Facile and Green Synthesis of Core–Shell Structured Magnetic

Aug 22, 2013 - (3, 4) Among these magnetic composites, submicrometer-sized ones are ... Ltd. (Zhejiang, China), with viscosity-average MW of about 40 ...
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Facile and Green Synthesis of Core−Shell Structured Magnetic Chitosan Submicrospheres and Their Surface Functionalization Yiya Li, Dongying Yuan, Mingjie Dong, Zhihua Chai, and Guoqi Fu* Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China

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S Supporting Information *

ABSTRACT: Submicrometer-sized magnetite colloid nanocrystal clusters (MCNCs) provide a new avenue for constructing uniformly sized and highly magnetic composite submicrospheres. Herein, a facile and eco-friendly method is described for the synthesis of Fe3O4@poly(acrylic acid) (PAA)/chitosan (CS) core−shell submicrospheres using MCNCs bearing carboxyl groups as the magnetic cores. It is based on the self-assembly of positively charged CS chains on the surface of the oppositely charged MCNCs dispersed in the aqueous solution containing acrylic acid (AA) and a cross-linker N,N′-methylenebis(acrylamide) (MBA), followed by radical induced cross-linking copolymerization of AA and MBA along the CS chains. The resulting polymer shell comprises a medium shell of cross-linked PAA/CS polyelectrolyte complexes and an outer shell of protonated CS chains. It was found that the shell thickness could be tuned by varying either the concentration of radical initiator or the molar ratio of AA to aminoglucoside units of CS. To the surface of thus obtained Fe3O4@ PAA/CS particles, Au nanoparticles, a variety of functional groups such as fluorescein, carboxyl, quaternary ammonium, and aliphatic bromide, and even functional polymer chains were successfully introduced. Therefore, such Fe3O4@PAA/CS submicrospheres may be used as versatile magnetic functional scaffolds in biorelated areas like bioseparation and medical assay, considering the unique features of CS like nontoxicity and biocompatibility.

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INTRODUCTION

which are necessary for rapid and homogeneous separation in an applied magnetic field. On the other hand, Deng et al.12 along with other groups13,14 have recently developed a facile method for the preparation of monodisperse or narrowly dispersed magnetite colloid nanocrystal clusters (MCNCs) by one-pot solvothermal reduction of FeCl3 with ethylene glycol. Thus, obtained MCNCs are spherical in shape, and the sizes can be readily tailored in a wide submicrometer range (100−800 nm). Each MCNC is composed of many interconnected primary Fe3O4 nanocrystals of ∼10 nm in size and minor amount of organic components, which render the resulting clusters superparamagnetism and also high magnetization. These MCNCs provide a new avenue for constructing functional composite submicrospheres such as highly magnetic silica, titania, and carbon particles.15−17 A variety of approaches have also been reported for coating MCNCs with different synthetic polymers.18−24 However, natural polymers are rarely employed to fabricate core−shell magnetic microspheres based on such MCNCs. Chitosan (CS) is the naturally unique alkaline polysaccharide composed of β-1,4-linked glucosamine. It is easily obtained by N-deacetylation of the natural polymer of chitin, the second-

Core−shell structured nano- and microspheres have recently been subject to extensive research for the combined functionalities of cores and shells which endow them with great application potentials in various fields.1,2 As an important kind of core−shell structures, magnetic polymer particles consisting of an iron oxide core and a polymer shell have attracted particular attention for their unique magnetic responsiveness, good dispersivity, and availability of surface functional groups. They have shown great potential in wide areas such as target drug delivery, enhanced magnetic resonance imaging, separation or purification of bioentities, immunoassay tests, immobilization of enzymes, and catalysis.3,4 Among these magnetic composites, submicrometer-sized ones are particularly suitable for the applications such as bioseparation and immunoassay due to their faster magnetic separation than the nanoparticles and also larger specific surface area for ligand coupling together with weaker sedimentation than the micrometer-sized controls.5,6 These magnetic polymer submicrospheres are usually prepared by various heterogeneous polymerization techniques like emulsion and miniemulsion polymerization with iron oxide nanoparticles of ∼10 nm in size as the seeds.7,8 However, it is still a substantial challenge to produce the magnetic polymer particles with high and uniform magnetic content and narrow particle size distribution,9−11 © 2013 American Chemical Society

