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Synthesis and characterization of magnetic Fe3O4-silica-poly(γ-benzyl-l -glutamate) composite microspheres
Reactive & Functional Polymers 71 (2011) 1040–1044
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Reactive & Functional Polymers journal homepage: www.elsevier.com/locate/react
Synthesis and characterization of magnetic Fe3O4-silica-poly(c-benzyl-L-glutamate) composite microspheres Dong Liu, Yi Li, Jianping Deng ⇑, Wantai Yang ⇑ State Key Laboratory of Chemical Resource Engineering, College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China
a r t i c l e
i n f o
Article history: Received 18 March 2011 Received in revised form 24 June 2011 Accepted 18 July 2011 Available online 23 July 2011 Keywords: Magnetic property Composite microspheres Fe3O4 Poly(c-benzyl-L-glutamate)
a b s t r a c t This article reports a novel type of composite microspheres showing magnetic properties. The composite microspheres consist of Fe3O4, silica, and poly(c-benzyl-L-glutamate) (PBLG). For preparing such composite microspheres, Fe3O4 nanoparticles were fabricated by the solvothermal method and then coated with a silica shell by tetraethoxysilane. Subsequently, amino groups were introduced on the above Fe3O4-silica spheres by using 3-aminopropyltriethoxysilane. Finally, the obtained spheres were used as initiator for polymerizing c-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), providing the anticipated magnetic composite microspheres. Such microspheres were characterized by FT-IR, TEM, large-angle powder XRD, and vibrating sample magnetometer. FT-IR spectra demonstrated that the PBLG chains adopted a-helical conformations. The magnetic composite microspheres showed a high saturation magnetization of 34.1 emu/g and the expected rapid magnetic responsivity. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction Magnetic nano- and micro-spheres display interesting features such as superparamagnetism, high field irreversibility, high saturation field, and large specific surface area [1]. Particularly magnetic spheres combined with other functional groups have attracted much attention because of their potential applications in biotechnology and biomedicine areas such as separation and purification of biochemical products [2], magnetic resonance imaging contrast agents [3], targeted drug delivery labeling [4], enzyme immobilization [5], and hyperthermia treatment cancers [6]. However, magnetic nanoparticles must be stabilized by protective agents against the tendency towards agglomeration. Silica is extensively used as such a protective agent [7,8]. The magnetic/silica composite microspheres with a magnetic core and a silica layer have been intensively studied for their magnetic responsivity, low cytotoxicity, good stability, and chemically modifiable surface [8]. Nevertheless, novel type of magnetic spheres are still expected to satisfy some special applications. The present study thus deals with a novel type of magnetic spheres simultaneously showing interesting properties from PBLG. Polypeptides are gathering ever-increasing attention for their unique secondary structures, significant functionality, and superior biocompatibility [9]. Particularly, the secondary structures of polypeptides (a-helix, b-sheet, etc.) play important roles in their ⇑ Corresponding authors. Tel./fax: +86 10 6443 5128. E-mail addresses: [email protected] (J. Deng), [email protected] (W. Yang).
unique properties [10]. For example, the self-assembly behavior of polypeptide copolymers is significantly affected by the conformations of polypeptide [11]. Additionally, the conformation transitions of polypeptide-based materials provide some intriguing functionality, which cannot be observed in usual polymers [12]. Such conformation transitions can be induced by pH [13], temperature [14,15], and solvent [16]. Polypeptides also show an ample chemistry with interesting physical and optical properties [17]. However, up to now only few reports were devoted to polypeptides grafted on colloid particles surface [18–21]. In principle, grafting polypeptides on particles surface will undoubtedly give rise to a number of advanced materials. Magnetic spheres coated with homopolypeptides have hardly been studied yet. In theory, such magnetic composite spheres are expected to show not only the magnetic properties derived from Fe3O4, but also certain versatile properties from the polypeptides. Moreover, this novel type of magnetic composite particles may find significant applications in chiral recognition, chiral separation, chiral catalysis, etc. Herein, we report a facile synthesis of well-defined magnetic microspheres consisting of Fe3O4@SiO2@poly(c-benzyl-L-glutamate) (PBLG). The reason for using PBLG as a model of synthetic polypeptides is that PBLG has been well investigated. Moreover, PBLG demonstrates a variety of intriguing properties and has found some important applications [10]. The as-prepared composite microspheres demonstrate both unique properties derived from PBLG and magnetism originating from the Fe3O4 cores. Following the same strategy, a great number of other novel composite microspheres can be prepared next.
