- Email: [email protected]
Amphiphilic comblike polymers enhance the colloidal stability of Fe3O4 nanoparticles
Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Amphiphilic comblike polymers enhance the colloidal stability of Fe3 O4 nanoparticles Myeongjin Kim a , Jaeyeon Jung a , Jonghwan Lee a , Kyunga Na a,b , Subeom Park a , Jinho Hyun a,∗ a b
Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 151-742, Republic of Korea Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 July 2009 Received in revised form 14 October 2009 Accepted 27 October 2009 Available online 1 November 2009 Keywords: Amphiphilic Chemical coprecipitation Colloidal stability Magnetite Nanoparticles
a b s t r a c t Stable colloidal dispersions of magnetite (Fe3 O4 ) nanoparticles (MNPs) were obtained with the inclusion of an amphiphilic comblike polyethylene glycol derivative (CL-PEG) as an amphiphilic polymeric surfactant. Both the size and morphology of the resulting CL-PEG-modified MNPs could be controlled and were characterized by transmission electron microscopy (TEM). The interaction between MNPs and CL-PEG was confirmed by the presence of characteristic infrared absorption peaks, and the colloidal stability of the nanoparticle dispersion in water was evaluated by long-term observation of the dispersion using UV-visible spectroscopy. SQUID measurements confirmed the magnetization of CL-PEG-modified MNPs. The zeta potential of the CL-PEG-modified MNPs showed a dramatic conversion from positive to negative in response to the pH of the surrounding aqueous medium due to the presence of carboxyl groups at the surface. These carboxyl groups can be used to functionalize the MNPs with biomolecules for biotechnological applications. However, regardless of surface electrostatics, the flexible, hydrophilic side chains of CL-PEG-modified MNPs prevented the approach of adjacent nanoparticles, thereby resisting aggregation and resulting in a stable aqueous colloid. The cytotoxicity of MNPs and CL-PEG-modified MNPs was evaluated by a MTT assay. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Magnetite (Fe3 O4 ) nanoparticles (MNPs) have recently been evaluated for applications in biomedical imaging [1,2], immunomagnetic separations [3–5], and as agents for drug delivery [6–8]. Due to their susceptibility to aggregation [9,10], the colloidal stability of MNPs is critical in these types of systems. This paper describes a method for the preparation of well dispersed MNP suspensions using a polymeric surfactant. This technique further allows longterm immobilization of proteins on the MNP surface. Polyethylene glycol (PEG) is a polymer commonly used to coat MNPs for biomedical applications because of its biocompatibility and nonadhesive properties that arise from steric stabilizing effects at bio-interfaces, highly dynamic motion, and extended chain conformation [11,12]. Furthermore, MNPs modified with PEG have demonstrated a relatively long circulation time in the bloodstream and are non-immunogenic, nonantigenic, and resistant to protein binding [13–15]. However, under biological conditions, a risk exists that the PEG coating may deteriorate due to relatively weak interactions with the particle [16]. Therefore, the development of a reliable
∗ Corresponding author. Tel.: +82 2 880 4624; fax: +82 2 873 2285. E-mail address: [email protected] (J. Hyun). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.10.042
method for protecting MNPs against this deterioration is of great importance. PEG-containing, comblike polymer (CL-PEG) consists of relatively hydrophilic side groups attached along a hydrophobic backbone. This material is effective in coating hydrophobic nanoparticles because the backbone interacts with the hydrophobic surface of the particle while the hydrophilic PEG side chains provide water solubility and enhance colloidal stability. The relative hydrophilicity of the polymer can be tailored by controlling the size of the PEG side chains or by chemical functionalization [17]. The hydrophilic surface of CL-PEG-modified MNPs enables dispersion in pure water and allows convenient one-step syntheses for chemical modification.
