Magnetic poly(glycidyl methacrylate) microspheres containing maghemite prepared by emulsion polymerization

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Journal of Magnetism and Magnetic Materials 306 (2006) 241–247 www.elsevier.com/locate/jmmm

Magnetic poly(glycidyl methacrylate) microspheres containing maghemite prepared by emulsion polymerization E. Pollerta,, K. Knı´ zˇeka, M. Marysˇ koa, K. Za´veˇtaa,b, A. Lancˇoka,b, J. Boha´cˇekc, D. Hora´kd, M. Babicˇd a Institute of Physics AS CR, 162 53 Praha 6, Cukrovarnicka´ 10, Czech Republic Joint Laboratory of Moessbauer Spectroscopy, Faculty of Mathematics and Physics, Charles University, 180 00 Praha 8, Czech Republic c Institute of Inorganic Chemistry, AS CR, 250 68 Rˇezˇ u Prahy, Czech Republic d Institute of Macromolecular Chemistry, AS CR, 162 06 Praha 6, Czech Republic

b

Received 4 October 2005; received in revised form 30 January 2006 Available online 9 May 2006

Abstract Magnetic poly(glycidyl methacrylate) (PGMA) microspheres were prepared by emulsion polymerization of glycidyl methacrylate (GMA) in the presence of (carboxymethyl)dextran (CM-dextran)-stabilized iron oxide colloid. Scanning electron microscope (SEM) and transmission electron microscope (TEM) microstructural studies revealed a spherical shape of the particles in 72–84 nm size range. Magnetic iron oxide nanoparticles (o 10 nm) were partly inside the PGMA microspheres and partly adhering to the microsphere surface. X-ray phase analysis revealed spinel structure of the iron oxide particles. According to the Mo¨ssbauer spectra, iron oxide consisted mainly of the maghemite phase. Temperature evolution of the spectra completed by the magnetization measurements confirmed superparamagnetic behavior at room temperature and the transition to the ordered state at lower temperatures. As expected, saturated magnetization increased with increasing content of the maghemite phase in the magnetic PGMA microspheres. r 2006 Elsevier B.V. All rights reserved. Keywords: Magnetic microspheres; Nanoparticles; Maghemite; Magnetic properties

1. Introduction Magnetic polymer microspheres find widespread and diverse use, such as environment protection (removal of toxic and radioactive waste solutions), with the emphasis laid mainly on biomedical applications, both therapeutic (controlled drug targeting, hyperthermia) and diagnostics (ELISA, NMR imaging) [1,2]. They play important role in isolation and purification of biomolecules (enzymes, antibodies, peptides and nucleic acids), cells, bacteria and viruses from complex biological material, determination of pathogens in foodstuff and microchip DNA preparation [3]. Magnetic polymer microspheres consist of magnetic core surrounded in ideal case by a polymer shell. They are Corresponding author. Tel.: +420 22031841; fax: +420 233343184.

E-mail addresses: [email protected], [email protected] (E. Pollert). 0304-8853/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2006.03.069

obtained by various techniques, such as coating of maghemite (g-Fe2O3) or magnetite (Fe3O4) particles by solvent evaporation [4], precipitation of iron oxide on the polymer microspheres or heterogeneous polymerization methods including activated swelling [5], suspension [6,7], dispersion [8], miniemulsion [9] and emulsion [10] polymerization in the presence of iron oxide colloid. The polymer shell has several aims, e.g., it prevents particles from the aggregation and enables covalent attachment of target biomolecules (drugs, proteins, antibodies, etc.). The present report is aimed at the synthesis and study of the magnetic properties and microstructure of new poly (glycidyl methacrylate) (PGMA) microspheres developed by emulsion polymerization in the presence of the iron oxide colloid. The study is oriented to a design of new microspheres for the magnetic separation.

