Thus an adsorbed layer thickness determined by electrokinetic means should be similar to other hydrodynamic measurements, assuming that the adsorbed layer does not alter the degree of ionization of the underlying surface. The decay of electrostatic potential ψ within the double layer around a charged sphere of radius a is approximated by54 ψðrÞ ¼ ψo

a exp½ - Kðr - aÞ r

ð1Þ

where r is the radial distance from the center of particles, ψo is the surface potential, and κ-1 is the Debye screening length: !1=2 2ce2 z2 -1 ð2Þ K ¼ εr ε0 kT Here c is the electrolyte concentration, ε0 is the vacuum permittivity, and εr is the relative permittivity of water. The charged surface underlying the adsorbed polymer layer has a potential ψo that here is equated to ζbare, the ζ-potential of the uncoated particle. The hydrodynamic thickness of the adsorbed polymer layer is then simply equated to the distance δ=r-a from the underlying charged surface at which ψ (r)= ζafter, the ζ-potential measured after adsorption in the same ionic strength solution. For the layer thickness determination, we conducted ζ-potential measurements at a low ionic strength (1 mM NaCl) before and after adsorption in order to maintain colloidal stability for the bare particles. The calculated layer thickness for Pluronic F127 (ζbare = -12.6 mV, ζafter = -3.5 mV) is 9.3 nm. For Pluronic F68 (ζbare =-12.6 mV, ζafter =-4.7 mV), the thickness is estimated to be 7.1 nm. These layer thicknesses are comparable to those estimated by DLS: 13 nm for F127 and 9.4 nm for F68. (54) Hunter, R. J. Foundations of Colloid Science; Clarendon Press: Oxford, U.K., 1986; Vol. I.

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UvdW ¼ -Aeff ðr -a1 -a2 ÞH

  r a

ð4Þ

where Aeff is the effective Hamaker constant and "   r 1 2a1 a2 2a1 a2 ¼ þ H 2 2 2 a 6 r -ða1 þ a2 Þ r -ða1 - a2 Þ2 # r2 -ða1 þ a2 Þ2 þ ln r2 -ða1 - a2 Þ2

ð5Þ

The situation is more complex for core-shell particles. We used the approach developed by Viravathana and Marr,56 who used Hamaker theory57 to geometrically decompose the van der Waals attraction between core-shell particles in terms of van der Waals interactions among homogeneous spheres consisting of either the core or the shell material. Details are provided in the Supporting Information (Figure S3 and related explanation). For the gold-gold interaction in water, we used the literature value AAu-Au = 3  10-19 J,58 and we used the method of Mahanty and Ninham57 to calculate AFeox-Feox for the partially retarded iron oxide-iron oxide van der Waals interaction in water. The geometric mean mixing rule AFeox-Au = (AFeox-FeoxAAu-Au)1/2 was used to estimate the attraction between dissimilar materials. To calculate the magnetic attraction, the iron oxide cores of the two interacting particles were assumed to have their magnetic spins perfectly aligned. This provides the worst case, maximum conceivable magnetic attraction between the superparamagnetic particles. Thus the magnetic attraction between particles with iron oxide cores of radius r1 is calculated as59 Umag ¼ -

8πμ0 r1 6 Ms 2 9ðr -2r1 Þ3

ð6Þ

where μ0 is the permeability of free space (1.26  10-6 m kg s-2 A-2), and Ms is the measured saturation magnetization for the iron oxide nanoparticles (Ms = 2.86  105 A m-1).8 (55) Mefford, O. T.; Vadala, M. L.; Goff, J. D.; Carroll, M. R. J.; Mejia-Ariza, R.; Caba, B. L.; Pierre, T. G. St.; Woodward, R. C.; Davis, R. M.; Riffle, J. S. Langmuir 2008, 24, 5060. (56) Viravathana, P.; Marr, D. W. M. J. Colloid Interface Sci. 2000, 24, 301. (57) Russel,W. B.; Saville, D. A.; Schowalter, W. R. Colloidal Dispersions; Cambridge University Press: Cambridge, U.K., 1989. (58) Parsegian, V. A.; Weiss, G. H. J. Colloid Interface Sci. 1981, 81, 285. (59) Rosensweig, R. E. Ferrohydrodynamics; Cambridge University Press: New York, 1985.

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Figure 8. Extended DLVO interaction potential between two Fe/Au nanoparticles. (Left) Total and contributing interactions for two particles with 20 nm iron oxide cores, 15 nm thick gold shells, and a 13.4 nm thick adsorbed Pluronic F127 layer. (Right) Comparison of total interaction energy between particles with 20 nm iron oxide cores and varying gold shell thicknesses. All have a 13.4 nm thick Pluronic F127 layer.

