Stabilization of Magnetic Iron Oxide Nanoparticles in Biological Media

Dec 20, 2010 - As a control, gel electrophoresis was also carried out for neat ... using Mascot Daemon software from Matrix Science (www.matrixscience...
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Stabilization of Magnetic Iron Oxide Nanoparticles in Biological Media by Fetal Bovine Serum (FBS) Hilda T. R. Wiogo,† May Lim,† Volga Bulmus,‡ Jimmy Yun,§ and Rose Amal*,† †

ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, University of New _ South Wales, Sydney NSW 2052, Australia, ‡Department of Chemical Engineering, Izmir Institute of Technology, G€ ulbahc-e Urla 35430, Turkey, and §NanoMaterials Technology Pte. Ltd., Block 28, Ayer Rajah Crescent No. 03-03, 139959, Singapore Received October 26, 2010. Revised Manuscript Received December 6, 2010

A facile method of stabilizing magnetic iron oxide nanoparticles (MNPs) in biological media (RPMI-1640) via surface modification with fetal bovine serum (FBS) is presented herein. Dynamic light scattering (DLS) shows that the size of the MNP aggregates can be maintained at 190 ( 2 nm for up to 16 h in an RPMI 1640 culture medium containing g4 vol % FBS. Under transmission electron microscopy (TEM), a layer of protein coating is observed to cover the MNP surface following treatment with FBS. The adsorption of proteins is further confirmed by X-ray photoelectron spectroscopy (XPS). Gel electrophoresis and LC-MS/MS studies reveal that complement factor H, antithrombin, complement factor I, R-1-antiproteinase, and apolipoprotein E are the proteins most strongly attached to the surface of an MNP. These surface-adsorbed proteins serve as a linker that aids the adsorption of other serum proteins, such as albumin, which otherwise adsorb poorly onto MNPs. The size stability of FBS-treated MNPs in biological media is attributed to the secondary adsorbed proteins, and the size stability in biological media can be maintained only when both the surfaceadsorbed proteins and the secondary adsorbed proteins are present on the particle’s surface.

Introduction One key limitation to the application of magnetic nanoparticles in biomedicine is the tendency of the particles to aggregate, particularly in high-ionic-strength solutions such as biological media. Therefore, a major research direction in this field is the stabilization of particles against aggregation. Magnetic nanoparticle aggregation occurs because of the strong magnetic attraction between the nanoparticles and the need to reduce the high surface energies that result from the high surface area to volume ratio.1 The high ionic strength in biological media further exacerbates the problem of aggregation by suppressing the double-layer repulsion between particles. Particle aggregates that are larger than 4 μm are undesirable in biological applications because they have a reduced surface area for functionalization and may cause a blockage of blood capillaries in vivo.2 In addition, large aggregates of nanoparticles may also be recognized as foreign by the body’s immune system and eliminated by the reticuloendothelial system (RES), reducing their circulation time in vivo.3 The key to stabilizing magnetic nanoparticles against aggregation in biological fluid is to overcome magnetic and van der Waals attraction forces, which act to attract the particles to one another. Most research on this is focused on modifying the surfaces of *Corresponding author. Tel: þ6129385 4361. Fax: þ6129385 5966. E-mail: [email protected]. (1) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N. Chem. Rev. 2008, 108, 2064–2110. (2) Neuberger, T.; Sch€opf, B.; Hofmann, H.; Hofmann, M.; Von Rechenberg, B. J. Magn. Magn. Mater. 2005, 293, 483–496. (3) Fukumori, Y.; Ichikawa, H. Adv. Powder Technol. 2006, 17, 1–28. (4) Bhattarai, S. R.; Kc, R. B.; Kim, S. Y.; Sharma, M.; Khil, M. S.; Hwang, P. H.; Chung, G. H.; Kim, H. Y. J. Nanobiotechnol. 2008, 6, 1–9. (5) Kawaguchi, T.; Hanaichi, T.; Hasegawa, M.; Maruno, S. J. Mater. Sci.: Mater. Med. 2001, 12, 121–127. (6) Ma, H.; Qi, X.; Maitani, Y.; Nagai, T. Int. J. Pharm. 2007, 333, 177–186. (7) Barrera, C.; Herrera, A. P.; Rinaldi, C. J. Colloid Interface Sci. 2009, 329, 107–113. (8) Gu, L.; Shen, Z.; Feng, C.; Li, Y.; Lu, G.; Huang, X.; Wang, G.; Huang, J. J. Mater. Chem. 2008, 18, 4332–4340.