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2. Experimental

2.4. Characterization

2.1. Chemicals

Iron oxide nanoparticles and polymer microspheres were observed by Philips EM 201 and JEOL JEM 200 CX transmission electron microscope, respectively. The size and polydispersity index (PDI ¼ Dw/Dn, where Dw and Dn are weight- and number-average microsphere diameter, respectively) were determined from photographs (using at least 500 particles) obtained by scanning electron microscope (SEM, JEOL JSM 6400) and analyzed with image analysis software Atlas (Tescan, Brno, Czech Republic). For the SEM viewing, a drop of dilute microsphere dispersion in water was spread on the glass surface and dried in a dust-free environment at room temperature. The dried sample was then sputtered under vacuum with a thin layer (4 nm) of platinum. The amount of iron in the CMdextran-stabilized iron oxide particles and in the magnetic PGMA microspheres was analyzed by AAS (Perkin–Elmer 3110) of an extract from the sample obtained with dilute HCl (1:1) at 80 1C for 1 h. The phase composition of the magnetic iron oxide particles/PGMA composite microspheres and the crystallite size of magnetic particles were determined by X-ray powder diffraction using Bruker D8 diffractometer (Cu Ka radiation, Sol-X energy dispersive detector). X-ray diffraction (XRD) patterns were analyzed with the Rietveld method using the FULLPROF program (Version 2.20-Sep 2002-LLB JRC). The crystallite size delimits XRD coherence length and thus contributes to peak width. Thompson–Cox–Hastings pseudo-Voigt profile was used to resolve instrumental, strain and size contributions to peak broadening. Instrumental resolution was determined by measuring strain-free tungsten powder with crystallite size 9.4 mm. Magnetic properties were measured by SQUID magnetometer (MPMS5 by Quantum Design, USA) in the temperature range 5–295 K in the fields up to 5 T. The Mo¨ssbauer spectra of pure magnetic particles were acquired in the transmission mode with 57Co diffused into Rh matrix as the source moving with constant acceleration. The spectrometer was calibrated by means of a standard aFe foil and the isomer shift was expressed with respect to this standard at 293 K. The samples were measured in a Janis cryosystem at the temperatures of 120 and 295 K and during warming up of the sample volume between these two temperatures. The spectra were fitted with the help of the NORMOS program [12].

Glycidyl methacrylate (GMA) supplied from Ro¨hm, Darmstadt, Germany, was purified by vacuum distillation. (Carboxymethyl)dextran (CM-dextran) was obtained by the reaction of dextran T 40 (M w ¼ 33; 000; M w =M n ¼ 1:4; from Dextran Products Lmt., Scarborough, Canada) with chloroacetic acid [11]. Na salt of poly(oxyethylene) alkylaryl ether sulfate (Disponils AES 60) was from Henkel, Du¨sseldorf, Germany, 25% ammonium hydroxide solution from Lachema, Neratovice, Czech Republic, sodium hypochlorite solution was from Bochemie, Bohumı´ n, Czech Republic, 4,40 -azobis(4-cyanovaleric acid) (ACVA) and all other reagent grade chemicals were purchased from Aldrich and used as received. 2.2. Synthesis of CM-dextran-stabilized maghemite nanoparticles CM-dextran aqueous solution (10 ml of 50 wt% ) was mixed under stirring with 10 ml of water solution of 1.51 g FeCl3
6H2O and 0.64 g FeCl2
4 H2O. NH4OH solution (15 ml of 7.5%) was drop-wise added until pH 12 was reached and the mixture heated to 60 1C for 15 min. Large aggregates were destroyed by sonication (W 385 Sonicator; Cole-Palmer Instruments, USA; output 40%) for 5 min. To remove unreacted iron salts, the colloid was washed by dialysis against water, using molecular weight cut-off 14,000 Visking membrane (Carl Roth GmbH, Karlsruhe, Germany), for 24 h at room temperature, changing water five times (2 l each time) until pH 6 was reached. Finally, colloid was oxidized with sodium hypochlorite solution. The above-described washing procedure was repeated and the volume reduced by evaporation. Pure iron oxide nanoparticles were obtained analogously to the above procedure, the only difference being room temperature during the synthesis and that the nanoparticles were repeatedly washed by magnetic separation in water. 2.3. Preparation of magnetic PGMA microspheres Magnetic polymer microspheres were prepared by emulsion polymerization of GMA in the presence of CMdextran-stabilized iron oxide nanoparticles. Briefly, a 100 ml reactor was loaded with 60 ml water containing 1–3 g iron oxide nanoparticles, 0.9 g Disponil AES 60, 9 g GMA and 0.18 g ACVA in 1.5 ml 1 M NaOH. The reaction mixture was bubbled with nitrogen for 10 min and polymerized under stirring (600 rpm) at 70 1C for 20 h. All products were purified by dialysis using the abovedescribed membrane and at least two cycles of ultracentrifugation (Beckman model L8-55, rotor SW 27; 5 h at 15,000 rpm), decantation and redispersion in water. Finally, the microspheres were freeze-dried.