Received: June 17, 2013 Revised: August 17, 2013 Published: August 22, 2013 11770

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Beijing), and all the other chemicals were used as received unless otherwise stated. Synthesis of Fe3O4@PAA/CS Submicropheres. The MCNCs bearing carboxylic groups were prepared according to the method described previously.14 Briefly, FeCl3 (3.9 g), trisodium citrate dehydrate (1.8 g), and sodium acetate (7.2 g) were dissolved in ethylene glycol (120 mL). The resultant mixture was sealed in a Teflon-lined stainless-steel autoclave, heated to 200 °C and maintained for 10 h, and then cooled to room temperature. The black particles were thoroughly washed sequentially with ethanol and deionized water and finally dried under vacuum at 50 °C. The Fe3O4@PAA/CS particles were synthesized by copolymerization of AA and MBA in CS solution mixed with the above-obtained MCNCs. Typically, CS (250 mg) and MBA (11 mg) was added to 10 mL of aqueous AA solution with the molar ratio of 1:1 ([aminoglucoside units]:[AA]) and then intensively stirred overnight for thorough CS dissolution. This solution was diluted with 70 mL of deionized water and mixed with 100 mg of MCNCs. The dispersion was sonicated under mechanical agitation for 30 min for sufficient preassembly and then heated to 70 °C together with continued stirring and driving out air using nitrogen stream. After adding 44 mg of KPS dissolved in 2 mL of deionized water, the polymerization was carried out for 5 h under a nitrogen atmosphere and mechanical agitation. The resulting magnetic polymer particles were enriched with the help of a magnet, washed sequentially with HAc solution (1 wt %) and deionized water, and finally freeze-dried. Loading of Au Nanoparticles with Fe3O4@PAA/CS Submicropheres as Support. 3 mg of the Fe3O4@PAA/CS particles obtained with AA/CS = 1:1 was mixed with 3 mL of distillated water and 155 μL of aqueous HAuCl3 solution (1 mM). After shaking for 1 h, 155 μL of aqueous NaBH4 solution (10 mM) was added, and the suspension was shaken for 2 h to complete the reduction. The resulting particles (denoted as Fe3O4@PAA/CS−Au) were isolated with a magnet, washed with deionized water, and finally freeze-dried. Surface Chemical Modification of Fe3O4@PAA/CS Submicropheres. The surface of the Fe3O4@PAA/CS submicropheres was allowed to be modified with FITC, succinic anhydride, glycidyltrimethylammonium chloride, 2-bromoisobutyryl bromide, and PDMAEMA. The reaction procedures are described as follows. As for coupling FITC, the Fe3O4@PAA/CS particles (5 mg) were suspended in 1 mL of phosphate buffer solution (10 mM, pH 8.2), mixed with 1 mg of FITC dissolved in 1 mL of dehydrated methanol, and then incubated for 24 h in the dark at room temperature. After the reaction, the resulting FITC-labeled particles (denoted as Fe3O4@ PAA/CS−FITC) were collected by magnetic separation, washed extensively with methanol for the removal of the unconjugated FITC, and finally dispersed in deionized water. For reaction with succinic anhydride, the Fe3O4@PAA/CS particles (0.2 g) were mixed with DMF (15 mL) and succinic anhydride (310 mg), and the dispersion was stirred at 60 °C overnight. Finally, the product (denoted as Fe3O4@PAA/CS-COOH) was collected by magnetic separation, washed extensively with ethanol and water, and finally freeze-dried. When reacted with glycidyltrimethylammonium chloride, the Fe3O4@PAA/CS particles (15 mg) were dispersed in 5 mL of glycidyltrimethylammonium chloride aqueous solution (0.1 g/mL), and then the reaction was carried out at 60 °C for 24 h. Finally, the product (denoted as Fe3O4@PAA/CS-N(CH3)3+) was collected by magnetic separation, washed extensively with water, and finally freezedried. For modification with 2-bromoisobutyryl bromide, the Fe3O4@ PAA/CS (0.2 g) was added to 15 mL of DMF containing 0.5 mL of triethylamine. To this mixture cooled with an ice bath, 0.3 mL of 2bromoisobutyryl bromide was dropped slowly under stirring, and then the reaction was allowed to proceed at room temperature for 8 h. The resultant particles (denoted as Fe3O4@PAA/CS-Br) were collected by magnetic separation, washed sufficiently with DMF and then with ethanol, and finally dried under vacuum at room temperature. Grafting of PDMAEMA from the Fe3O4@PAA/CS particles was achieved via surface-initiated ATRP of DMAEMA with the Fe3O4@