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magnetic nanoparticles with natural4-6 or synthetic polymers7-12 or with inorganic materials such as silica or gold.13 These surface modifications introduce steric and/or electrostatic repulsions that prevent the magnetic particles from aggregating. Although considerable success has been achieved in stabilizing magnetic nanoparticles in water, the challenge in producing magnetite nanoparticles with long-term aggregation stability in high-ionic-strength media, particularly in biological fluid, remains. Our group had successfully synthesized magnetite core-gold shell nanoparticles, which can be maintained at 200 nm in a 10 mM NaCl solution at pH 7 with the aid of poly(ethyleneimine) (PEI).14 Lutz et al. have reported the use of poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid) to modify the surface of 10 nm iron oxide nanoparticles for use as an MRI contrast agent.15 They reported that the modified iron oxide nanoparticles were stable in Tris-buffered saline solution but did not provide any long-term stability data. Tural et al. reported the use of poly(methacrylic acid) (PMAA) to modify and functionalize the surface of magnetite nanoparticles with Ni(II)-nitrilotriacetic acid (Ni-NTA). They claimed that the modified nanoparticles would be stable in biological environments because of the highly negative zeta potential of the modified nanoparticles at neutral pH but did not provide any experimental evidence.16 A more (9) Jain, N.; Wang, Y.; Jones, S. K.; Hawkett, B. S.; Warr, G. G. Langmuir 2010, 26, 4465–4472. (10) Kim, M.; Jung, J.; Lee, J.; Na, K.; Park, S.; Hyun, J. Colloids Surf., B 2010, 76, 236–240. (11) Lin, C. L.; Lee, C. F.; Chiu, W. Y. J. Colloid Interface Sci. 2005, 291, 411– 420. (12) Wan, S.; Huang, J.; Guo, M.; Zhang, H.; Cao, Y.; Yan, H.; Liu, K. J. Biol. Mater. Res., Part A 2007, 80, 946–954. (13) Wu, W.; He, Q.; Jiang, C. Nanoscale Res. Lett. 2008, 3, 397–415. (14) Goon, I. Y.; Lai, L. M. H.; Lim, M.; Munroe, P.; Gooding, J. J.; Amal, R. Chem. Mater. 2009, 21, 673–681. (15) Lutz, J. F.; Stiller, S.; Hoth, A.; Kaufner, L.; Pison, U.; Cartier, R. Biomacromolecules 2006, 7, 3132–3138. € (16) Tural, B.; Kaya, M.; Ozkan, N.; Volkan, M. J. Nanosci. Nanotechnol. 2008, 8, 695–701.

Published on Web 12/20/2010

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recent study by Lim et al. found that gold-coated magnetic iron oxide nanoparticles can be stabilized in phosphate-buffered saline (PBS), a high-ionic-strength solution, when pluronic copolymers or bovine serum albumin (BSA) were present on its surfaces.17 There have also been several recent studies on the effect of serum adsorption on the behavior of nanoparticles in biological systems, focusing mainly on its effect on cell uptake and the cytotoxicity of the particles.18-24 The adsorption of serum was shown to result in a significant reduction in the size of the magnetic nanoparticle aggregates. Petri-Fink et al., for instance, observed that iron oxide nanoparticles modified with poly(vinyl alcohol) (PVA) and PVA containing amine group (A-PVA) had a different degree of stability in media supplemented with fetal calf serum (FCS).24 Chen et al. subsequently showed the ability of FCS to stabilize magnetite nanoparticles functionalized with dimercaptosuccinic acid in RPMI-1640 over a period of five days, but they did not perform any studies on the factors responsible for the stability. Chen et al. later investigated the adsorption of proteins from fetal bovine serum (FBS) onto the surface of silanesmagnetite nanoparticles functionalized with either amino groups, poly(ethylene glycol), or carboxylic acid and found that protein adsorption was highest for the particles having carboxylic acid groups on the surface.19 A study by Mu et al. further confirmed that magnetic nanoparticles with carboxylic acid groups on their surfaces bind more serum protein compared to particles with aminated surfaces.23 The fact that serum can stabilize magnetic nanoparticles in biological fluid is of significant interest and warrants further investigation, particularly with respect to the identity of the proteins that contribute to the stabilization effect and the mechanisms by which the stabilization occurs. Herein, we provide a comprehensive study using magnetic nanoparticles (MNP) with a carboxyl group on the surface and RPMI-1640, a commercially available cell culture medium, as the model system. The magnetic nanoparticles were treated with fetal bovine serum (FBS), a widely available serum that is commonly used in cell culture studies to mimic the protein contents and properties of a real biological fluid. The FBS-MNP composite was investigated using transmission electron microscopy (TEM), and its stability in biological media was monitored by measuring the hydrodynamic diameter of the particles using dynamic light scattering (DLS). The interaction between the proteins in FBS and the MNP surface was investigated using X-ray photoelectron spectroscopy (XPS). The identification of proteins attached strongly to the MNP surface was also carried out using 1-D gel electrophoresis (SDSPAGE) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).