1381-5148/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2011.07.009
D. Liu et al. / Reactive & Functional Polymers 71 (2011) 1040–1044
2. Experimental 2.1. Measurements Powder X-ray diffraction (XRD) patterns were recorded on a D/ max2500 VB2+/PC X-ray diffractometer (Rigaku) using Cu Ka radiation in the 2h range 10–70°. Transmission electron microscopy (TEM) images were obtained on an H-800 (Hitachi) transmission electron microscope at an accelerating voltage of 200 kV. Samples dispersed in solution were cast onto a carbon-coated copper grid. Fourier transform infrared (FTIR) spectra were recorded with a Nicolet-50 DXC FTIR spectrophotometer. Dry samples were prepared as KBr pellets. Magnetic characterization was carried out on a vibrating sample magnetometer (VSM, Jilin University JDM13 VSM) at room temperature. 2.2. Materials FeCl36H2O, anhydrous sodium acetate (NaAc), polyethylene glycol (PEG1500), ethylene glycol, ethanol, ammonia solution (25%), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), toluene, CaH2, and tetraethoxysilane (TEOS P 28%) were purchased from Beijing Chemical Reagents Company (China) and used as received except for toluene, THF and DMF. DMF was distilled over CaH2 and stored under dry nitrogen. THF and toluene were dried using CaH2 and then distilled over sodium. 3-Aminopropyltriethoxysilane (APTES P 98%) was obtained from Alfa. c-Benzyl-L-glutamate (BLG) was purchased from Sigma–Aldrich. Triphosgene (98%) was received from Shandong Pingyuan Yongheng Chemical Co., Ltd. (China). Deionized water was used in all experiments. 2.3. Synthesis of Fe3O4 nanoparticles The Fe3O4 nanoparticles (NPs) were prepared according to a modified solvothermal reaction [22]. Typically, 1.35 g of FeCl36H2O, 3.6 g of NaAc (3.6 g), and 1.0 g of PEG1500 was dissolved in 40 ml of ethylene glycol. The mixture was stirred vigorously for 30 min, yielding a homogeneous yellow solution. Then the resultant solution was transferred into a Teflon-lined stainless-steel autoclave (50 ml capacity). The autoclave was heated to 200 °C, in which the reaction lasted for 10 h. After cooling to room temperature, the obtained black products were isolated, washed three times with ethanol, and dried at 60 °C for 12 h. 2.4. Synthesis of Fe3O4@SiO2 microspheres The Fe3O4@SiO2 microspheres were synthesized according to the reported method [23]. Briefly, 0.03 g Fe3O4 NPs (approx. 260 nm in diameter) was homogeneously dispersed in a mixture of ethanol (160 ml), deionized water (40 ml), and concentrated ammonia aqueous solution (10 ml, 28 wt.%). The dispersion mixture was sonicated for 30 min, followed by addition of 0.6 ml of tetraethoxysilane (TEOS). After mechanically stirring at room temperature for 12 h, the Fe3O4@SiO2 microspheres were isolated and washed with ethanol and water three times, respectively, and then dried under vacuum at 60 °C for 12 h. 2.5. Synthesis of amine-functionalized Fe3O4@SiO2 microspheres Amine-functionalized Fe3O4@SiO2 microspheres were prepared using 3-aminopropyltriethoxysilane (APTES) according to the reported method with some modifications [24]. Briefly, 24 ml of anhydrous DMF and 16 ml of anhydrous toluene were placed in a flask, in which 0.1 g Fe3O4@SiO2 microspheres was added. The solution was sonicated for ca. 30 min, in which 100 ll of APTES
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was dropwise added by using a syringe. The reaction was mechanically stirred for 24 h at room temperature with N2 gas flow. The amine-functionalized Fe3O4@SiO2 microspheres were washed four times with anhydrous toluene. The sample was then dried in an oven at 40 °C for 24 h. 2.6. Synthesis of c-Benzyl-L-glutamate N-carboxyanhydride (BLGNCA) BLG-NCA monomer was synthesized by the reaction of c-benzyl-L-glutamate (BLG) with triphosgene according to the reported approach [25]. Typically, 50 ml of anhydrous THF was added into a 100 ml three-neck flask, in which 5 g BLG was suspended. After heating to 50 °C, an equimol of triphosgene was added under stirring. When the suspension turned transparent, nitrogen was continuously bubbled to remove the excess phosgene. After 2 h the reaction mixture was poured into 200 ml of n-hexane, and the resulting suspension was stored at 4 °C overnight. The NCA was re-crystallized from THF/n-hexane three times. The yield of BLGNCA was 65%. The melting point of it was 91–92 °C, similar to the reported data 93–94 °C [26]. 2.7. Synthesis of the Fe3O4@SiO2@PBLG composite microspheres Anhydrous THF (50 ml) was added into a 100 ml three-neck flask equipped with a stirrer, a N2 inlet, and a reflux condenser, in which 0.5 g BLG-NCA was added. 0.1 g amine-functionalized Fe3O4@SiO2 was dispersed in 5 ml of N,N-dimethylformamide (DMF), and then injected in the flask. The reaction solution was allowed to mechanically stir for 3 days under N2 gas. The formed composite microspheres were separated by magnet and subsequently washed with DMF to remove the free polymers and the residue monomers. The obtained microspheres were dried up, weighed, and stored for characterizations. 