most abundant natural polymer after cellulose and the main structural component of marine crustaceans like crab and lobster. The presence of plentiful amino and hydroxyl groups in the macromolecular chains is highly advantageous for conducting modification reactions and for providing distinctive biological functions. Its other outstanding characteristics include biocompatibility, biodegradability, bioactivity, nontoxicity, mucus adhesion, good adsorption performance, potent antimicrobial activity, and low cost. Therefore, in recent years, this polycationic biopolymer is receiving a great deal of attention for biosensing, medical, pharmaceutical, catalytic, and separation applications.25 Inheriting these special properties, CS-based magnetic particles ranging from nanometer up to tens of micrometer size have attracted particular research interest, especially in the areas of biomedicine and biotechnology. Several methods have been developed for obtaining magnetic chitosan particles, such as reverse-phase suspension crosslinking method,26 in-situ microemulsion method,27 and magnetite synthesis by coprecipitation followed by chitosan coating.28 Usually, these preparation procedures are complex and need to use some organic solvents or surfactants and also face difficulty in obtaining the products with high and uniform magnetic content and uniform particle size distribution. In this work, we report a facile and green method for the fabrication of core−shell magnetic chitosan submicrospheres using high-magnetization and uniform-sized MCNCs as the core materials. On the basis of aqueous one-pot cross-linking copolymerization of acrylic acid (AA) and a cross-linker N,N′methylenebis(acrylamide) (MBA) in the presence of dispersed MCNCs bearing carboxylic groups and dissolved CS, each MCNC is covered by a polymer shell which is composed of a medium shell of cross-linked PAA/CS polyelectrolyte complexs and an outer shell of protonated CS chains. The medium layer can serve to protect the internal Fe3O4 from oxidization and low pH environment, and the outer CS chains can afford further surface modification of the composite particles. Asprepared Fe3O4@PAA/CS particles were characterized detailedly by a variety of approaches like TEM, DLS, and TGA. The potential in surface functionalization thanks to the outer CS chains was confirmed by a variety of chemical modification procedures.

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EXPERIMENTAL SECTION

Materials. CS powder was purchased from Yuhuan Biochemical Co., Ltd. (Zhejiang, China), with viscosity-average MW of about 40 000 and deacetylation degree of about 90%. AA (Tianjin Chemical Reagents Co., Tianjin) were purified by vacuum distillation. 2(Dimethylamino)ethyl methacrylate (DMAEMA, Alfa Aesar) was purified by passing through a basic alumina column. CuCl (Tianjin Chemical Reagents Co., Tianjin, AR) was purified by stirring it with acetic acid overnight, washing with ethanol and diethyl ether, and then dried under vacuum. Potassium persulfate (KPS, Sinopharm Chemical Regent Co. Ltd., Shanghai, AR) was recrystallized in water. CuCl2 (Tianjin Chemical Reagents Co., Tianjin, AR), ethylene glycol (Guangfu Chemical Reagents Co. Tianjin, AR), sodium acetate (Guangfu Chemical Reagents Co. Tianjin, AR), trisodium citrate dehydrate (Guangfu Chemical Reagents Co. Tianjin, AR), FeCl3 (Alfa Aesar, 99.9%), succinic anhydride (Tianjin Chemical Reagents Co., Tianjin, AR), glycidyltrimethylammonium chloride (Tianjin Chemical Reagents Co., Tianjin, AR), HAuCl3·4H2O (Sinopharm Chem Regent Co. Ltd., Shanghai, AR), ethyl 2-bromoisobutyrate (EBriB, Alfa Aesar, 98%), 2-bromo-2-methylpropionyl bromide (Aldrich, 98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, Aldrich, 99%), fluorescein isothiocyanate (FITC, Dingguo Biotech Co., Ltd. 11771