2. Experimental 2.1. Synthesis of the PEG comblike polymer CL-PEG was synthesized via a free radical polymerization [17] by mixing 15.6 mL of 2-hydroxyethyl methacrylate (HEMA, 100 mmol), 29.5 mL of hydroxy-poly(oxyethylene) methacrylate (HPOEM, 50 mmol), 0.87 mL of mercaptopropionic acid (MPA, 10 mmol), and 2.46 g of 2,2 -azo-bis-isobutyronitrile (AIBN, 15 mmol) in 500 mL of tetrahydrofuran (THF). The solution was
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
237
degassed by bubbling nitrogen through it for 20 min and the mixture was refluxed at 75 ◦ C for 5 h. The resulting copolymer was purified by two precipitation steps in 8:1 (v/v) petroleum ether:methanol and dried under vacuum at 25 ◦ C for 24 h. 2.2. Preparation of magnetite nanoparticles A colloidal suspension of magnetite nanoparticles was prepared in accordance with a previously published coprecipitation method [18]. First, 0.2 mol FeCl2 ·4H2 O (99%; Sigma–Aldrich, St. Louis, MO) and 0.4 mol FeCl3 ·6H2 O (98%; Sigma–Aldrich) were dissolved in 5 mL deionized water. A CL-PEG solution at concentrations of 1, 2, 3, and 4 mM was then added to prepare a precursor solution. The precursor solution was added drop-wise to an aqueous solution of ammonium hydroxide (28%, w/w (1.5 M); Duksan Pure Chemical Co., Ansan, Korea) with vigorous stirring. The precipitated MNPs were washed five times with deionized water by centrifugation and finally re-dispersed as a colloidal suspension in deionized water. 2.3. Characterization of the magnetite nanoparticles The morphology and size of MNPs were observed with a transmission electron microscope (TEM; JEM 1010; JEOL, Tokyo, Japan). The hydrodynamic diameter of the MNP particles in suspension (100 g/mL) was measured at 90◦ by dynamic laser light scattering spectroscopy (DLS; DLS-7000; Otsuka Electronics, Osaka, Japan). The surface chemistry of the nanoparticles was characterized by Fourier transform infrared spectroscopy (FTIR; M2000; Midac, Hamamatsu, Japan) from 4000 to 400 cm−1 using powdered samples pressed into KBr pellets. The long-term stability of MNP suspensions (10 g/mL, pH 8.3) was characterized by measuring the room-temperature optical density at 350 nm (OD350 ) of tightly sealed samples in a single cuvette over the course of 72 h at 1h intervals using an Optizen 2120UV UV-VIS spectrophotometer (Mecasys Co., Daejeon, Korea). The surface charge of MNPs was investigated by measuring zeta potentials (ELS; ELS-8000; Otsuka Electronics) as a function of pH from 4.0 to 12.0. Each measurement was done in triplicate. The degree of magnetization in MNPs was measured with a superconducting quantum interference device
Scheme 1. A schematic diagram shows magnetite nanoparticles modified with CLPEG. The inset presents the chemical structure of CL-PEG.
Fig. 1. FT-IR spectra of (A) magnetite nanoparticles, (B) magnetite nanoparticles modified with CL-PEG, and (C) CL-PEG.
(SQUID)-based magnetometer (MPMS-XL; Quantum Design, USA) at room temperature on MNP powders that had been freeze-dried overnight. Cell viability was assessed by a 3-(4,5-dimethylthiazol)-2diphenyltertrazolium bromide (MTT) assay. A 90-L aliquot of NIH 3T3 fibroblast cells (2.5 × 105 cell/mL) was seeded into 96-well tissue culture plates and 10 L of MNP suspension was added to each of the wells. The cells were then incubated for 24 h at 37 ◦ C in a humidified incubator maintained at 5% CO2 /95% air. Cells were also incubated in the absence of MNPs as a negative control and in the presence of 0.1% Triton X-100 as a positive control.
Fig. 2. (A) The TEM image shows magnetite nanoparticles after modification with CL-PEG; the scale bar corresponds to 20 nm. (B) The size distribution of the modified magnetite nanoparticles was determined from the data in (A).
238
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
Fig. 3. Normalized OD350 readings are shown for pure and CL-PEG-modified MNPs in water. The MNP suspensions were stored inside the UV spectrometer during the 72-h course of the measurement.
2.4. Atomic force microscopy (AFM) The AFM topographic images were collected in contact mode using silicon nitride cantilevers (PSIA, Korea, spring constant 0.6 N/m; tip radius <10 nm) using an XE100 (PSIA, Korea) in air. We used AFM in force spectrometry mode to measure interaction forces between either pure MNPs or CL-PEG-modified MNPs and a cantilever tip that was functionalized with either CH3 thiol or COOH thiol. 3. Results and discussion Under biological conditions, MNPs coated with what are generally considered biocompatible surfactants may become incompatible due to weak anchoring of surface modifiers, nonspecific adsorption, and colloidal instability. CL-PEG contains continuous hydrophobic components that exhibit stronger interactions with MNPs compared to short-chain surfactants or linear PEGs. In addition, the biocompatibility and hydrophilicity of CL-
Fig. 4. Surface characterization of MNPs by probing with surface modified cantilever tips in air. Larger adhesive interaction forces were observed when pure MNPs were probed with a hydrophobically modified AFM tip indicating the surface of pure MNPs was relatively hydrophobic. CL-PEG-modified MNPs showed larger interaction with hydrophilically modified AFM tip indicating the surface of CL-PEG-modified MNPs was relatively hydrophilic.
Fig. 5. Variations in the zeta potentials of pure and CL-PEG-modified MNPs are shown as a function of the pH of the surrounding medium.
PEG-modified MNPs allow even dispersion in biological fluids and relatively easy functionalization with established NHS/EDC chemistries due to the presence of surface carboxyl groups (Scheme 1). The presence of organic molecules on the particle surface was determined by comparing IR absorption peaks of modified and uncoated MNPs. For the uncoated MNPs, the characteristic absorption peak of Fe–O vibrations in Fe3 O4 was observed at 580.46 cm−1 (Fig. 1A). This band was blue-shifted to 595.89 cm−1 for CL-PEGmodified nanoparticles (Fig. 1B), suggesting the formation of new bonds between the MNP and CL-PEG [19,20]. The FT-IR spectra in Fig. 1B and C confirm the presence of absorption bands representing –C–O–C–, –CH2 –, and C O groups and imply the formation of CL-PEG-modified MNPs as presented in Scheme 1. The immobilization of CL-PEG on the MNP surface was robust and able to withstand ultrasonication and ultracentrifugation at 10,000 rpm for 15 min in water. Fig. 2A shows a TEM image of CL-PEG-modified MNPs. The modified particles were spherical with a relatively uniform diameter of 16 ± 2 nm which was about 6 nm larger than uncoated MNPs (10 ± 2 nm). The size distribution was calculated according to a lognormal function using the TEM images in Fig. 2B.