3. Results and discussion The synthesis procedure for the magnetic Fe3O4-silica-PBLG composite microspheres is schematically illustrated in Scheme 1. There are totally four major steps in this synthesis procedure. Firstly, magnetic Fe3O4 NPs were prepared via a solvothermal reaction. Secondly, the magnetic NPs were encapsulated with a silica shell by using TEOS through a sol–gel process. Thirdly, the Fe3O4@SiO2 microspheres were modified by APTES, yielding the aminefunctionalized microspheres (Fe3O4@SiO2–NH2). Finally, the Fe3O4@SiO2–NH2 microspheres initiated the open-ring polymerization of BLG-NCA monomer. The aforementioned synthesis process proved to be highly effective and facile for preparing PBLG coated Fe3O4@SiO2 composite microspheres. Magnetic Fe3O4 particles of different sizes can be prepared by using diverse approaches, for instance solvothermal method [22], thermal decomposition [27], coprecipitation [28], etc. In our research, we adopted solvothermal method with iron (III) chloride hexahydrate as the iron source, ethylene glycol as both solvent and reducing agent, and NaAc as electrostatic stabilization. NaAc was used not only to prevent particle agglomeration, but also made contribution to ethylene glycol mediated reduction of FeCl36H2O to Fe3O4 [22]. This efficient approach can provide a one-pot, simple, and economical method for synthesis of Fe3O4 NPs with high magnetic saturation value (approx. 80 emu/g) and a large range of diameter (200–800 nm) [22]. The magnetic saturation value of Fe3O4 NPs was dependent on their average grain size [29] and the diameter of Fe3O4 NPs can be controlled by reaction time [8,22] or Fe3+ concentration [29]. Fig. 1a shows a typical TEM image of the Fe3O4 NPs prepared under the conditions: iron source con-
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D. Liu et al. / Reactive & Functional Polymers 71 (2011) 1040–1044
Scheme 1. Schematic illustration of the synthesis of Fe3O4@SiO2@PBLG composite microspheres.
Fig. 1. TEM images of particles: (a) Fe3O4, (b) Fe3O4@SiO2, and (c) Fe3O4@SiO2@PBLG.
centration (0.13 M), temperature, 200 °C, and reaction time, 10 h. The Fe3O4 NPs (ca. 260 nm in diameter) are spherical and uniform in size. The saturation magnetization value of the Fe3O4 NPs was approx. 77.1 emu/g (see below), which is slightly higher than the reported 75.8 emu/g [8]. Fe3O4 particles tend to aggregate together because of anisotropic dipolar attraction [28], which affects their dispersion and magnetic properties. In order to protect magnetic particles from aggregation, inert materials such as silica or polymers were used to coat magnetic particles. In the present study, Fe3O4 NPs were encapsulated with silica by the hydrolysis and condensation of TEOS in ethanol/ammonia mixture. Fig. 1b shows the TEM micrographs for such Fe3O4@SiO2 microspheres. The silica shells thickness is found to be 50 nm. The silica layer plays an important role in improving the dispersion and further modification of Fe3O4 NPs. To make Fe3O4@SiO2 microspheres surface-amine-func-
tionalized, the spheres were modified by APTES. After modification, the amine-functionalized Fe3O4@SiO2–NH2 microspheres were produced. FT-IR spectrum further verified the formation of Fe3O4@SiO2–NH2 microspheres, as discussed later. To obtain the designed magnetic composite particles, polypeptide (PBLG) was grafting polymerized on the surface of Fe3O4@SiO2–NH2 microspheres. Fig. 1c presents a typical TEM image of the Fe3O4@SiO2@PBLG composite microspheres. The obtained microspheres (ca. 340 nm in diameter) are considerably larger than the corresponding Fe3O4@SiO2 microspheres (ca. 310 nm in diameter). This observation indicates that PBLG was successfully grafted on the surface of Fe3O4@SiO2 microspheres. As mentioned above, pure Fe3O4 particles tend to aggregate together. After coated with silica and grafted PBLG, the PBLG coated Fe3O4@SiO2 composite microspheres can be dispersed in solvent (e.g. DMF). The dispersion kept stable for at least 3 days,
D. Liu et al. / Reactive & Functional Polymers 71 (2011) 1040–1044
indicating the largely improved stability of the composite microspheres. Fig. 2 displays the XRD patterns of the Fe3O4 NPs and Fe3O4@SiO2@PBLG composite microspheres. All detected diffraction peaks [(1 1 1), (2 0 0), (3 1 1), (2 2 2), (4 0 0), (4 2 2), (5 1 1), and (4 4 0)] of Fe3O4 in face centered cubic crystal (JCPDS card No. 19-629) can be observed in Fe3O4 NPs and the final composite microspheres. The intensities of all Fe3O4 diffraction peaks in Fe3O4@SiO2@PBLG are weaker than those in pure Fe3O4 NPs, most likely due to the amorphous materials (silica and PBLG) on the surface of Fe3O4 NPs. All the samples of the original Fe3O4 NPs and Fe3O4 NPs encapsulated with silica, modified with APTES, and grafted with PBLG were characterized by the Fourier transform infrared (FT-IR) spectroscopy. The resulting spectra are described in Fig. 3. The spectrum of Fe3O4 NPs (Fig. 3a) shows strong peaks at 583 