The steric repulsion between two particles of radius r2 with adsorbed polymer layers was modeled as60 8 >  < ¥;  r < 2r2 9 1 1 Usteric ¼ U0 -lnðyÞ - ð1 -yÞ þ ð1 -y3 Þ - ð1 -y6 Þ ; > 5 3 30 : 0; r > 2ðr2 þ LÞ

where r -2r2 y ¼ 2L and U0 ¼

! π3 Lσp kT aL2 12Np l 2

ð8Þ

ð9Þ

Here L is the thickness of the absorbed polymer layer (taken to be 13.4 nm for Pluronic F127 based on DLS), Np is the number of segments in polymer chain, l is the segment length, and σp is the surface density of adsorbed chains. Here the expected conformation of an adsorbed amphiphilic triblock copolymer is assumed. Each adsorbed Pluronic F127 polymer is assumed to be anchored by the central PPO block that is not extended, while two PEO blocks extend into solution. Each triblock copolymer then presents two unadsorbed PEO tails (each containing Np = 100 segments, with a segment length l=2.91 A˚61). The surface concentration of Pluronic F127 on gold has been reported to be 0.5 molecules/nm2.62 Since each adsorbed molecule presents two extended PEO tails, we set σp =1.0 nm-2. The electrostatic double layer repulsion originates from the surface underlying the adsorbed polymer layer and is calculated as55 Uelec ¼ 2πr2 εε0 ζ2 ln½1 þ expð -KhÞ

ð10Þ

(60) Genz, U.; D’Aguanno, B.; Mewis, J.; Klein, R. Langmuir 1994, 10, 2206. (61) Lam, Y. M.; Goldbeck-Wood, G. Polymer 2003, 44, 3593. (62) Glomm, W. R.; Bidegain, B. F.; Volden, S.; Sjoblom, J. J. Dispersion Sci. Technol. 2006, 27, 651.

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2r2 < r < 2ðr2 þ LÞ

ð7Þ

where ζ is approximated by the zeta potential of the coated coreshell nanoparticle (-3.5 mV at 150 mM ionic strength), and κ-1 is the Debye length (0.8 nm at 150 mM ionic strength). The strong screening makes this interaction unimportant at elevated ionic strength. The predicted interaction energy between Fe/Au NPs with 20 nm diameter iron oxide cores surrounded by 15 nm thick gold shells and stabilized by adsorbed Pluronic F127 in 154 mM PBS is plotted in Figure 8. Steric repulsion is the main factor to prevent the particles from falling into a deep attractive well. The well is only about -0.4 kT deep. Aggregation is expected to be weak for well depths less than ∼1.5 kT.63 Hence, any weak aggregation should be disrupted by thermal motion. This is consistent with the observation that Pluronic F127 coated particles were still unflocculated after 45 days in PBS (see Supporting Information for DLS data). Coating the iron oxide particle with a gold shell having a large Hamaker constant comes at the risk of increasing the van der Waals attraction between particles. Figure 8 shows that the depth of the attractive well in Utot does increase with increasing gold shell thickness, but the well depth is still less than 1 kT for shells as thick as 20 nm. The desirability of strong steric repulsion is evident in preventing particle access to deeper attractive wells.

Conclusions Superparamagnetic nanoparticles with iron oxide cores and contiguous gold shells can be stabilized in media of physiologically relevant ionic strengths (150 mM) for at least 5 days by adsorption of macromolecules from solution. BSA and Pluronic F127 and F68 are better stabilizing agents than PEG homopolymers spanning from 6000 to 100 000 in molecular weight. Insights obtained from the combination of DLS and spectrophotometric (63) Napper, D. H. Polymeric Stabilization of Colloidal Dispersions; Academic Press: New York, 1983.

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measurement of the plasmonic band indicates that dextran-coated particles aggregated to small clusters, but these did not grow to large flocs. Those polymers that did stabilize core/shell nanoparticles were not effective stabilizers for the iron oxide nanoparticles. This demonstrates that the gold shell is clearly advantageous for colloidal stability. Gold provides a superior platform for adsorbing robust macromolecular layers for steric stabilization. The stabilizing effectiveness of the Pluronics was consistent with layer thickness measurements that indicated the development of wellextended chain conformations on the particle surface and with corresponding extended DLVO theory calculations that indicate only weak attractive wells in the interparticle potential. Pluronic coated iron oxide core/gold shell nanoparticles were stable for as long as 45 days in pH 7.4, 150 mM PBS. Exposure of the Pluronicstabilized nanoparticles to concentrated albumin solutions did not compromise their colloidal stability.

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Acknowledgment. This material is based in part on work supported by the National Science Foundation (Grant No. CBET-0853963). Jit Kang Lim gratefully acknowledges the financial support of the Dowd-ICES fellowship of Carnegie Mellon University. Supporting Information Available: Intensity weighted hydrodynamic diameter distribution for bare iron oxide core/gold shell nanoparticles (Fe/Au NPs) in deionized water, available surface area calculation for macromolecule adsorption, color change upon flocculation of Fe/Au NPs, calculation of the van der Waals attraction between Fe/Au NPs in water, and hydrodynamic diameter distribution of Pluronic F127-coated Fe/Au NPs after 1, 5, and 45 days in PBS. This material is available free of charge via the Internet at http://pubs.acs.org.

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