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Scheme 1. Schematic Presentation of the Synthetic Route for the Fabrication of Fe3O4@PAA/CS Submicrospheres

PAA/CS-Br particles as initiator. The initiator particles (50 mg), EBriB (0.0015 mmol) as co-initiator, DMAEMA (1.5 mL), acetone (1.5 mL), CuCl2 (0.03 mmol), and CuCl (0.03 mmol) were added to a dry flask, and the mixture was degassed by three freeze−pump−thaw cycles. PMDETA (0.18 mmol) was added, and the flask was degassed by another two freeze−pump−thaw cycles. Afterward, the suspension was stirred at 35 °C in a nitrogen atmosphere for 24 h. The resulting particles (denoted as Fe3O4@PAA/CS−PDMAEMA) were collected by magnetic separation, extensively washed with abundant methanol, and dried under vacuum. The supernatant was dialyzed (MWCO of 1000) against distillated water for 3 days and then lyophilized to obtain the PDMAEMA hompolymer. Characterizations. Transmission electronic microscopy (TEM) images were taken using a Technai G2 20-S-TWIN TEM operated at 200 kV. Samples were dispersed in ethanol at an appropriate concentration, cast onto a carbon-coated copper grids, and then dried under vacuum. Hydrodynamic diameters (Dh) and zeta potentials of the particles were measured by dynamic light scattering (DLS) with a Malvern ZEN3600 Zetasizer Nano instrument using a He−Ne laser at a wavelength of 632.8 nm. Fourier-transformation infrared (FT-IR) spectra were determined on a Bio-Rad FTS 135 FTIR spectrometer over KBr pellets. The magnetic properties were studied with a vibrate sample magnetometer (VSM, 9600, BOJ Electronics) at room temperature. Thermogravimetric analysis (TGA) was carried out for the particle samples (∼10 mg) using a NETZSCH TG 209 thermogravimetric analyzer under a nitrogen atmosphere with a heating rate of 10 °C/min up to 900 °C. Powder X-ray diffraction (XRD) patterns were recorded on a D/max 2500 V X-ray diffractometer using Cu Kα radiation at 40 kV and 100 mA. The apparent molecular weight and polydispersity of the PDMAEMA homopolymer were determined by gel permeation chromatography (GPC) with a CoMetre 6000 LDI pump and Schambeck SFD GmbH RI2000 refractive index detector. DMF with 0.01 M LiBr was used as the mobile phase at a flow rate of 1 mL/min. Poly(methyl methacrylate) calibration kit was used as the calibration standard. The sample polymer solution was injected through two PLgel columns (PL1110-6130 and PL1110-6140) at 70 °C.