Fig. 6. Magnetic hysteresis curves of pure and CL-PEG-modified MNPs.
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
Fig. 3 presents the time-dependent OD350 of a MNP suspension that was allowed to settle for a total of 72 h. The OD350 of the uncoated MNP suspension decreased about 45% after 72 h while the OD350 of the modified MNP suspension decreased by only 11.21%. This difference is due to the inherent colloidal instability of the uncoated particles in water and consequent aggregation and precipitation. The CL-PEG coating prevented aggregation and enabled a more stable aqueous dispersion. Based on the structure of the CLPEG, the methyl methacrylate backbone is likely adsorbed to the surface of the nanoparticle while the relatively hydrophilic PEGcontaining side chains extend out into the surrounding solution. The preferred binding of a hydrophobic backbone of CLPEG molecules was investigated by measuring put-off forces of molecules immobilized on the colloidal surface using surfacemodified AFM tips. The tips were coated with gold and immersed in either COOH-terminated or CH3 -terminated thiols for the preparation of hydrophilically and hydrophobically modified tips,
239
respectively. Hydrophobically modified tips showed adhesive interactions with the uncoated MNPs that were about four times stronger than with the CL-PEG modified MNPs (Fig. 4). It provided the possibility that the surface of MNPs was relatively hydrophobic even though a certain amount of oxide groups might exist on the surface of MNPs. Further experiments with hydrophilically modified tips showed relatively stronger adhesive interactions with the CL-PEG modified MNPs than with the uncoated MNPs as shown in Fig. 4. It resulted from the preferred attraction of hydrophobic backbone to the MNPs and extension of hydrophilic side chains out into the surrounding environment as shown in Scheme 1. The magnitude of the zeta potential is proportional to the amount of charge on the nanoparticle surface. Due to the presence of surface-bound carboxyl groups, the zeta potential of modified MNPs changed drastically from a positive (26.05 mV) to a negative value (−39.39 mV) as a function of the pH of the surrounding
Fig. 7. Cell morphologies of the (A) negative (complete culture medium) and (B) positive control groups (Triton X-100), and after incubation with (C) pure and (D) CL-PEGmodified MNP suspensions. The concentration of the MNP suspensions was 20 g/mL. (E) Cell viability was quantified with a MTT assay following incubation for 24 h at 37 ◦ C. Error bars indicate standard error (n = 10). * Significant (p < 0.05, ANOVA) difference in comparison to negative control or MNPs-CL-PEG.
240
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
solution (Fig. 5). In contrast, the zeta potential of uncoated MNPs was negative over the entire range of pH values. Moreover, the colloidal stability of the modified MNPs was high even at pH 8.5, when the zeta potential approached zero. While the uncoated nanoparticles aggregated regardless of zeta potential, suspensions of coated MNPs were stable from pH 4.0 to 12.0 and showed no significant signs of aggregation. Thus, the resistance to aggregation was not due to electrostatic forces alone and was due in part to steric repulsion of PEG side chains. This phenomenon is manifested in the use of steric stabilizers, which commonly consist of block copolymers with lyophilic and lyophobic components. The lyophobic component attaches to the particle surface via van der Waals interactions while the lyophilic chain extends into the surrounding medium. In an appropriate solvent system, the lyophilic chains of adjacent particles do not interpenetrate but instead induce interparticle repulsion. However, in poor solvent systems, the interpenetration of stabilizer chains occurs until it is prevented by elastic repulsion. For these reasons, the observed colloidal stability was achieved with CL-PEG-modified MNPs in a hydrophilic medium. To investigate the influence of the CL-PEG surface coating on the magnetic behavior of the nanoparticles, magnetization measurements were performed using SQUID. Fig. 6 shows the hysteresis curves obtained with uncoated and CL-PEG-modified MNPs. Saturation magnetization was attained at 87.32 and 56.36 emu/g, respectively, and confirmed the characteristic superparamagnetic properties