Experimental Section Materials. Magnetite nanoparticles having carboxyl groups on their surfaces (i.e., MNPs) with a primary size of between 10 and 20 nm were provided by NanoMaterials Pte. Ltd. Biological media, RPMI-1640 (cat. no. 21870-092), and fetal bovine serum (17) Lim, J. K.; Majetich, S. A.; Tilton, R. D. Langmuir 2009, 25, 13384–13393. (18) Casey, A.; Davoren, M.; Herzog, E.; Lyng, F. M.; Byrne, H. J.; Chambers, G. Carbon 2007, 45, 34–40. (19) Chen, Z. P.; Xu, R. Z.; Zhang, Y.; Gu, N. Nanoscale Res. Lett. 2009, 4, 204–209. (20) Chen, Z. P.; Zhang, Y.; Xu, K.; Xu, R. Z.; Liu, J. W.; Gu, N. J. Nanosci. Nanotechnol. 2008, 8, 6260–6265. (21) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Nano Lett. 2006, 6, 662–668. (22) Deng, Z. J.; Mortimer, G.; Schiller, T.; Musumeci, A.; Martin, D.; Minchin, R. F. Nanotechnology 2009, 20, 455101. (23) Mu, Q.; Li, Z.; Li, X.; Mishra, S. R.; Zhang, B.; Si, Z.; Yang, L.; Jiang, W.; Yan, B. J. Phys. Chem. C 2009, 113, 5390–5395. (24) Petri-Fink, A.; Chastellain, M.; Juillerat-Jeanneret, L.; Ferrari, A.; Hofmann, H. Biomaterials 2005, 26, 2685–2694.

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(FBS, cat. no. 10437) were purchased from Invitrogen. The pH of RPMI-1640 and FBS was 7.3 and 7.1, respectively. (The complete RPMI-1640 formulation is available online from http://www. invitrogen.com/site/us/en/home/support/Product-TechnicalResources/media_formulation.121.html.) Heat inactivation of FBS was conducted by heating FBS to 55 °C for 30 min prior to use in the experiment. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. The NuPAGE Novex 4-12% bis-tris precast protein gel (cat. no. NP0322) and kit, NuPAGE 2-(N-morpholino) ethane sulfonic acid (MES) running buffer, NuPAGE 3-(Nmorpholino) propane sulfonic acid (MOPS) running buffer, NuPAGE lithium dodecyl sulfate (LDS) sample buffer, and SeeBlue Plus2 protein molecular weight standard were purchased from Invitrogen. Gel code blue used for protein staining was purchased from Fisher Scientific. Stabilization of MNP with FBS. The adsorption of serum proteins onto the MNP surface was carried out by transferring MNP (125 μL, 50 mg/mL) into an RPMI-1640 solution containing varying amount of FBS (ranging from 1 to 10% by volume, 49.875 mL). The serum-MNP suspension was sonicated for 30 s with an ultrasonic probe (Misonix Sonicator S-4000) to disperse the particles and then mixed in a rotating wheel for 24 h. The final MNP concentration was 125 mg/L. Serum-free suspensions of MNP at the same concentration in Milli-Q water, PBS, or RPMI1640 solution were also prepared by adding MNP (20 μL, 50 mg/mL) to the medium of interest for comparison (8 mL). Samples of serum-free MNP in RPMI-1640 solution containing 5% BSA and the serum-MNP composite prepared by mixing MNP with 10% FBS in RPMI-1640 solution, followed by three cycles of washing with Milli-Q water and resuspension in just RPMI-1640 solution or RPMI-1640 solution containing 5% BSA, were also prepared for further analysis. Particle Characterization. The hydrodynamic diameter of the various MNP suspensions prepared previously was monitored using dynamic light scattering (DLS, Brookhaven BI-90 PALS) over a period of 16 h. The samples were sonicated with an ultrasonic probe for 30 s prior to the first measurement and left undisturbed for subsequent measurements. The zeta potential of particles was measured by phase analysis light scattering (PALS, Brookhaven BI-90 PALS). Concurrent with the size analysis, a drop of MNPs suspended in the various media was also deposited onto a carbon-coated copper grid and dried at room temperature for visualization under a transmission electron microscope (TEM, JEOL JEM 1400) operating with an acceleration voltage of 100 kV and a beam current of 55 μA. The surface of an MNP before and after treatment with 10% FBS was analyzed using X-ray photoelectron spectroscopy (XPS). The sample consisting of MNPs that has been treated with 10% FBS, magnetically separated, and then washed three times with Milli-Q water was also analyzed using the same technique. XPS analyses were performed using EscaLab 220-IXL (Fisons Surface Science at VG Scientific, U.K.), which operates with monochromated Al KR radiation at 1486.60 eV and a source power of 120 W. A spot size of approximately 0.5 mm in diameter with a pass energy of 100 eV was used for wide scans, and a pass energy of 20 eV was used for narrow scans of particular elemental peaks.

Separation of the Serum Protein Bound to MNPs by OneDimensional Gel Electrophoresis. Magnetic nanoparticles (MNPs) that were suspended in RPMI-1640 containing 10% FBS were magnetically separated and washed three times with Milli-Q water. Any serum proteins that remained attached to the MNP surface were then released by immersing the washed FBSMNP composite in protein-solubilizing solution consisting of an SDS solution (5%) containing an LDS sample buffer for 1 h and holding the mixture at 95 °C for 5 min, after which the particles were separated from the liquor by centrifugation, as has been shown in the literature.23,25 The supernatant of RPMI-1640 (25) Gessner, A.; Lieske, A.; Paulke, B. R.; M€uller, R. H. J. Biomed. Mater. Res., Part A 2003, 65, 319–326.