cm 1, a characteristic of Fe–O stretching vibration [30]. Fig. 3b illustrates the spectrum of Fe3O4@SiO2 spheres. A new strong band appears around 1088 cm 1 due to the bending vibration of Si–O band of silica [31,32]. This result further confirms that silica is coated on Fe3O4 NPs. Fig. 3c presents the spectrum of amine-functionalized Fe3O4@SiO2 microspheres, in which the characteristic peaks of primary amine ( NH2) groups can be observed at 1569 and 1331 cm 1 [24]. The peaks observed at 2980 and 2855 cm 1 correspond with the C H stretching vibrations of the alkyl group of APTES. Fig. 3d shows the spectrum of Fe3O4@SiO2@PBLG composite microspheres. New absorptions at 1652 and 1550 cm 1 are derived from amideI and amideII, respectively. Reportedly, the conformation (a-helices and b-sheets) of polypeptides can be characterized by FT-IR [19,33–35,21,36–38]. The peaks at 1650–1658 cm 1 (amideI) and 1548–1550 cm 1 (amideII) reflect the a-helices in PBLG, while for b-sheets, peaks should appear at 1626–1630 cm 1 (amideI) and 1520 cm 1 (amideII) [38]. Accordingly, the FT-IR spectrum in Fig. 3d indicates that PBLG is successfully grafted onto the composite microspheres and furthermore the PBLG chains adopt a-helices. We also tried to confirm the a-helical conformations of the grafted PBLG via measuring CD spectroscopy. Unfortunately, the CD signals were too complex to provide clear information. This observation should be caused by the presence of Fe3O4. Additionally, the secondary conformations of polypeptides were not uniform for surface grafted polypeptides [10]. However, the FT-IR spectrum in Fig. 3d clearly conformed that the surface-grafted PBLG chains on microspheres are majorly in a-helical conformation. To ensure the consideration that PBLG was chemically grafted on the microspheres, the Fe3O4@SiO2@PBLG composite microspheres were further treated under strict conditions. Before testing FT-IR, we sonicated the microspheres with DMF for 36 h. Then the
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Fig. 3. FT-IR spectra of particles (a) Fe3O4, (b) Fe3O4@SiO2, (c) Fe3O4@SiO2–NH2, and (d) Fe3O4@SiO2@PBLG.
Fig. 4. The hysteresis loops of: (a) Fe3O4, (b) Fe3O4@SiO2, and (c) Fe3O4@SiO2@PBLG.
Fig. 5. Separability of Fe3O4@SiO2@PBLG by placing an external magnetic field. The time from state (A) to state (B) is within 10 s.
Fig. 2. XRD patterns of: (a) Fe3O4 and (b) Fe3O4@SiO2@PBLG.
microspheres were washed with THF three times to obtain dry samples. The FT-IR spectrum in this case was in accordance with the spectrum presented in Fig. 3d. Therefore the PBLG is chemically grafted on the microspheres. Further, the grafting efficiency of PBLG was determined to be ca. 73% by gravimetric method. Fig. 4 shows the varied VSM magnetization curves for pure Fe3O4 NPs (Fig. 4a), Fe3O4@SiO2 microspheres (Fig. 4b), and Fe3O4@SiO2@PBLG composite microspheres (Fig. 4c). The saturation magnetization of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@PBLG
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D. Liu et al. / Reactive & Functional Polymers 71 (2011) 1040–1044
are 77.1, 44.3, and 34.1 emu/g, respectively. It is therefore indicated that the Fe3O4@SiO2@PBLG composite microspheres still possess high magnetization. Fig. 5 shows that the Fe3O4@SiO2@PBLG composite microspheres dispersed in aqueous respond quickly to an external magnet. The time from state (A) to state (B) is within 10 s. When the external magnet was taken off, the accumulation particles can be re-dispersed in a solvent, e.g., DMF, just by shaking. This quick responsivity of Fe3O4@SiO2@PBLG composite microspheres is very important for applications in separation. 4. Conclusions PBLG-coated Fe3O4@SiO2 composite microspheres with a diameter of 340 nm were successfully synthesized. The microspheres exhibited high magnetization (34.1 emu/g). FT-IR spectra demonstrated that the surface-grafted PBLG adopted a-helical second structures. The magnetic Fe3O4@SiO2@PBLG composite microspheres could be separated quickly by using an external magnetic field. Such novel composite microspheres are expected to exhibit some interesting functions, which are under investigation at present. Acknowledgments The Project was supported by the ‘‘Program for New Century Excellent Talents in University’’ (NCET-06-0096), the ‘‘Fundamental Research Funds for the Central Universities’’ (ZZ1117), the ‘‘National Natural Science Foundation of China’’ (20974007), and the ‘‘Project of Polymer Chemistry and Physics, Beijing Municipal Commission of Education’’. References [1] P. Tartaj, M.P. Morales, S. Veintemillas-Verdaguer, T. González-Carreño, C.J. Serna, J. Phys. D: Appl. Phys. 36 (2003) 182–197. [2] C. Wilhelm, C. Billotey, J. Roger, J.N. Pons, J.-C. Bacri, F. Gazeau, Biomaterials 24 (2003) 1001–1011. [3] Y.W. Jun, Y.M. Huh, J.S. Choi, J.H. Lee, H.T. Song, S. Kim, S. Yoon, K.S. Kim, J.S. Shin, J.S. Suh, J. Cheon, J. Am. Chem. Soc. 127 (2005) 5732–5733. [4] S. Mornet, S. Vasseur, F. Grasset, E. Duguet, J. Mater. Chem. 14 (2004) 2161– 2175.