of the magnetite nanoparticles. As the magnetic field decreased, the degree of magnetization decreased to zero. This implies that MNPs that have been separated from a suspension by their superparamagnetic properties can be rapidly re-dispersed when the magnetic field is removed. The test of cytotoxicity is the initial phase in evaluating biocompatibility of biomaterials. Fig. 7 shows in vitro cytotoxicity tests after incubation with uncoated and CL-PEG-modified MNPs respectively. After a 24 h incubation, the negative control group incubated in the complete culture medium without MNPs grew healthily and attached well to the surface of the well plate (Fig. 7A). In contrast, the positive controls where cells were exposed to toxic Triton X-100 (0.1%) showed significant detachment of cells from the surface of the well plate inferring the high toxicity to cells (Fig. 7B). Similar to the negative control group, cells incubated in a suspension of CL-PEG-modified MNPs spread and attached well to the plate surface even after 72 h incubation (Fig. 7D). However, cells incubated with uncoated MNPs did not spread properly and dead cells detached from the surface of the well plate (Fig. 7C) as the positive control group showed in Fig. 7B. Fig. 7E shows the quantitative viability of cells measured by the MTT assay revealing a significant difference between uncoated and CL-PEG-modified MNPs. The level of MTT was lower for the medium containing uncoated MNPs (54%, p < 0.05) compared with CL-PEG-modified MNPs (94%, p < 0.05) after a 24 h incubation. Based on the results of cell morphology and MTT assay, it could be determined that
the modification of MNPs with CL-PEG significantly improved the biocompatibility of a material. 4. Conclusions CL-PEG-modified MNPs were synthesized by a chemical coprecipitation method from a ferrous/ferric salt solution and an aqueous ammonium hydroxide solution containing CL-PEG at room temperature. The presence of CL-PEG on the surface of the MNPs was confirmed by characteristic peaks in the FT-IR spectra of the modified particles, and a stable colloidal dispersion of the modified MNPs was indicated by optical density analyses. Surface charge measurements revealed zeta potentials for both the uncoated and CL-PEG-modified particles as a function of the pH of the surrounding medium. The CL-PEG modification resulted in a well dispersed suspension of particles throughout an aqueous solution due to the presence of hydrophilic side chains at the surface of the MNPs. The biocompatibility of MNPs modified with CL-PEG was confirmed by relative cell morphology observations and MTT assays. Acknowledgment This work was supported by Grant R01-2006-000-10217 from the Basic Research Program of the Korea Science & Engineering Foundation. References [1] R.Y. Hong, B. Feng, L.L. Chen, G.H. Liu, H.Z. Li, Y. Zheng, D.G. Wei, Biochem. Eng. J. 42 (2008) 290. [2] D.K. Kim, M. Mikhaylova, Y. Zhang, M. Muhammed, Chem. Mater. 15 (2003) 1617. [3] M. Takahashi, Y. Akiyama, J. Ikezumi, T. Nagata, T. Yoshino, A. Lizuka, K. Yamaguchi, T. Matsunaga, Bioconjugate Chem. 20 (2009) 304. [4] Y.C. Liu, Y.H. Che, Y.B. Li, Sens. Actuators B: Chem. 72 (2001) 214. [5] K.S. Lee, S.S. Ryu, C. Kim, B.K. Ju, S.K. Lee, J.Y. Kang, Biochip J. 1 (2007) 165. [6] S.W. Cao, Y.J. Zhu, J. Phys. Chem. C 112 (2008) 12149. [7] S.S. Guo, C.C. Zuo, W.H. Huang, C. Peroz, Y. Chen, Microelectron. Eng. 83 (2006) 1655. [8] Y.N. Xia, P.D. Yang, Y.G. Sun, Y.Y. Wu, B. Mayers, B. Gates, Y.D. Yin, F. Kim, Y.Q. Yan, Adv. Mater. 15 (2003) 353. [9] A.H. Lu, E.L. Salabas, F. Schuth, Angew. Chem. Int. Ed. 46 (2007) 1222. [10] M.A. Morales, T.K. Jain, V. Labhasetwar, D.L. Leslie-Pelecky, J. Appl. Phys. 97 (10) (2005) Q905. [11] S.X. Wang, Y. Zhou, S.C. Yang, B.J. Ding, Colloid Surf. B: Biointerfaces 67 (2008) 122. [12] R.R. Seigel, P. Harder, R. Dahint, M. Grunze, F. Josse, M. Mrksich, G.M. Whitesides, Anal. Chem. 69 (1997) 3321. [13] F.X. Hu, K.G. Neoh, L. Cen, E.T. Kang, Biomacromolecules 7 (2006) 809. [14] A.K. Gupta, M. Gupta, Biomaterials 26 (2005) 3995. [15] M.Q. Zhang, T. Desai, M. Ferrari, Biomaterials 19 (1998) 953. [16] W.W. Yu, E. Chang, J.C. Falkner, J.Y. Zhang, A.M. Al-Somali, C.M. Sayes, J. Johns, R. Drezek, V.L. Colvin, J. Am. Chem. Soc. 129 (2007) 2871. [17] J. Jung, K. Na, B. Shin, O. Kim, J. Lee, K. Yun, J. Hyun, J. Biomater. Sci. Polym. Ed. 19 (2008) 161. [18] J. Sun, S.B. Zhou, P. Hou, Y. Yang, J. Weng, X.H. Li, M.Y. Li, J. Biomed. Mater. Res. Part A 80 (2007) 333. [19] Z.M. Gao, T.H. Wu, S.Y. Peng, Acta Phys. Chim. Sin. 11 (1995) 395. [20] M. Ma, Y. Zhang, W. Yu, H.Y. Shen, H.Q. Zhang, N. Gu, Colloid Surf. A: Physicochem. Eng. Asp. 212 (2003) 219.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Amphiphilic comblike polymers enhance the colloidal stability of Fe3 O4 nanoparticles Myeongjin Kim a , Jaeyeon Jung a , Jonghwan Lee a , Kyunga Na a,b , Subeom Park a , Jinho Hyun a,∗ a b
Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 151-742, Republic of Korea Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 July 2009 Received in revised form 14 October 2009 Accepted 27 October 2009 Available online 1 November 2009 Keywords: Amphiphilic Chemical coprecipitation Colloidal stability Magnetite Nanoparticles
a b s t r a c t Stable colloidal dispersions of magnetite (Fe3 O4 ) nanoparticles (MNPs) were obtained with the inclusion of an amphiphilic comblike polyethylene glycol derivative (CL-PEG) as an amphiphilic polymeric surfactant. Both the size and morphology of the resulting CL-PEG-modified MNPs could be controlled and were characterized by transmission electron microscopy (TEM). The interaction between MNPs and CL-PEG was confirmed by the presence of characteristic infrared absorption peaks, and the colloidal stability of the nanoparticle dispersion in water was evaluated by long-term observation of the dispersion using UV-visible spectroscopy. SQUID measurements confirmed the magnetization of CL-PEG-modified MNPs. The zeta potential of the CL-PEG-modified MNPs showed a dramatic conversion from positive to negative in response to the pH of the surrounding aqueous medium due to the presence of carboxyl groups at the surface. These carboxyl groups can be used to functionalize the MNPs with biomolecules for biotechnological applications. However, regardless of surface electrostatics, the flexible, hydrophilic side chains of CL-PEG-modified MNPs prevented the approach of adjacent nanoparticles, thereby resisting aggregation and resulting in a stable aqueous colloid. The cytotoxicity of MNPs and CL-PEG-modified MNPs was evaluated by a MTT assay. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Magnetite (Fe3 O4 ) nanoparticles (MNPs) have recently been evaluated for applications in biomedical imaging [1,2], immunomagnetic separations [3–5], and as agents for drug delivery [6–8]. Due to their susceptibility to aggregation [9,10], the colloidal stability of MNPs is critical in these types of systems. This paper describes a method for the preparation of well dispersed MNP suspensions using a polymeric surfactant. This technique further allows longterm immobilization of proteins on the MNP surface. Polyethylene glycol (PEG) is a polymer commonly used to coat MNPs for biomedical applications because of its biocompatibility and nonadhesive properties that arise from steric stabilizing effects at bio-interfaces, highly dynamic motion, and extended chain conformation [11,12]. Furthermore, MNPs modified with PEG have demonstrated a relatively long circulation time in the bloodstream and are non-immunogenic, nonantigenic, and resistant to protein binding [13–15]. However, under biological conditions, a risk exists that the PEG coating may deteriorate due to relatively weak interactions with the particle [16]. Therefore, the development of a reliable
∗ Corresponding author. Tel.: +82 2 880 4624; fax: +82 2 873 2285. E-mail address: [email protected] (J. Hyun). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.10.042
method for protecting MNPs against this deterioration is of great importance. PEG-containing, comblike polymer (CL-PEG) consists of relatively hydrophilic side groups attached along a hydrophobic backbone. This material is effective in coating hydrophobic nanoparticles because the backbone interacts with the hydrophobic surface of the particle while the hydrophilic PEG side chains provide water solubility and enhance colloidal stability. The relative hydrophilicity of the polymer can be tailored by controlling the size of the PEG side chains or by chemical functionalization [17]. The hydrophilic surface of CL-PEG-modified MNPs enables dispersion in pure water and allows convenient one-step syntheses for chemical modification.