3. Results and discussion 3.1. Preparation of magnetic PGMA microspheres First, magnetic iron oxide colloid was prepared by chemical coprecipitation of Fe(II) and Fe(III) salts with ammonium hydroxide in CM-dextran solution and subsequent oxidation with sodium hypochlorite to avoid uncontrolled oxidation of primarily formed magnetite by

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243

Fig. 1. TEM of (carboxymethyl)dextran-stabilized maghemite.

air oxygen. Advantage of the precipitation method consists in its capability to produce very fine nanoparticles. CMdextran provides a sterical stabilization of magnetic nanoparticles. While COOH group possesses affinity to Fe3+ ions, the dextran part allows the dispersion in water and prevents aggregation of the particles forming a stable colloid. Colloidal magnetic phase precipitated in the presence of CM-dextran formed ‘‘chains’’ of fine particles (Fig. 1) ca. 5 nm in diameter and with narrow size distribution (PDI ¼ 1.06; Table 1). Second, magnetic polymer microspheres were prepared by emulsion polymerization. GMA was selected as a starting monomer, because oxirane groups of its polymer (PGMA) can be easily modified, e.g., hydrolyzed to vicinal diols [13], transformed to amines or oxidized to aldehyde groups [14];  also –SO 3 , –N R3, chelating and other functional groups can be easily introduced [15]. Modifiability of polymer microspheres is an essential condition for any prospective attachment of target enzymes and other biologically active molecules required by the biomedical applications. Emulsion polymerization of GMA was carried out in the presence of CM-dextran-coated magnetic nanoparticles using ACVA as an initiator. In emulsion polymerization, the surfactant plays an important role in the stability, rheology and control of the microsphere size of the resulting latexes. Disponil AES 60 was selected as the surfactant in this study because it is a well-balanced tenside having a high hydrophilicity (due to its poly(oxyethylene) chain) on one site and the alkylaryl group with the affinity to the organic liquid on the other site. Moreover, its anionic charge stemming from SO 3 boosts the emulsifier efficiency. As iron oxide/monomer ratio in the emulsion polymerization is the most effective parameter for controlling the

Table 1 Some characteristics of PGMA microspheres Sample

I II III IVa

Dn (nm)

72 84 75 5b

PDI

1.024 1.055 1.040 1.062

Maghemite (wt%) Feed

Polymer

1.4 2.9 4.3 56

0.8 6.2 7.6 25.2

Ms (emu/g)

0.5 2.96 3.84 17.42

Dn—diameter determined from SEM; Ms—saturation magnetization. a CM-dextran-stabilized maghemite. b Determined from TEM.

magnetic quality of the resulting microspheres, the polymerization of GMA in water medium was carried out according to a standard recipe in which all the reaction parameters were kept constant with the CM-dextrancoated maghemite content being the only variable. The monomer concentration was maintained at 15 wt%, the Disponil AES 60 concentration was 1.5 wt% (relative to water) and the initiator concentration (ACVA) was 2 wt% (relative to the monomer). 3.2. Microstructure analysis As confirmed by the SEM micrograph (Fig. 2), latex PGMA microspheres possessed discrete spherical shape and no marked aggregation of the latex particles occurred. Size of microspheres varied from 72 to 84 nm with a narrow size distribution characterized by PDI 1.05 (Table 1). The magnetic particles seemed to be mostly

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Fig. 2. SEM of magnetic PGMA microspheres, sample III, containing 7.6 wt% of maghemite.