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Contents lists available at ScienceDirect
Reactive & Functional Polymers journal homepage: www.elsevier.com/locate/react
Synthesis and characterization of magnetic Fe3O4-silica-poly(c-benzyl-L-glutamate) composite microspheres Dong Liu, Yi Li, Jianping Deng ⇑, Wantai Yang ⇑ State Key Laboratory of Chemical Resource Engineering, College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China
a r t i c l e
i n f o
Article history: Received 18 March 2011 Received in revised form 24 June 2011 Accepted 18 July 2011 Available online 23 July 2011 Keywords: Magnetic property Composite microspheres Fe3O4 Poly(c-benzyl-L-glutamate)
a b s t r a c t This article reports a novel type of composite microspheres showing magnetic properties. The composite microspheres consist of Fe3O4, silica, and poly(c-benzyl-L-glutamate) (PBLG). For preparing such composite microspheres, Fe3O4 nanoparticles were fabricated by the solvothermal method and then coated with a silica shell by tetraethoxysilane. Subsequently, amino groups were introduced on the above Fe3O4-silica spheres by using 3-aminopropyltriethoxysilane. Finally, the obtained spheres were used as initiator for polymerizing c-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), providing the anticipated magnetic composite microspheres. Such microspheres were characterized by FT-IR, TEM, large-angle powder XRD, and vibrating sample magnetometer. FT-IR spectra demonstrated that the PBLG chains adopted a-helical conformations. The magnetic composite microspheres showed a high saturation magnetization of 34.1 emu/g and the expected rapid magnetic responsivity. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction Magnetic nano- and micro-spheres display interesting features such as superparamagnetism, high field irreversibility, high saturation field, and large specific surface area [1]. Particularly magnetic spheres combined with other functional groups have attracted much attention because of their potential applications in biotechnology and biomedicine areas such as separation and purification of biochemical products [2], magnetic resonance imaging contrast agents [3], targeted drug delivery labeling [4], enzyme immobilization [5], and hyperthermia treatment cancers [6]. However, magnetic nanoparticles must be stabilized by protective agents against the tendency towards agglomeration. Silica is extensively used as such a protective agent [7,8]. The magnetic/silica composite microspheres with a magnetic core and a silica layer have been intensively studied for their magnetic responsivity, low cytotoxicity, good stability, and chemically modifiable surface [8]. Nevertheless, novel type of magnetic spheres are still expected to satisfy some special applications. The present study thus deals with a novel type of magnetic spheres simultaneously showing interesting properties from PBLG. Polypeptides are gathering ever-increasing attention for their unique secondary structures, significant functionality, and superior biocompatibility [9]. Particularly, the secondary structures of polypeptides (a-helix, b-sheet, etc.) play important roles in their ⇑ Corresponding authors. Tel./fax: +86 10 6443 5128. E-mail addresses: [email protected] (J. Deng), [email protected] (W. Yang).
unique properties [10]. For example, the self-assembly behavior of polypeptide copolymers is significantly affected by the conformations of polypeptide [11]. Additionally, the conformation transitions of polypeptide-based materials provide some intriguing functionality, which cannot be observed in usual polymers [12]. Such conformation transitions can be induced by pH [13], temperature [14,15], and solvent [16]. Polypeptides also show an ample chemistry with interesting physical and optical properties [17]. However, up to now only few reports were devoted to polypeptides grafted on colloid particles surface [18–21]. In principle, grafting polypeptides on particles surface will undoubtedly give rise to a number of advanced materials. Magnetic spheres coated with homopolypeptides have hardly been studied yet. In theory, such magnetic composite spheres are expected to show not only the magnetic properties derived from Fe3O4, but also certain versatile properties from the polypeptides. Moreover, this novel type of magnetic composite particles may find significant applications in chiral recognition, chiral separation, chiral catalysis, etc. Herein, we report a facile synthesis of well-defined magnetic microspheres consisting of Fe3O4@SiO2@poly(c-benzyl-L-glutamate) (PBLG). The reason for using PBLG as a model of synthetic polypeptides is that PBLG has been well investigated. Moreover, PBLG demonstrates a variety of intriguing properties and has found some important applications [10]. The as-prepared composite microspheres demonstrate both unique properties derived from PBLG and magnetism originating from the Fe3O4 cores. Following the same strategy, a great number of other novel composite microspheres can be prepared next.