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Scheme 1. Sample Preparation of the Magnetic Nanoparticles for 1-D Gel Electrophoresis Identification of Surface-Adsorbed Serum Protein

Figure 1. Light-scattering average hydrodynamic diameter of MNPs suspended in (a) water, (b) phosphate-buffered saline (PBS), and RPMI-1640 solution containing (c) 0% FBS, (d) 1% FBS, (e) 2% FBS, (f) 4% FBS, (g) 8% FBS, and (h) 10% FBS. *The measurement was not continued for a longer period of time because of the settling of particles. Table 1. Zeta Potential of MNPs in Different Media media

containing 10% FBS that had been contacted with MNP (S1) and supernatants from Milli-Q water washing (S2-S4) and SDS washing steps (S5) as well as neat RPMI-1640 containing 10% FBS (S0) were collected and analyzed using 1-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A schematic of the sample-preparation method is shown in Scheme 1. As a control, gel electrophoresis was also carried out for neat RPMI1640 solution, FBS, and RPMI-1640 solution that had been in contact with MNPs. In a typical 1-D gel electrophoresis analysis, an aliquot of the supernatant (30 μL) was mixed with LDS buffer (10 μL), except for S5, which already contained LDS buffer, and held at 95 °C for 10 min before being loaded onto a NuPAGE Novex 4-12% bistris precast protein gel with either 2-(N-morpholino) ethane sulfonic acid (MES) or 3-(N-morpholino) propane sulfonic acid (MOPS) as the running buffer. A constant voltage of 170 V applied across the gel for 65 min separated the serum proteins according to their molecular weight. The protein bands were stained using gel code blue for visualization purposes, and SeeBlue Plus2 was used as the molecular weight marker. The position of the protein in the gel was fixed by immersing the gel in a solution containing 10% acetic acid, 50% methanol, and 40% water for 20 min. The resulting gel was then washed with water for at least 1 h in a rocking platform; the water was changed every 15 min, after which the gel was stained with gel code blue overnight.

Protein Digestion and Identification by Liquid Chromatograph-Mass Spectrometer/Mass Spectrometer (LCMS/MS) Analysis. The protein band of interest was sliced from the 1-D gel using a clean blade, and the proteins were extracted for LC-MS/MS analysis (Ultimate 3000 HPLC and an autosampler system from Dionex, Amsterdam, The Netherlands, coupled with an LTQ FT ultra mass spectrometer from Thermo Electron, Bremen, Germany). Reverse-phase C18 material was used for the HPLC column. Peptides from the digested proteins were resolved with a linear gradient from 100% buffer A (2% acetonitrile and 0.1% formic acid in Milli-Q water) to 45% buffer B (80% acetonitrile and 0.1% formic acid in Milli-Q water) for 30 min at room temperature. The eluent was run at 250 nL/min. Prior to protein extraction, the stain from each gel band was removed by washing with ammonium bicarbonate (NH4HCO3, 100 mM) for 20 min, followed by incubation with NH4HCO3/CH3CN (1:1, 250 μL, 25 mM) until the gel became clear Langmuir 2011, 27(2), 843–850

zeta potential (mV)

water PBS RPMI-1640

-32 ( 5 -22 ( 5 -20 ( 5

(approximately 1-14 h). Aliquots of CH3CN (50 μL, 25 mM) were then added to the samples, and after 10 min, the samples were left to dry under vacuum. Gel pieces were reduced and alkylated before protein digestion to increase the sensitivity of the analysis. Proteins were reduced by immersing the gel in a solution of dithiothreitol (10 mM) in NH4HCO3 (50 mM) at 37 °C for 30 min, followed by alkylation in iodoacetamide (25 mM) in NH4HCO3 (50 mM) at 37 °C for 30 min. The gel was then washed three times with water, followed by washing with NH4HCO3 (10 mM) and then twice washing with CH3CN, and dried under vacuum. To digest the protein, an aliquot of trypsin (5-10 ng/μL) in NH4HCO3 (25 μL, 10 mM) was added per gel band and left at 37 °C for 14 h. An aliquot of formic acid (25 μL, 1%) was then added and left for 15 min, followed by the addition of CH3CN (25 μL) for another 15 min to extract the protein from the gel band. The liquid was then transferred to a new Eppendorf tube and then dried using a SpeedVac. The dried digested protein was dissolved in a solution containing formic acid (1%) and heptafluorobutyric acid (0.05%) in water (50 μL). The peptide detected via LC-MS/MS was matched with protein from the NCBInr database using Mascot Daemon software from Matrix Science (www.matrixscience.com), with a peptide mass tolerance of (4 ppm and a fragment mass tolerance of (0.4 Da.