2. Experimental 2.1. Synthesis of the PEG comblike polymer CL-PEG was synthesized via a free radical polymerization [17] by mixing 15.6 mL of 2-hydroxyethyl methacrylate (HEMA, 100 mmol), 29.5 mL of hydroxy-poly(oxyethylene) methacrylate (HPOEM, 50 mmol), 0.87 mL of mercaptopropionic acid (MPA, 10 mmol), and 2.46 g of 2,2 -azo-bis-isobutyronitrile (AIBN, 15 mmol) in 500 mL of tetrahydrofuran (THF). The solution was
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
237
degassed by bubbling nitrogen through it for 20 min and the mixture was refluxed at 75 ◦ C for 5 h. The resulting copolymer was purified by two precipitation steps in 8:1 (v/v) petroleum ether:methanol and dried under vacuum at 25 ◦ C for 24 h. 2.2. Preparation of magnetite nanoparticles A colloidal suspension of magnetite nanoparticles was prepared in accordance with a previously published coprecipitation method [18]. First, 0.2 mol FeCl2 ·4H2 O (99%; Sigma–Aldrich, St. Louis, MO) and 0.4 mol FeCl3 ·6H2 O (98%; Sigma–Aldrich) were dissolved in 5 mL deionized water. A CL-PEG solution at concentrations of 1, 2, 3, and 4 mM was then added to prepare a precursor solution. The precursor solution was added drop-wise to an aqueous solution of ammonium hydroxide (28%, w/w (1.5 M); Duksan Pure Chemical Co., Ansan, Korea) with vigorous stirring. The precipitated MNPs were washed five times with deionized water by centrifugation and finally re-dispersed as a colloidal suspension in deionized water. 2.3. Characterization of the magnetite nanoparticles The morphology and size of MNPs were observed with a transmission electron microscope (TEM; JEM 1010; JEOL, Tokyo, Japan). The hydrodynamic diameter of the MNP particles in suspension (100 g/mL) was measured at 90◦ by dynamic laser light scattering spectroscopy (DLS; DLS-7000; Otsuka Electronics, Osaka, Japan). The surface chemistry of the nanoparticles was characterized by Fourier transform infrared spectroscopy (FTIR; M2000; Midac, Hamamatsu, Japan) from 4000 to 400 cm−1 using powdered samples pressed into KBr pellets. The long-term stability of MNP suspensions (10 g/mL, pH 8.3) was characterized by measuring the room-temperature optical density at 350 nm (OD350 ) of tightly sealed samples in a single cuvette over the course of 72 h at 1h intervals using an Optizen 2120UV UV-VIS spectrophotometer (Mecasys Co., Daejeon, Korea). The surface charge of MNPs was investigated by measuring zeta potentials (ELS; ELS-8000; Otsuka Electronics) as a function of pH from 4.0 to 12.0. Each measurement was done in triplicate. The degree of magnetization in MNPs was measured with a superconducting quantum interference device
Scheme 1. A schematic diagram shows magnetite nanoparticles modified with CLPEG. The inset presents the chemical structure of CL-PEG.
Fig. 1. FT-IR spectra of (A) magnetite nanoparticles, (B) magnetite nanoparticles modified with CL-PEG, and (C) CL-PEG.
(SQUID)-based magnetometer (MPMS-XL; Quantum Design, USA) at room temperature on MNP powders that had been freeze-dried overnight. Cell viability was assessed by a 3-(4,5-dimethylthiazol)-2diphenyltertrazolium bromide (MTT) assay. A 90-L aliquot of NIH 3T3 fibroblast cells (2.5 × 105 cell/mL) was seeded into 96-well tissue culture plates and 10 L of MNP suspension was added to each of the wells. The cells were then incubated for 24 h at 37 ◦ C in a humidified incubator maintained at 5% CO2 /95% air. Cells were also incubated in the absence of MNPs as a negative control and in the presence of 0.1% Triton X-100 as a positive control.
Fig. 2. (A) The TEM image shows magnetite nanoparticles after modification with CL-PEG; the scale bar corresponds to 20 nm. (B) The size distribution of the modified magnetite nanoparticles was determined from the data in (A).
238
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
Fig. 3. Normalized OD350 readings are shown for pure and CL-PEG-modified MNPs in water. The MNP suspensions were stored inside the UV spectrometer during the 72-h course of the measurement.
2.4. Atomic force microscopy (AFM) The AFM topographic images were collected in contact mode using silicon nitride cantilevers (PSIA, Korea, spring constant 0.6 N/m; tip radius <10 nm) using an XE100 (PSIA, Korea) in air. We used AFM in force spectrometry mode to measure interaction forces between either pure MNPs or CL-PEG-modified MNPs and a cantilever tip that was functionalized with either CH3 thiol or COOH thiol. 3. Results and discussion Under biological conditions, MNPs coated with what are generally considered biocompatible surfactants may become incompatible due to weak anchoring of surface modifiers, nonspecific adsorption, and colloidal instability. CL-PEG contains continuous hydrophobic components that exhibit stronger interactions with MNPs compared to short-chain surfactants or linear PEGs. In addition, the biocompatibility and hydrophilicity of CL-
Fig. 4. Surface characterization of MNPs by probing with surface modified cantilever tips in air. Larger adhesive interaction forces were observed when pure MNPs were probed with a hydrophobically modified AFM tip indicating the surface of pure MNPs was relatively hydrophobic. CL-PEG-modified MNPs showed larger interaction with hydrophilically modified AFM tip indicating the surface of CL-PEG-modified MNPs was relatively hydrophilic.
Fig. 5. Variations in the zeta potentials of pure and CL-PEG-modified MNPs are shown as a function of the pH of the surrounding medium.
PEG-modified MNPs allow even dispersion in biological fluids and relatively easy functionalization with established NHS/EDC chemistries due to the presence of surface carboxyl groups (Scheme 1). The presence of organic molecules on the particle surface was determined by comparing IR absorption peaks of modified and uncoated MNPs. For the uncoated MNPs, the characteristic absorption peak of Fe–O vibrations in Fe3 O4 was observed at 580.46 cm−1 (Fig. 1A). This band was blue-shifted to 595.89 cm−1 for CL-PEGmodified nanoparticles (Fig. 1B), suggesting the formation of new bonds between the MNP and CL-PEG [19,20]. The FT-IR spectra in Fig. 1B and C confirm the presence of absorption bands representing –C–O–C–, –CH2 –, and C O groups and imply the formation of CL-PEG-modified MNPs as presented in Scheme 1. The immobilization of CL-PEG on the MNP surface was robust and able to withstand ultrasonication and ultracentrifugation at 10,000 rpm for 15 min in water. Fig. 2A shows a TEM image of CL-PEG-modified MNPs. The modified particles were spherical with a relatively uniform diameter of 16 ± 2 nm which was about 6 nm larger than uncoated MNPs (10 ± 2 nm). The size distribution was calculated according to a lognormal function using the TEM images in Fig. 2B.