1381-5148/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2011.07.009
D. Liu et al. / Reactive & Functional Polymers 71 (2011) 1040–1044
2. Experimental 2.1. Measurements Powder X-ray diffraction (XRD) patterns were recorded on a D/ max2500 VB2+/PC X-ray diffractometer (Rigaku) using Cu Ka radiation in the 2h range 10–70°. Transmission electron microscopy (TEM) images were obtained on an H-800 (Hitachi) transmission electron microscope at an accelerating voltage of 200 kV. Samples dispersed in solution were cast onto a carbon-coated copper grid. Fourier transform infrared (FTIR) spectra were recorded with a Nicolet-50 DXC FTIR spectrophotometer. Dry samples were prepared as KBr pellets. Magnetic characterization was carried out on a vibrating sample magnetometer (VSM, Jilin University JDM13 VSM) at room temperature. 2.2. Materials FeCl36H2O, anhydrous sodium acetate (NaAc), polyethylene glycol (PEG1500), ethylene glycol, ethanol, ammonia solution (25%), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), toluene, CaH2, and tetraethoxysilane (TEOS P 28%) were purchased from Beijing Chemical Reagents Company (China) and used as received except for toluene, THF and DMF. DMF was distilled over CaH2 and stored under dry nitrogen. THF and toluene were dried using CaH2 and then distilled over sodium. 3-Aminopropyltriethoxysilane (APTES P 98%) was obtained from Alfa. c-Benzyl-L-glutamate (BLG) was purchased from Sigma–Aldrich. Triphosgene (98%) was received from Shandong Pingyuan Yongheng Chemical Co., Ltd. (China). Deionized water was used in all experiments. 2.3. Synthesis of Fe3O4 nanoparticles The Fe3O4 nanoparticles (NPs) were prepared according to a modified solvothermal reaction [22]. Typically, 1.35 g of FeCl36H2O, 3.6 g of NaAc (3.6 g), and 1.0 g of PEG1500 was dissolved in 40 ml of ethylene glycol. The mixture was stirred vigorously for 30 min, yielding a homogeneous yellow solution. Then the resultant solution was transferred into a Teflon-lined stainless-steel autoclave (50 ml capacity). The autoclave was heated to 200 °C, in which the reaction lasted for 10 h. After cooling to room temperature, the obtained black products were isolated, washed three times with ethanol, and dried at 60 °C for 12 h. 2.4. Synthesis of Fe3O4@SiO2 microspheres The Fe3O4@SiO2 microspheres were synthesized according to the reported method [23]. Briefly, 0.03 g Fe3O4 NPs (approx. 260 nm in diameter) was homogeneously dispersed in a mixture of ethanol (160 ml), deionized water (40 ml), and concentrated ammonia aqueous solution (10 ml, 28 wt.%). The dispersion mixture was sonicated for 30 min, followed by addition of 0.6 ml of tetraethoxysilane (TEOS). After mechanically stirring at room temperature for 12 h, the Fe3O4@SiO2 microspheres were isolated and washed with ethanol and water three times, respectively, and then dried under vacuum at 60 °C for 12 h. 2.5. Synthesis of amine-functionalized Fe3O4@SiO2 microspheres Amine-functionalized Fe3O4@SiO2 microspheres were prepared using 3-aminopropyltriethoxysilane (APTES) according to the reported method with some modifications [24]. Briefly, 24 ml of anhydrous DMF and 16 ml of anhydrous toluene were placed in a flask, in which 0.1 g Fe3O4@SiO2 microspheres was added. The solution was sonicated for ca. 30 min, in which 100 ll of APTES
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was dropwise added by using a syringe. The reaction was mechanically stirred for 24 h at room temperature with N2 gas flow. The amine-functionalized Fe3O4@SiO2 microspheres were washed four times with anhydrous toluene. The sample was then dried in an oven at 40 °C for 24 h. 2.6. Synthesis of c-Benzyl-L-glutamate N-carboxyanhydride (BLGNCA) BLG-NCA monomer was synthesized by the reaction of c-benzyl-L-glutamate (BLG) with triphosgene according to the reported approach [25]. Typically, 50 ml of anhydrous THF was added into a 100 ml three-neck flask, in which 5 g BLG was suspended. After heating to 50 °C, an equimol of triphosgene was added under stirring. When the suspension turned transparent, nitrogen was continuously bubbled to remove the excess phosgene. After 2 h the reaction mixture was poured into 200 ml of n-hexane, and the resulting suspension was stored at 4 °C overnight. The NCA was re-crystallized from THF/n-hexane three times. The yield of BLGNCA was 65%. The melting point of it was 91–92 °C, similar to the reported data 93–94 °C [26]. 2.7. Synthesis of the Fe3O4@SiO2@PBLG composite microspheres Anhydrous THF (50 ml) was added into a 100 ml three-neck flask equipped with a stirrer, a N2 inlet, and a reflux condenser, in which 0.5 g BLG-NCA was added. 0.1 g amine-functionalized Fe3O4@SiO2 was dispersed in 5 ml of N,N-dimethylformamide (DMF), and then injected in the flask. The reaction solution was allowed to mechanically stir for 3 days under N2 gas. The formed composite microspheres were separated by magnet and subsequently washed with DMF to remove the free polymers and the residue monomers. The obtained microspheres were dried up, weighed, and stored for characterizations. 3. Results and discussion The synthesis procedure for the magnetic Fe3O4-silica-PBLG composite microspheres is schematically illustrated in Scheme 1. There are totally four major steps in this synthesis procedure. Firstly, magnetic Fe3O4 NPs were prepared via a solvothermal reaction. Secondly, the magnetic NPs were encapsulated with a silica shell by using TEOS through a sol–gel process. Thirdly, the Fe3O4@SiO2 microspheres were modified by APTES, yielding the aminefunctionalized microspheres (Fe3O4@SiO2–NH2). Finally, the Fe3O4@SiO2–NH2 microspheres initiated the open-ring polymerization of BLG-NCA monomer. The aforementioned synthesis process proved to be highly effective and facile for preparing PBLG coated Fe3O4@SiO2 composite microspheres. Magnetic Fe3O4 particles of different sizes can be prepared by using diverse approaches, for instance solvothermal method [22], thermal decomposition [27], coprecipitation [28], etc. In our research, we adopted solvothermal method with iron (III) chloride hexahydrate as the iron source, ethylene glycol as both solvent and reducing agent, and NaAc as electrostatic stabilization. NaAc was used not only to prevent particle agglomeration, but also made contribution to ethylene glycol mediated reduction of FeCl36H2O to Fe3O4 [22]. This efficient approach can provide a one-pot, simple, and economical method for synthesis of Fe3O4 NPs with high magnetic saturation value (approx. 80 emu/g) and a large range of diameter (200–800 nm) [22]. The magnetic saturation value of Fe3O4 NPs was dependent on their average grain size [29] and the diameter of Fe3O4 NPs can be controlled by reaction time [8,22] or Fe3+ concentration [29]. Fig. 1a shows a typical TEM image of the Fe3O4 NPs prepared under the conditions: iron source con-
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Scheme 1. Schematic illustration of the synthesis of Fe3O4@SiO2@PBLG composite microspheres.