Results and Discussion Stability and Surface Charge of MNP in Biological Media. Figure 1 shows that as-received MNPs (magnetic nanoparticles with carboxyl groups on their surfaces) can be maintained at 55 ( 1 nm in water at pH of 7.4 for at least 16 h, and their stability in water was also indicated by the intensity-weighted size distribution that did not broaden significantly over time (Supporting Information Figure S1). The observed stability of MNPs in water is due to the presence of highly negatively charged carboxyl groups on the surface, as indicated by the highly negative zeta potential (Table 1), which induces a strong electrostatic repulsion force between magnetic particles. Figure 1 also shows that the as-received MNPs aggregated and settled very quickly when transferred to a high-ionic-strength DOI: 10.1021/la104278m

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Figure 2. TEM images of MNPs suspended in RPMI-1640 solution containing (a) 0% FBS, (b) 1% FBS, (c) 2% FBS, (d) 4% FBS, (e) 8% FBS, and (f) 10% FBS. All scale bars represent 100 nm.

medium such as phosphate-buffered saline (PBS) or RPMI-1640 solution because of the suppression of the double layer, which reduces the electrostatic repulsion barrier. A drop in the magnitude of the zeta potential from -32 to approximately -20 mV was also observed for particles suspended in PBS and RPMI-1640 (Table 1), and the settling of these particles was confirmed by a decrease in the scattering intensity of the laser beam (Supporting Information Figure S4) and a shift in the size distribution of the particles toward a larger size (Supporting Information Figure S2, left). In the presence of FBS, however, MNPs show increased stability against aggregation in the RPMI-1640 solution with an increasing amount of FBS. Moreover, the size of MNP aggregates could be maintained at approximately 180 nm for at least 16 h when the FBS concentration was 4% or higher. The intensityweighted size distribution of MNP in RPMI-1640 containing 846 DOI: 10.1021/la104278m

10% FBS also did not show any significant broadening over time, which indicated that MNP was not aggregating in the presence of FBS in the solution (Supporting Information Figure S2, right). TEM imaging confirms that the size of the MNP aggregates decreased after MNPs were treated with FBS. TEM imaging also shows that when the FBS concentration in the RPMI 1640 solution was 4% or higher (Figure 2d-f), a thick layer of substance, probably serum protein, can be seen to cover the MNP surface. The adsorption of protein onto the surfaces of nanoparticles has previously been shown to proceed immediately upon contact with the physiological environment, forming a “corona” around the particles.26 This protein corona will determine the particles’ behavior and interaction with living cells. The presence of a (26) Lynch, I.; Dawson, K. A. Nano Today 2008, 3, 40–47.

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Figure 3. Wide XPS scan of (a) MNP, (b) FBS-MNP, and (c) washed FBS-MNP.

protein corona was not seen for MNPs incubated in RPMI-1640 containing 0% FBS (Figure 2a) and was thinner for MNPs incubated in RPMI-1640 containing 1 or 2% FBS (Figure 2b,c). Surface Functional Groups Analysis on MNP Surfaces (As Received, FBS-MNP Composite, and Washed FBSMNP Composite). Figure 3a shows the wide XPS spectra of the as-received MNPs, which consists of Fe 2p, C 1s, and O 1s peaks. The atomic ratios of C to Fe and O to Fe are 1.6 and 4.3, respectively, as listed in Table 2. The deconvolution of the Fe 2p spectra, according to the approach used by Lin et al., shows that it is composed of signals from both Fe2þ and Fe3þ 27 (Supporting Information C). This is expected because magnetite is a mixed oxide consisting of FeO and Fe2O3. The O 1s spectra can be decomposed to a major peak at 529.8 eV, which can be assigned to an oxygen atom in iron oxide,27 and a minor peak at 531.5 eV, which can be assigned to oxygen in OH and CdO.28 The C 1s spectra for the untreated MNP consisted of two peaks located at 285.0 and 288.7 eV. The first peak corresponds to C bound only to a C or H single bond, and the second peak at 288.7 eV can be attributed to C in COOH groups.29 The existence of peaks corresponding to C-(C, H) and CdO confirms the presence of carboxyl functional groups on the surfaces of as-received MNPs. The spectra of the unwashed and washed FBS-MNP (Figure 3b,c, respectively) have the same set of Fe 2p, C 1s, and O 1s peaks as well as an additional N 1s peak at approximately 400 eV. The elemental N 1s narrow scan peak (Supporting Information Figure S5) showed a symmetrical peak that is attributed to either the protonated or nonprotonated nitrogen from the amide or amine groups29 from the serum proteins. The position of the C 1s peak remained unchanged following treatment with FBS, with an additional peak appearing at 286.4 eV attributed to a C-(O, N) single bond29 from the serum protein (Supporting Information Figure S5). The atomic ratio of C to Fe on the surface of the MNP increased significantly from 1.6 to 70.3 after FBS treatment. In contrast, the Fe 2p peak for MNP after incubation with FBS showed a weaker signal compared to that of the original MNP (Figure 3b), with the total number of Fe atoms detected for the MNP to decrease from approximately 14 to 1 atom %. This is due to the shielding of the Fe 2p signal by the adsorbed material on the MNP surface because the XPS penetration depth is approximately 5 nm. Treatment with FBS also resulted in the suppression of the major O 1s peak at 529.8 eV (assigned to the oxygen atom in iron oxide), whereas the peak at 531.4 (attributed to the OdC double bond that is present in both COOH and NCO functional groups) became more dominant (Supporting Information Figure S5).29 An additional new peak (27) Lin, T. C.; Seshadri, G.; Kelber, J. A. Appl. Surf. Sci. 1997, 119, 83–92. (28) Boonaert, C. J. P.; Rouxhet, P. G. Appl. Environ. Microbiol. 2000, 66, 2548– 2554. (29) Pradier, C. M.; Karman, F.; Telegdi, J.; Kalman, E.; Marcus, P. J. Phys. Chem. B 2003, 107, 6766–6773.