Fig. 6. Magnetic hysteresis curves of pure and CL-PEG-modified MNPs.
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
Fig. 3 presents the time-dependent OD350 of a MNP suspension that was allowed to settle for a total of 72 h. The OD350 of the uncoated MNP suspension decreased about 45% after 72 h while the OD350 of the modified MNP suspension decreased by only 11.21%. This difference is due to the inherent colloidal instability of the uncoated particles in water and consequent aggregation and precipitation. The CL-PEG coating prevented aggregation and enabled a more stable aqueous dispersion. Based on the structure of the CLPEG, the methyl methacrylate backbone is likely adsorbed to the surface of the nanoparticle while the relatively hydrophilic PEGcontaining side chains extend out into the surrounding solution. The preferred binding of a hydrophobic backbone of CLPEG molecules was investigated by measuring put-off forces of molecules immobilized on the colloidal surface using surfacemodified AFM tips. The tips were coated with gold and immersed in either COOH-terminated or CH3 -terminated thiols for the preparation of hydrophilically and hydrophobically modified tips,
239
respectively. Hydrophobically modified tips showed adhesive interactions with the uncoated MNPs that were about four times stronger than with the CL-PEG modified MNPs (Fig. 4). It provided the possibility that the surface of MNPs was relatively hydrophobic even though a certain amount of oxide groups might exist on the surface of MNPs. Further experiments with hydrophilically modified tips showed relatively stronger adhesive interactions with the CL-PEG modified MNPs than with the uncoated MNPs as shown in Fig. 4. It resulted from the preferred attraction of hydrophobic backbone to the MNPs and extension of hydrophilic side chains out into the surrounding environment as shown in Scheme 1. The magnitude of the zeta potential is proportional to the amount of charge on the nanoparticle surface. Due to the presence of surface-bound carboxyl groups, the zeta potential of modified MNPs changed drastically from a positive (26.05 mV) to a negative value (−39.39 mV) as a function of the pH of the surrounding
Fig. 7. Cell morphologies of the (A) negative (complete culture medium) and (B) positive control groups (Triton X-100), and after incubation with (C) pure and (D) CL-PEGmodified MNP suspensions. The concentration of the MNP suspensions was 20 g/mL. (E) Cell viability was quantified with a MTT assay following incubation for 24 h at 37 ◦ C. Error bars indicate standard error (n = 10). * Significant (p < 0.05, ANOVA) difference in comparison to negative control or MNPs-CL-PEG.
240
M. Kim et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 236–240
solution (Fig. 5). In contrast, the zeta potential of uncoated MNPs was negative over the entire range of pH values. Moreover, the colloidal stability of the modified MNPs was high even at pH 8.5, when the zeta potential approached zero. While the uncoated nanoparticles aggregated regardless of zeta potential, suspensions of coated MNPs were stable from pH 4.0 to 12.0 and showed no significant signs of aggregation. Thus, the resistance to aggregation was not due to electrostatic forces alone and was due in part to steric repulsion of PEG side chains. This phenomenon is manifested in the use of steric stabilizers, which commonly consist of block copolymers with lyophilic and lyophobic components. The lyophobic component attaches to the particle surface via van der Waals interactions while the lyophilic chain extends into the surrounding medium. In an appropriate solvent system, the lyophilic chains of adjacent particles do not interpenetrate but instead induce interparticle repulsion. However, in poor solvent systems, the interpenetration of stabilizer chains occurs until it is prevented by elastic repulsion. For these reasons, the observed colloidal stability was achieved with CL-PEG-modified MNPs in a hydrophilic medium. To investigate the influence of the CL-PEG surface coating on the magnetic behavior of the nanoparticles, magnetization measurements were performed using SQUID. Fig. 6 shows the hysteresis curves obtained with uncoated and CL-PEG-modified MNPs. Saturation magnetization was attained at 87.32 and 56.36 emu/g, respectively, and confirmed the characteristic superparamagnetic properties of the magnetite nanoparticles. As the magnetic field decreased, the degree of magnetization decreased to zero. This implies that MNPs that have been