chains and an inner shell of PAA/CS polyelectrolyte complexes. The outer CS chains can be cross-linked with glutaraldehyde to stabilize the nanostructures. Moreover, inorganic components such as Fe3O4 and Au nanoparticles can be in-site embedded in the shell, resulting in multifunctional hybrid nanostructures.31,32 For endowment of the polymer particles with magnetic property, the authors proposed an approach to prepare magnetic PAA/CS hollow nanospheres. A minor amount of poly(vinyl alcohol) stabilized Fe3O4 nanoparticles were encapsulated in the inner shell of PAA/CS complex by their interaction with CS and AA (or PAA) via hydrogen bonds.31 On the basis of the strong electrostatic interaction of cationic CS and negatively charged Fe3O4 nanopaticles stabilized by sodium citrate, Wu et al.33 fabricated magnetic PAA/CS solid submicrospheres with higher magnetic content. Inspired by the aforementioned studies, we developed a novel method for the synthesis of narrowly dispersed and highly magnetic PAA/CS submicrospheres (denoted as Fe3O4@PAA/CS) by utilization of sodium citrate stabilized MCNCs as the magnetic cores. The procedure is illustrated in Scheme 1. At first, CS is dissolved in an aqueous AA solution containing MBA as a cross-linker. Then the carboxyl-bearing MCNCs are thoroughly dispersed in this solution for selfassembly. After adding a radical initiator KPS to the mixture, the cross-linking copolymerization of AA with MBA proceeds, finally leading to the formation of a polymer shell over the MCNCs. According to the structure of the hollow PAA/CS nanospheres and its formation mechanism proved by Jiang and co-workers,30,31 it is safe to say that the polymer shell generated over the MCNCs is composed of a medium shell of crosslinked PAA/CS polyelectrolyte complexes and outer shell of protonated CS chains. The presence of the outer CS chains will be confirmed by a variety of surface modification of the Fe3O4@PAA/CS particles in the following sections. This approach for the synthesis of the Fe3O4@PAA/CS particles presents the following features: (1) the obtained core−shell particles can inherit the advantages of the MCNCs, that is, high-magnetization, uniform or narrow size distribution, and easy size regulation; (2) instead of using glutaraldehyde for cross-linking the outer CS chains, utilization of the cross-linking monomer MBA for coplymerization with AA not only makes the synthesis process in one step but also reserves a larger amount of active amino groups for further surface functionalization; (3) the two parts of the formed polymer shell can function differently, with the relatively compact medium layer of cross-linked PAA/CS polyelectrolyte complexes serving as protection for the internal Fe3O4 and the outer CS chains bearing amino and hydroxyl groups affording facile surface modification. Previously, several research groups34−36 fabricated CS containing MCNCs by dissolving it together with FeCl3 and sodium acetate in ethylene glycol during the solvothermal

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RESULTS AND DISCUSSION Previously, Jiang’s group29,30 developed a core-template-free strategy for the preparation of hollow nanospheres and hollow composite nanospheres of PAA/CS in completely aqueous solution of AA and CS without the aid of surfactants and other additive substances. This approach is based on the observed fact that core−shell micelles can be formed above certain CS concentration, with the cores being mainly composed of polyion complexes of positively charged protonated CS chains and negatively charged dissociated AA, whereas the swelling shells consisting of protonated CS chains. Initiation of the polymerization of AA with KPS leads to the formation of hollow nanospheres. Based on the cut-section TEM image of the resultant nanoparticles after microtoming, the hollow structure is confirmed, and it is also concluded that the hollow nanospheres are composed of an outer shell of protonated CS 11772

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increasing AA/CS ratio from 0.6:1 to 1:1. This is reasonable since larger amount of AA is advantageous for immobilization of the preadsorpted CS on the surface of the MCNCs by the formation of the MBA cross-linked PAA/CS complexes. Moreover, higher concentration of AA naturally favors grafting of more PAA onto the MCNCs. It is worth noting that the amount of grafted polymer was negligible when AA/CS ratio was less than 0.4:1. On the other hand, Table 1 shows that the polymer graft percentage also increases with the increase in KPS concentration within the range covered. This may be explained by assuming that relatively high initiator concentration can promote the copolymerization of AA and MBA along the CS chains. Based on the graft percentage listed in Table 1 and the polymerization recipe, the amounts of the polymer coated onto the MCNCs were estimated to be less than 15% the total feeding amounts of monomers and chitosan. Such relatively low graft amounts can be accounted for by the observed fact that a lot of homogeneous polymer nanoparticles were also generated in the final polymerization system. The core−shell particles obtained with different AA/CS ratios together with the bare MCNCs were further characterized. TEM was employed to observe the morphology and structure of these particles. As shown in Figure 1a, the original MCNCs are spherical-shaped and nearly monodisperse with an avenge diameter of ∼200 nm. High-resolution TEM image (inset in Figure 1a) shows that each MCNC is composed of many minute nanocrystals less than 10 nm. A well-defined core−shell structure can be distinguished from the TEM images of the Fe3O4@PAA/CS particles prepared with increasing AA/CS molar ratio (see Figure 1b−d). The largest thickness of the PAA/CS particles with AA/CS = 1:1 was estimated to be ∼16 nm. Figure 2A shows the TG curves of these particles. Up to ∼900 °C, the Fe3O4@PAA/CS particles exhibit significantly larger weight loss than the bare MCNCs,