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also emerged at 532.7 eV and is attributed to the O-C single bond.29 The emergence of the N 1s peaks and the increase in the amount of carbon on the particle surfaces (Table 2) suggest that the materials that adsorbed onto the MNP surface are protein molecules originating from FBS. The types of bonds identified from the chemical state analysis further support this inference. XPS analysis of the washed MNP showed the re-establishment of the Fe 2p peak and O 1s peak at 530 eV (Figure 3c and Supporting Information Figure S5). The C to Fe ratio and the N to Fe ratio also decreased from 70.7 to 15.7 and from 6.1 to 1.9, respectively. These results indicate a thinning of the protein layer due to the washing process, although the presence of the N 1s peak after washing suggests that not all of the adsorbed proteins were removed in the washing step. Identification of Proteins by One-Dimensional Gel Electrophoresis and LC-MS/MS. Figure 4 shows the gel electrophoresis result of the supernatant obtained from MNP that was incubated in RPMI-1640 solution (A) and MNP that was incubated in RPMI-1640 containing 10% FBS (B and C) following Scheme 1. Lanes A2 and A3 in Figure 4A show the protein bands that were obtained from neat RPMI-1640 before and after contact with MNP, respectively. The absence of protein bands in these lanes indicates the absence of protein molecules in the neat RPMI-1640 solution. In addition, there was no protein detected from SDS solution that was used to leach any protein that may be present on the MNP surface after contact with RPMI-1640 (lane A4). This result confirms the absence of detectable protein molecules on the surfaces of as-received MNPs. In contrast, lanes B1 and B3 in Figure 4B show the presence of protein components with molecular weights of approximately 14, 25, 49-65, and 98 kDa in RPMI-1640 solution containing 10% FBS, before and after coming into contact with MNP (S0 and S1, respectively). From the intensity ratio of the bands, it can be deduced that FBS mainly consists of proteins with a molecular weight of between 49 and 65 kDa. LC-MS/MS analysis further showed that a key component of the 49-65 kDa protein bands is bovine serum albumin (BSA). This is expected because BSA is the most abundant component in serum.30 Lanes B4, B5, and B6 in Figure 4B show the protein bands obtained from Milli-Q water that were used to wash FBS-MNP (S2-S4). The presence of strong protein bands at 49-65 and 98 kDa in lane B4 indicates that a significant amount of the proteins in these bands was removed from the MNP surface in the first washing cycle. In the second washing cycle, a small amount of protein with a molecular weight of between 49 and 65 kDa was still leached from the surface (lane B5). No bands were detected in lane B6, indicating that protein was no longer being leached from the MNP surface after three washing cycles. Lane B7 in Figure 4B shows the proteins in the SDS solution that were used to leach the proteins that remained strongly attached to the MNP surface after the three sequential washing cycles (S5). It can be seen that proteins with molecular weights of approximately 14, 19, 49, 65, and 98 kDa were strongly bound to the MNP surface (i.e., which could not be removed by the water washing process). The protein bands detected in the SDS solution were similar to those found in the original FBS solution (lane B1), with the exception of the protein with a molecular weight of approximately 19 kDa. This protein was, however, detected in a more concentrated FBS solution (Supporting Information Figure S6), which confirms that all proteins released from the surface of the FBS-MNP composite originated from the serum. (30) Blomb€ack, B., Hanson, L., Eds. Plasma Proteins; John Wiley & Sons: New York, 1979; pp 43, 55.

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Table 2. Surface Elemental Ratio and Functional Groups of the Neat MNP and FBS-MNP Composite before and after Three Washings with Milli-Q Water as Determined by XPS Elemental Scans sample MNP

element C 1s O 1s

FBS-MNP, unwashed

N 1s C 1s O 1s

FBS-MNP, washed

N 1s C 1s O 1s N 1s

ion/bond type

main peak ((0.2 eV)

ratio

C-(C, H) single bond COOH Fe-O OH and OdC

285.0 288.7 529.8 531.5

C/Fe = 1.6

C-(C, H) single bond C-(O, N) single bond COOH and CONH Fe-O OH and OdC O-C single bond amide bond C-(C, H) single bond C-(O, N) single bond COOH and CONH Fe-O OH and OdC O-C single bond amide bond

285.0 286.4 288.9 529.9 531.4 532.7 400.1 284.9 286.3 288.8 530.0 531.3 532.4 400.1

O/Fe = 4.3 N/Fe = 0 C/Fe = 70.7 O/Fe = 23.1 N/Fe = 6.1 C/Fe = 15.7 O/Fe = 10.4 N/Fe = 1.9

Table 3. Molecular Weights and Isoelectric Point of Proteins That Remained Strongly Attached to the Surface of MNP after Three Cycles of Washing with Milli-Q Water protein

molecular weight (kDa)a

complement factor H antithrombin complement factor I albumin R-1-antiproteinase apoliprotein E a Measured from gel electrophoresis.