Fig. 3. TEM of (a) sample I (0.8 wt% of g-Fe2O3) and (b) sample II magnetic PGMA microspheres (6.2 wt% of g-Fe2O3).

incorporated in the latex spheres and no free magnetic particles were discernible in SEM. A more detailed observation in TEM, however, revealed two kinds of the magnetic particles. They were only partly inside the PGMA microspheres, but predominantly adhering to the microsphere surface. This was especially pronounced at the higher concentration of the magnetic phase (Fig. 3). Moreover, magnetic PGMA microspheres were aggregated, which can be ascribed to the fact that TEM micrographs were taken of freeze-dried microspheres. Microsphere drying is always associated with difficulties, because of irreversible particle agglomeration and structure shrinkage.

3.3. Phase analysis XRD pattern of sample I showed PGMA shell and hardly discernible magnetic iron oxide core, because of its low content. An increase of the amount of CM-dextranstabilized magnetic phase in the feed, and consequently in the microspheres, led to a better resolution of the iron oxide spinel phase, either magnetite or maghemite. Nevertheless, because of nearly identical lattice constants of both these phases, it is difficult to decide which phase was present. Simultaneously, instead of PGMA patterns, those corresponding to CM-dextran phase developed (Fig. 4). This surprising finding can be presumably understood in

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terms of efficacy of the iron oxide encapsulation by the emulsion polymerization (see TEM of magnetic microspheres in Fig. 3). While the crystallites preferably entered

the interior of the PGMA microspheres (sample I) at the low amount of the magnetic phase in the feed, a significant part of the crystallites remained on the surface of the PGMA microspheres which had a tendency to agglomerate at an increased amount of CM-dextran-stabilized iron oxide in the feed. Consequently, patterns of CM-dextran were distinguished in the phase analysis of magnetic PGMA microspheres (samples II and III in Table 1).

8000

relative intensities

245

A

6000

3.4. Mo¨ssbauer spectra B

The spectra of pure iron oxide nanoparticles obtained at room temperature and at 120 K are shown in Fig. 5. The information derived from the spectra are summarized in Table 2. On the qualitative level it is obvious that at the higher temperature most of the particles are in the superparamagnetic state as witnessed both by the large relative area of the doublets and the broad sextet (see Table 2) and also by the rather broad distribution of the hyperfine fields of this sextet. On the other hand, the spectrum at 120 K can be fitted by two doublets, one rather broad sextet and two relatively narrow sextets (sextets 1 and 2 in Table 2), the latter ones corresponding to a magnetically ordered phase. It is evident how at lower temperatures the relative area of the components ascribed to the magnetically ordered phase grow at the expense of those attributed to the superparamagnetic one. The hyperfine fields for sextets 1 and 2 at 120 K shown in Table 2, as well as the isomer shift of at most 0.38 mm/s indicate that the magnetic particles predominantly consisted of the maghemite phase. The changes of the spectra with temperature are illustrated in Fig. 6. Though the lower quality of the spectra due to much shorter exposure times at intermediate temperatures did not enable their detailed analysis, it seems that the transition from ordered to superparamagnetic state with increasing temperature mostly takes place in the temperature interval between 170–230 K.

4000 C 2000

D E

0 20

30

40

50

60

70

2θ Fig. 4. X-ray phase analysis of (A) non-magnetic PGMA microspheres, (B) sample I, (C) sample II, (D) sample I, and (E) CM-dextran. Vertical bars—g-Fe2O3 standard.

Intensity (a.u.)

RT

120 K

3.5. Magnetic properties -10

-5

0 Velocity (mm/s)

5

10

Magnetization curves of the sample consisting of pure magnetic particles are plotted in Fig. 7. They were measured for several temperatures between 120 and 300 K, i.e., in the temperature range in which the

Fig. 5. Mo¨ssbauer spectra at room temperature and 120 K, pure g-Fe2O3 nanoparticles.

Table 2 Relative areas of the components of the Mo¨ssbauer spectra RT

Sextet 1 Sextet 2 Broad sextet Doublet 1 Doublet 2

120 K

Bhf (T)

ISO (mm/s)

r.a. (%)

Bhf (T)

ISO (mm/s)

r.a. (%)

40.3

0.38

14.4

25.9

0.29 0.37 0.18

76.0 5.0 4.6

47.9 44.0 26.4

0.13 0.10 0.34 ~0.0

38.3 41.8 16.7 3.2

Bhf—hyperfine field, ISO—isomer shift, r.a.—relative area.