Fig. 1. TEM images of particles: (a) Fe3O4, (b) Fe3O4@SiO2, and (c) Fe3O4@SiO2@PBLG.
centration (0.13 M), temperature, 200 °C, and reaction time, 10 h. The Fe3O4 NPs (ca. 260 nm in diameter) are spherical and uniform in size. The saturation magnetization value of the Fe3O4 NPs was approx. 77.1 emu/g (see below), which is slightly higher than the reported 75.8 emu/g [8]. Fe3O4 particles tend to aggregate together because of anisotropic dipolar attraction [28], which affects their dispersion and magnetic properties. In order to protect magnetic particles from aggregation, inert materials such as silica or polymers were used to coat magnetic particles. In the present study, Fe3O4 NPs were encapsulated with silica by the hydrolysis and condensation of TEOS in ethanol/ammonia mixture. Fig. 1b shows the TEM micrographs for such Fe3O4@SiO2 microspheres. The silica shells thickness is found to be 50 nm. The silica layer plays an important role in improving the dispersion and further modification of Fe3O4 NPs. To make Fe3O4@SiO2 microspheres surface-amine-func-
tionalized, the spheres were modified by APTES. After modification, the amine-functionalized Fe3O4@SiO2–NH2 microspheres were produced. FT-IR spectrum further verified the formation of Fe3O4@SiO2–NH2 microspheres, as discussed later. To obtain the designed magnetic composite particles, polypeptide (PBLG) was grafting polymerized on the surface of Fe3O4@SiO2–NH2 microspheres. Fig. 1c presents a typical TEM image of the Fe3O4@SiO2@PBLG composite microspheres. The obtained microspheres (ca. 340 nm in diameter) are considerably larger than the corresponding Fe3O4@SiO2 microspheres (ca. 310 nm in diameter). This observation indicates that PBLG was successfully grafted on the surface of Fe3O4@SiO2 microspheres. As mentioned above, pure Fe3O4 particles tend to aggregate together. After coated with silica and grafted PBLG, the PBLG coated Fe3O4@SiO2 composite microspheres can be dispersed in solvent (e.g. DMF). The dispersion kept stable for at least 3 days,
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indicating the largely improved stability of the composite microspheres. Fig. 2 displays the XRD patterns of the Fe3O4 NPs and Fe3O4@SiO2@PBLG composite microspheres. All detected diffraction peaks [(1 1 1), (2 0 0), (3 1 1), (2 2 2), (4 0 0), (4 2 2), (5 1 1), and (4 4 0)] of Fe3O4 in face centered cubic crystal (JCPDS card No. 19-629) can be observed in Fe3O4 NPs and the final composite microspheres. The intensities of all Fe3O4 diffraction peaks in Fe3O4@SiO2@PBLG are weaker than those in pure Fe3O4 NPs, most likely due to the amorphous materials (silica and PBLG) on the surface of Fe3O4 NPs. All the samples of the original Fe3O4 NPs and Fe3O4 NPs encapsulated with silica, modified with APTES, and grafted with PBLG were characterized by the Fourier transform infrared (FT-IR) spectroscopy. The resulting spectra are described in Fig. 3. The spectrum of Fe3O4 NPs (Fig. 3a) shows strong peaks at 583 cm 1, a characteristic of Fe–O stretching vibration [30]. Fig. 3b illustrates the spectrum of Fe3O4@SiO2 spheres. A new strong band appears around 1088 cm 1 due to the bending vibration of Si–O band of silica [31,32]. This result further confirms that silica is coated on Fe3O4 NPs. Fig. 3c presents the spectrum of amine-functionalized Fe3O4@SiO2 microspheres, in which the characteristic peaks of primary amine ( NH2) groups can be observed at 1569 and 1331 cm 1 [24]. The peaks observed at 2980 and 2855 cm 1 correspond with the C H stretching vibrations of the alkyl group of APTES. Fig. 3d shows the spectrum of Fe3O4@SiO2@PBLG composite microspheres. New absorptions at 1652 and 1550 cm 1 are derived from amideI and amideII, respectively. Reportedly, the conformation (a-helices and b-sheets) of polypeptides can be characterized by FT-IR [19,33–35,21,36–38]. The peaks at 1650–1658 cm 1 (amideI) and 1548–1550 cm 1 (amideII) reflect the a-helices in PBLG, while for b-sheets, peaks should appear at 1626–1630 cm 1 (amideI) and 1520 cm 1 (amideII) [38]. Accordingly, the FT-IR spectrum in Fig. 3d indicates that PBLG is successfully grafted onto the composite microspheres and furthermore the PBLG chains adopt a-helices. We also tried to confirm the a-helical conformations of the grafted PBLG via measuring CD spectroscopy. Unfortunately, the CD signals were too complex to provide clear information. This observation should be caused by the presence of Fe3O4. Additionally, the secondary conformations of polypeptides were not uniform for surface grafted polypeptides [10]. However, the FT-IR spectrum in Fig. 3d clearly conformed that the surface-grafted PBLG chains on microspheres are majorly in a-helical conformation. To ensure the consideration that PBLG was chemically grafted on the microspheres, the Fe3O4@SiO2@PBLG composite microspheres were further treated under strict conditions. Before testing FT-IR, we sonicated the microspheres with DMF for 36 h. Then the
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Fig. 3. FT-IR spectra of particles (a) Fe3O4, (b) Fe3O4@SiO2, (c) Fe3O4@SiO2–NH2, and (d) Fe3O4@SiO2@PBLG.
Fig. 4. The hysteresis loops of: (a) Fe3O4, (b) Fe3O4@SiO2, and (c) Fe3O4@SiO2@PBLG.
Fig. 5. Separability of Fe3O4@SiO2@PBLG by placing an external magnetic field. The time from state (A) to state (B) is within 10 s.
Fig. 2. XRD patterns of: (a) Fe3O4 and (b) Fe3O4@SiO2@PBLG.
microspheres were washed with THF three times to obtain dry samples. The FT-IR spectrum in this case was in accordance with the spectrum presented in Fig. 3d. Therefore the PBLG is chemically grafted on the microspheres. Further, the grafting efficiency of PBLG was determined to be ca. 73% by gravimetric method. Fig. 4 shows the varied VSM magnetization curves for pure Fe3O4 NPs (Fig. 4a), Fe3O4@SiO2 microspheres (Fig. 4b), and Fe3O4@SiO2@PBLG composite microspheres (Fig. 4c). The saturation magnetization of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@PBLG
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are 77.1, 44.3, and 34.1 emu/g, respectively. It is therefore indicated that the Fe3O4@SiO2@PBLG composite microspheres still possess high magnetization. Fig. 5 shows that the Fe3O4@SiO2@PBLG composite microspheres dispersed in aqueous respond quickly to an external magnet. The time from state (A) to state (B) is within 10 s. When the external magnet was taken off, the accumulation particles can be re-dispersed in a solvent, e.g., DMF, just by shaking. This quick responsivity of Fe3O4@SiO2@PBLG composite microspheres is very important for applications in separation. 4. Conclusions PBLG-coated Fe3O4@SiO2 composite microspheres with a diameter of 340 nm were successfully synthesized. The microspheres exhibited high magnetization (34.1 emu/g). FT-IR spectra demonstrated that the surface-grafted PBLG adopted a-helical second structures. The magnetic Fe3O4@SiO2@PBLG composite microspheres could be separated quickly by using an external magnetic field. Such novel composite microspheres are expected to exhibit some interesting functions, which are under investigation at present. Acknowledgments The Project was supported by the ‘‘Program for New Century Excellent Talents in University’’ (NCET-06-0096), the ‘‘Fundamental Research Funds for the Central Universities’’ (ZZ1117), the ‘‘National Natural Science Foundation of China’’ (20974007), and the ‘‘Project of Polymer Chemistry and Physics, Beijing Municipal Commission of Education’’. References [1] P. Tartaj, M.P. Morales, S. Veintemillas-Verdaguer, T. González-Carreño, C.J. Serna, J. Phys. D: Appl. Phys. 36 (2003) 182–197. [2] C. Wilhelm, C. Billotey, J. Roger, J.N. Pons, J.-C. Bacri, F. Gazeau, Biomaterials 24 (2003) 1001–1011. [3] Y.W. Jun, Y.M. Huh, J.S. Choi, J.H. Lee, H.T. Song, S. Kim, S. Yoon, K.S. Kim, J.S. Shin, J.S. Suh, J. Cheon, J. Am. Chem. Soc. 127 (2005) 5732–5733. [4] S. Mornet, S. Vasseur, F. Grasset, E. Duguet, J. Mater. Chem. 14 (2004) 2161– 2175.
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