97 80 66 60 53 39

pI 6.534 5.035 5.536 4.637 5.038 5.238

Figure 4. One-dimensional gel electrophoresis analysis of (A) control MNP incubated in RPMI-1640 without any FBS run with MES buffer: lane A1, molecular weight marker; lane A2, neat RPMI-1640; lane A3, supernatant of the RPMI-1640 solution after contact with MNP; lane A4, SDS supernatant solution after contact with MNP. (B) MNP treated with 10% FBS in RPMI1640 run with MES buffer: lane B1, RPMI-1640 þ 10% FBS; lane B2, molecular weight marker; lane B3, supernatant of the RPMI1640 þ 10% FBS solution after contact with MNP (S1); lane B4, supernatant of FBS-MNP after one cycle of washing with water (S2); lane B5, supernatant of FBS-MNP after two cycles of washing with water (S3); lane B6, supernatant of FBS-MNP after three cycles of washing with water (S4); lane B7, SDS supernatant washing solution of FBS-MNP after washing with water three times (S5). (C) Protein released from the FBS-MNP composite after three cycles of washing with Milli-Q water and after running with MOPS buffer: lane C1, molecular weight marker; lane C2, SDS supernatant washing solution of FBS-MNP after washing with water three times (S5). Each number on the image indicates the approximate molecular weight of each protein band in kDa.

Figure 5. Light-scattering average hydrodynamic diameter of (a) neat MNP suspended in RPMI-1640 solution containing 5% BSA, (b) the FBS-MNP composite that has been washed three times with Milli-Q water suspended in neat RPMI-1640 solution, and (c) the FBSMNP composite that has been washed three times with Milli-Q water and then resuspended in RPMI-1640 solution containing 5% BSA.

To provide better separation of the bands, a second electrophoresis study was carried out on the SDS washing solution (S5) using MOPS running buffer (Figure 4C). It can be seen that the majority of the strongly bounded proteins had molecular weights of approximately 97, 80, 66, 60, 53, and 39 kDa. LC-MS/MS analysis identified these proteins as complement factor H, antithrombin, complement factor I, bovine serum albumin (BSA), R-1antiproteinase, and apolipoprotein E (Table 3).

It is noteworthy that the band intensity ratio between the strongly attached proteins (i.e., those that were released after SDS solution washing) and the proteins contained in the original serum is quite different. For instance, the intensity of BSA bands was less than or similar to those of the other proteins, even though BSA is a major component of FBS, which comprises 50-60% of the total serum protein.29 This suggests that the adsorption of protein onto the MNP surface was not solely driven by

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Figure 6. TEM images of (a) neat MNP suspended in RPMI-1640 solution containing 5% BSA, (b) the FBS-MNP composite that has been washed three times with Milli-Q water suspended in neat RPMI-1640 solution, and (c) the FBS-MNP composite that has been washed three times with Milli-Q water suspended in RPMI-1640 solution containing 5% BSA. All scale bars represent 100 nm.

concentration. Instead, it was determined by the acidity or basicity of the particle surface in solution, which in turn is dependent on the functional groups that are present on the MNP surface.31 The adsorption of protein onto the surface of charged particles had been shown to be determined by both electrostatic and nonelectrostatic factors. The electrostatic interaction causes the adsorption of protein on oppositely charged surfaces,32 and the amphiphilic character of the protein and the ion-exchange effect govern the adsorption of protein on similarly charged surfaces as previously shown by Witteman and Ballouf for polyelectrolytemediated protein adsorption.31 The MNPs used in this study have a Br€onsted acid character that is due to the presence of dissociable carboxyl groups on its surfaces. The acidic surface interacts strongly with basic amino groups of the protein molecules, resulting in the chemical adsorption of the protein onto the particle surface.33 The fact that most of the proteins that remain strongly attached to the MNP surface after three cycles of washing with Milli-Q water have an isoelectric point (pI) of 5.0 or more34-38 (Table 3) further supports the postulation that the strong attachment of protein to the MNP surface is caused by this mechanism. This result also concurs with the study carried out by Gessner et al., which showed that proteins with a pI of greater than 5.5 prefer to bind to nanoparticles with an acidic surface whereas proteins with a pI of less than 5.5 are more likely to bind to latex nanoparticles with basic surfaces after the particles have been in contact with human serum for 5 min.25 Complement factor H, albumin, and R-1-antiproteinase have also been detected on the surfaces of carboxyl-modified latex and iron oxide nanoparticles in previous serum-nanoparticle interaction studies.23,39 However, it is also interesting that the other proteins that were found on the MNP surfaces (i.e., antithrombin, complement factor I, and apolipoprotein E) were not detected by other investigators for a similar particle-serum system.23 The differences can be explained by the type of suspension media, the (31) Wittemann, A.; Ballauff, M. Phys. Chem. Chem. Phys. 2006, 8, 5269–5275. (32) Ionov, L.; Houbenov, N.; Sidorenko, A.; Stamm, M.; Minko, S. Biointerphases 2009, 4, FA45–FA49. (33) Tavolaro, P.; Tavolaro, A.; Martino, G. Colloids Surf., B 2009, 70, 98–107. (34) Seya, T.; Okada, M.; Nishino, H.; Atkinson, J. P. J. Biochem. 1990, 107, 310–315. (35) Casanovas, A.; Carrascal, M.; Abian, J.; Lopez-Tejero, M. D.; Llobera, M. J. Proteome Res. 2008, 7, 4173–4177. (36) Yamane, K.; Minamoto, A.; Yamashita, H.; Takamura, H.; MiyamotoMyoken, Y.; Yoshizato, K.; Nabetani, T.; Tsugita, A.; Mishima, H. K. Mol. Cell. Proteomics 2003, 2, 1177–1187. (37) Kubota, Y.; Ueki, H. J. Biochem. 1968, 64, 405–406. (38) Hochstrasser, D. F.; Frutiger, S.; Wilkins, M. R.; Hughes, G.; Sanchez, J. C. FEBS Lett. 1997, 416, 161–163. (39) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14265–14270. (40) Gessner, A.; Waicz, R.; Lieske, A.; Paulke, B. R.; M€ader, K.; M€uller, R. H. Int. J. Pharm. 2000, 196, 245–249.