separated from a suspension by their superparamagnetic properties can be rapidly re-dispersed when the magnetic field is removed. The test of cytotoxicity is the initial phase in evaluating biocompatibility of biomaterials. Fig. 7 shows in vitro cytotoxicity tests after incubation with uncoated and CL-PEG-modified MNPs respectively. After a 24 h incubation, the negative control group incubated in the complete culture medium without MNPs grew healthily and attached well to the surface of the well plate (Fig. 7A). In contrast, the positive controls where cells were exposed to toxic Triton X-100 (0.1%) showed significant detachment of cells from the surface of the well plate inferring the high toxicity to cells (Fig. 7B). Similar to the negative control group, cells incubated in a suspension of CL-PEG-modified MNPs spread and attached well to the plate surface even after 72 h incubation (Fig. 7D). However, cells incubated with uncoated MNPs did not spread properly and dead cells detached from the surface of the well plate (Fig. 7C) as the positive control group showed in Fig. 7B. Fig. 7E shows the quantitative viability of cells measured by the MTT assay revealing a significant difference between uncoated and CL-PEG-modified MNPs. The level of MTT was lower for the medium containing uncoated MNPs (54%, p < 0.05) compared with CL-PEG-modified MNPs (94%, p < 0.05) after a 24 h incubation. Based on the results of cell morphology and MTT assay, it could be determined that
the modification of MNPs with CL-PEG significantly improved the biocompatibility of a material. 4. Conclusions CL-PEG-modified MNPs were synthesized by a chemical coprecipitation method from a ferrous/ferric salt solution and an aqueous ammonium hydroxide solution containing CL-PEG at room temperature. The presence of CL-PEG on the surface of the MNPs was confirmed by characteristic peaks in the FT-IR spectra of the modified particles, and a stable colloidal dispersion of the modified MNPs was indicated by optical density analyses. Surface charge measurements revealed zeta potentials for both the uncoated and CL-PEG-modified particles as a function of the pH of the surrounding medium. The CL-PEG modification resulted in a well dispersed suspension of particles throughout an aqueous solution due to the presence of hydrophilic side chains at the surface of the MNPs. The biocompatibility of MNPs modified with CL-PEG was confirmed by relative cell morphology observations and MTT assays. Acknowledgment This work was supported by Grant R01-2006-000-10217 from the Basic Research Program of the Korea Science & Engineering Foundation. References [1] R.Y. Hong, B. Feng, L.L. Chen, G.H. Liu, H.Z. Li, Y. Zheng, D.G. Wei, Biochem. Eng. J. 42 (2008) 290. [2] D.K. Kim, M. Mikhaylova, Y. Zhang, M. Muhammed, Chem. Mater. 15 (2003) 1617. [3] M. Takahashi, Y. Akiyama, J. Ikezumi, T. Nagata, T. Yoshino, A. Lizuka, K. Yamaguchi, T. Matsunaga, Bioconjugate Chem. 20 (2009) 304. [4] Y.C. Liu, Y.H. Che, Y.B. Li, Sens. Actuators B: Chem. 72 (2001) 214. [5] K.S. Lee, S.S. Ryu, C. Kim, B.K. Ju, S.K. Lee, J.Y. Kang, Biochip J. 1 (2007) 165. [6] S.W. Cao, Y.J. Zhu, J. Phys. Chem. C 112 (2008) 12149. [7] S.S. Guo, C.C. Zuo, W.H. Huang, C. Peroz, Y. Chen, Microelectron. Eng. 83 (2006) 1655. [8] Y.N. Xia, P.D. Yang, Y.G. Sun, Y.Y. Wu, B. Mayers, B. Gates, Y.D. Yin, F. Kim, Y.Q. Yan, Adv. Mater. 15 (2003) 353. [9] A.H. Lu, E.L. Salabas, F. Schuth, Angew. Chem. Int. Ed. 46 (2007) 1222. [10] M.A. Morales, T.K. Jain, V. Labhasetwar, D.L. Leslie-Pelecky, J. Appl. Phys. 97 (10) (2005) Q905. [11] S.X. Wang, Y. Zhou, S.C. Yang, B.J. Ding, Colloid Surf. B: Biointerfaces 67 (2008) 122. [12] R.R. Seigel, P. Harder, R. Dahint, M. Grunze, F. Josse, M. Mrksich, G.M. Whitesides, Anal. Chem. 69 (1997) 3321. [13] F.X. Hu, K.G. Neoh, L. Cen, E.T. Kang, Biomacromolecules 7 (2006) 809. [14] A.K. Gupta, M. Gupta, Biomaterials 26 (2005) 3995. [15] M.Q. Zhang, T. Desai, M. Ferrari, Biomaterials 19 (1998) 953. [16] W.W. Yu, E. Chang, J.C. Falkner, J.Y. Zhang, A.M. Al-Somali, C.M. Sayes, J. Johns, R. Drezek, V.L. Colvin, J. Am. Chem. Soc. 129 (2007) 2871. [17] J. Jung, K. Na, B. Shin, O. Kim, J. Lee, K. Yun, J. Hyun, J. Biomater. Sci. Polym. Ed. 19 (2008) 161. [18] J. Sun, S.B. Zhou, P. Hou, Y. Yang, J. Weng, X.H. Li, M.Y. Li, J. Biomed. Mater. Res. Part A 80 (2007) 333. [19] Z.M. Gao, T.H. Wu, S.Y. Peng, Acta Phys. Chim. Sin. 11 (1995) 395. [20] M. Ma, Y. Zhang, W. Yu, H.Y. Shen, H.Q. Zhang, N. Gu, Colloid Surf. A: Physicochem. Eng. Asp. 212 (2003) 219.