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246

12 RT 10 M [emu/g]

Intensity (a.u.)

275 K 230 K 170 K

8

6

140 K

ZFC FC

4

120 K 2 0 -10

-5

0 Velocity (mm/s)

5

10

Fig. 6. Temperature evolution of the Mo¨ssbauer spectra between 120 K and room temperature, pure g-Fe2O3 nanoparticles.

80 120 140 170 230 275 300

M [emu/g]

40

K K K K K K

0

-40

-80 -6

-4

-2

0 H [T]

2

4

6

Fig. 7. Magnetization curves of the sample used in the Mo¨ssbauer spectroscopy, pure g-Fe2O3 nanoparticles. Note the maximum field of 5 T.

Mo¨ssbauer spectra were studied. The saturation magnetization at room temperature was 55.9 emu/g. Employing previously reported data on the evolution of the magnetization with maghemite particle dimensions [16,17], the size of pure magnetic nanoparticles can be roughly estimated to be 9 nm. This is in agreement with the size of the magnetic particles Dno10 nm determined by TEM (Fig. 1). In order to get deeper insight of the transition from the ordered or frozen magnetic state of the particles to a superparamagnetic one, the temperature dependences of magnetization after cooling in zero (MZFC) and non-zero field (MFC) were measured from 10 to 300 K in the field of 10 mT (Fig. 8). If the onset of the blocking process is defined as the temperature at which these two curves draw apart, then it would start at around 200 K and proceed

50

100

150 T [K]

200

250

300

Fig. 8. Temperature dependence of magnetization after cooling in zero (ZFC) and non-zero (FC) magnetic field, pure g-Fe2O3 nanoparticles.

down to low temperatures. As the blocking temperature of a magnetic particle is rather sensitive to its size, in particular to its volume, any distribution of the particle sizes results in a distribution of blocking temperatures. A more detailed description of the blocking process may be received if we accept that the derivative by temperature of the difference of these two magnetizations d(MFC–MZFC )/ dT gives the distribution of blocking temperatures [18] of individual particles. This distribution starts from above at about 120 K and passes a maximum just below 40 K. Let us note that the results obtained elsewhere [19] show the onset of the blocking process of a system of g-Fe2O3 particles with mean size of 9–10 nm at a temperature ca. 100–120 K and the maximum of MZFC curve, measured in the field of 1 mT and denoted here as blocking temperature TB, at 75 K. From our Fig. 8 and its analysis it is obvious, why the magnetization curves in Fig. 7 are rather similar and certainly do not indicate any marked change of the magnetic state (i.e., from ordered to superparamagnetic) in the given temperature range. It is worth noting that the concept of the superparamagnetic state and the transition from the ordered to superparamagnetic state heavily depends on the characteristic time of the relevant observation: this time is in the order of 107 s in Mo¨ssbauer spectroscopy of 57Fe, while it amounts to at least units of seconds with the DC magnetic measurements. In agreement with this fact, the transition to superparamagnetism is indicated at much higher temperatures, as inferred from the Mo¨ssbauer spectra. The full discussion of the transition to the superparamagnetic state is beyond the scope of the present study and will be investigated in more detail in near future. Magnetization measurements of magnetic PGMA microspheres showed an expected gradual increase of the saturated magnetization with increasing content of the maghemite phase in the microspheres as determined by chemical analysis (see Table 1 and Fig. 9).

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seconds in the superparamagnetic state down to about 120 K. A full elucidation of the found difference will be a subject of a study in near future.

sample I

4

247

sample II sample III

Acknowledgment 0

20 M [emu/g]

M [emu/g]

2

-2

10 0 -10

sample IV

-20

-4

-2

-1

0

1

2

This study was performed under support of the Academy of Sciences of the Czech Republic, Project 1QS100100553: New hybride magnetic nanocomposite materials for selected applications in medicine, magnetic resonance imaging and magnetic hyperthermia.