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incubation time, and the size and curvature of the particles affecting the preferential binding of different proteins onto the surfaces of nanoparticles.24,25,39-41 Cedervall et al. also showed that the rate of the association and dissociation of protein onto and from the particle surface is a dynamic process that depends on the type of protein and the particles.42 Proteins that have a high affinity for the particle surface would form a “hard” corona, and proteins that are not attached to the particle surface may then interact with the hard protein corona, forming secondary protein coverage (a “soft” corona).26,39 To determine how the presence of soft and hard protein coronas contributes to the stability of MNPs in biological fluids, a sample of the three-times-washed FBS-MNP composite was resuspended in protein-free RPMI-1640 solution, as was RPMI1640 containing 5% BSA. Neat MNP was also suspended in RPMI-1640 solution containing 5% BSA for comparison. The size measurement results in Figure 5a show that the presence of 5% BSA alone could not prevent the aggregation of neat MNP in the RPMI-1640 solution, which can be seen by a shift of the size distribution toward larger size (Supporting Information Figure S3, left), and the average hydrodynamic diameter of MNPs increased from approximately 200 to 900 nm over a period of 16 h. The presence of a strongly attached protein on the surface of washed FBS-MNP could not prevent aggregation in neat RPMI-1640 solution (Figure 5b). TEM imaging also showed the absence of a thick protein layer on the surfaces of both MNP samples (Figure 6a,b). It is only when the washed FBSMNP was resuspended in RPMI-1640 solution containing 5% BSA that the aggregation stability could be retained over a period of 16 h (Figure 5c and Supporting Information Figure S3, right) and a thick layer of protein coverage could be seen on the aggregate surface (Figure 6c). These results strongly suggest that both the hard and soft protein coronas play distinct roles in maintaining the stability of MNPs in biological media. The hard corona formed by the adsorbed serum proteins (e.g., complement factor H, antithrombin, complement factor I, R-1-antiproteinase, and apolipoprotein E) functioned as a linker for the formation of the soft corona, which presumably is made up of serum components that do not adsorb well on MNP (e.g., BSA). It is this soft corona that provides the steric hindrance to overcome the magnetic and van der Waals attraction forces between the particles.

Conclusions Complement factor H, antithrombin, complement factor I, R-1-antiproteinase, and apolipoprotein E were shown to bind (41) Lundqvist, M.; Sethson, I.; Jonsson, B. H. Langmuir 2004, 20, 10639–10647. (42) Cedervall, T.; Lynch, I.; Foy, M.; Bergga˚rd, T.; Donnelly, S. C.; Cagney, G.; Linse, S.; Dawson, K. A. Angew. Chem. 2007, 119, 5856–5858.

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strongly to the MNP surface, whereas the binding affinity of BSA, a major component of FBS, was weaker compared to that of other proteins in the serum. The incubation of MNPs with FBS was shown to prevent the aggregation of the particles in biological media. It was shown that strongly bound surface proteins act as linkers that bind a second layer of protein around the nanoparticles. It is this second layer of protein that prevents the magnetic nanoparticles from aggregating by providing steric hindrance. These results provide new insight into how serum proteins can improve the stability of magnetic nanoparticles in biological media. The proven biocompatibility of serum in general enables the use of these findings to design safe, stable magnetic nanoparticles for biomedical and biotechnology applications.

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Acknowledgment. This work was financially supported by the Australian Research Council through the ARC Centre of Excellence program. We thank Dr. Ling Zhong from the Bioanalytical Mass Spectrometry Facility (BMSF, UNSW) for her assistance with LC-MS/MS analysis and Dr. Christopher Marquis and Miss Roslyn Tedja from the School of Biotechnology and Biomolecular Sciences (BABS, UNSW) for their instruction in protein gel electrophoresis. Supporting Information Available: Detailed characterization via DLS, XPS, and gel electrophoresis analysis. This material is available free of charge via the Internet at http:// pubs.acs.org.

Langmuir 2011, 27(2), 843–850