H [T]

-2.0

-1.5

-1.0

-0.5

0.0 H [T]

0.5

1.0

1.5

2.0

Fig. 9. Magnetization curves of magnetic PGMA microspheres, samples I–III and CM-dextran-stabilized maghemite, sample IV.

In addition, the magnetization curve is plotted in the inset of Fig. 9. 4. Conclusions Magnetic nanoparticles/PGMA composite microspheres prepared by emulsion polymerization of GMA in the presence of magnetic iron oxide colloid stabilized by CMdextran exhibit a spherical shape and a size in the range 72–84 nm. Narrow particle size distribution was characterized by PDI 1.05. Iron oxide magnetic nanoparticles (Dno10 nm) were found partly inside the PGMA microspheres and partly on the microsphere surface. According to the Mo¨ssbauer spectroscopy, iron-oxide nanoparticles mainly consisted of the maghemite phase possessing spinel structure. An expected increase of the saturated magnetization was observed with increasing content of the maghemite phase in the magnetic PGMA microspheres. Mo¨ssbauer spectroscopy revealed partially blocked magnetic moments of the maghemite nanoparticles at a time scale of hundreds of nanoseconds in the temperature range 230–170 K. In contrast, magnetization measurements showed that the system of nanoparticles is at a time scale of

References [1] T. Neuberger, B. Scho¨pf, H. Hofmann, M. Hofmann, B. von Rechenberg, J. Magn. Magn. Mater. 293 (2005) 483. [2] S. Mornet, S. Vasseur, F. Grasset, E. Duguet, J. Mater. Chem. 14 (2004) 2161. [3] M. Megens, M. Prins, J. Magn. Magn. Mater. 293 (2005) 702. [4] R.V. Ramanujak, W.T. Chong, J. Mater. Sci. Mater. Med. 15 (2004) 901. [5] J. Ugelstad, T. Ellingsen, A. Berge, et al., PCT Int. Appl., WO Patent 8,303,920, 1983. [6] C. Yang, H. Liu, Y. Guan, J. Xing, J. Liu, G. Shan, J. Magn. Magn. Mater. 293 (2005) 187. [7] Y. Lee, J. Rho, B. Jung, J. Appl. Polym. Sci. 89 (2003) 2058. [8] D. Hora´k, N. Semenyuk, F. Lednicky´, J. Polym. Sci. Polym. Chem. Ed. 41 (2003) 1848. [9] X. Liu, Y. Guan, H. Liu, Z. Ma, Y. Yang, X. Wu, J. Magn. Magn. Mater. 293 (2005) 111. [10] A. Kondo, H. Kamura, K. Higashitani, Appl. Microbiol. Biotechnol. 41 (1994) 99. [11] R. Huynh, F. Chaubet, J. Jozefonvicz, Angew. Makromol. Chem. 254 (1998) 61. [12] R.A. Brand, NORMOS PROGRAMS, Duisburg University, 1989. [13] T.B. Tennikova, D. Hora´k, F. Sˇvec, J. Kola´rˇ , J. Cˇoupek, S.V. Trushin, A.G. Maltsev, B.G. Belenkii, J. Chromatogr. 435 (1988) 357. [14] J. Lenfeld, F. Sˇvec, J. Ka´lal, Acta Polym. 37 (1986) 377. [15] E.R. Kenawy, F.I. Abdel-Hay, A.R. El-Shanshoury, M.H. El Neseny, J. Control. Release 50 (1998) 145. [16] D.H. Han, J.P. Wang, H.L. Luo, J. Magn. Magn. Mater. 136 (1994) 176. [17] X.N. Xu, Y.Y. Wolfus, A. Shaulov, Y. Yeshurun, I. Felner, I. Nowik, Yu. Koltypin, A. Gedanken, J. Appl. Phys. 91 (2002) 4611. [18] B. Martı´ nez, X. Obradors, L1. Balcells, A. Rouanet, C. Monty, Phys. Rev. Lett. 80 (1998) 181. [19] J.J. Lu, H.Y. Deng, H.L. Huang, J. Magn. Magn. Mater. 209 (2000) 37.