reduction process. However, thus-synthesized MCNCs are not core−shell structured, though there are also some CS moieties residual on their surface. Herein, the synthesis, characterization, and surface modification of the Fe3O4@PAA/CS particles were studied in detail as follows. Synthesis and Characterization of Fe3O4@PAA/CS Particles. We examined the influence of some polymerization parameters on the formation of the PAA/CS shell over the MCNCs. The molar ratio of AA/CS and the KPS concentration were found to exhibit a significant effect on polymer graft percentage, which is defined as the polymer percent content relative to the initial amount of the MCNCs employed and determined from TG measurements. As listed in Table 1, the graft percentage increases from 33.2 to 54.1 with Table 1. Effect of Polymerization Conditions on the Polymer Graft Percentage and Hydrodynamic Diameters of the Resulting Fe3O4@PAA/CS Submicrospheres samples 1 2 3 5 6 MCNCs

AA/CSa (molar ratio)

KPS concn (mol/L)

graft percentageb (%)

0.6:1 0.8:1 1:1 1:1 1:1

2.03 2.03 2.03 1.78 1.27

33.2 39.7 54.1 41.6 33.3

Dhc (nm) 229.1 252.9 262.7 256.1 233.6 195.6

shell thicknessd (nm) 16.8 28.7 33.6 30.3 19.0

a Molar ratio of AA to aminoglucoside units. bThe polymer graft percentage of the Fe3O4@PAA/CS particles was calculated based on the polymer percent content relative to the initial amount of the MCNCs employed. The polymer content was determined by TGA. c Average hydrodynamic diameter determined by DLS in ethanol. d Calculated according to [Dh(Fe3O4@PAA/CS) − Dh(MCNCs)]/2.

Figure 1. TEM images of (a) MCNCs, the Fe3O4@PAA/CS particles with AA/CS molar ratio of (b) 0.6:1, (c) 0.8:1, and (d) 1:1, and Au nanoparticle loaded Fe3O4@PAA/CS particles (AA/CS = 1:1). 11773

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Figure 2. TG curves (A) and hydrodynamic diameter distribution (B) of different magnetic particles: MCNCs (a), Fe3O4@PAA/CS particles with AA/CS molar ratios of 0.6:1 (b), 0.8:1 (c), and 1:1 (d).

the same pH varying range. It should be noted that all of the Fe3O4@PAA/CS particles demonstrate approximate zeta potential values, though fabricated with different AA/CS ratios. This result implies the similar chemical composition on the surface of these Fe3O4@PAA/CS particles, i.e., tethered CS chains which are not complexed with the oppositely charged PAA as illustrated in Scheme 1. As an alkaline polysaccharide with a pKa of 6.5,29 CS itself should not show negative zeta potentials even at high pH values. However, the Fe3O4@PAA/ CS particles show negatively charged when pH >5.8, as seen in Figure 3. This may be ascribed to the ionized carboxyl groups on the surface of the MCNCs and those of the PAA (pKa = 4.75)29 in the polymer shell. These negatively charged groups may contribute to the overall charges of the particles, though not located on the outer surface. Wu et al.33 also observed the negative zeta potentials of the core−shell magnetic PAA/CS particles which were synthesized with citrate stabilized Fe3O4 nanopaticles as the cores. Loading of Au Nanoparticles with Fe3O4@PAA/CS Particles as Magnetic Scaffold. CS of different physical forms, such as (nano)particles, free outstanding or supported films, and fibers, has been widely used as a support for metallic nanoparticles for catalysis and other applications.37,38 For proof of the existence of free CS chains on the Fe3O4@PAA/CS particles’ surface, we tried to immobilize Au nanoparticles onto the magnetic particles by their first sorption of Au3+ in the aqueous solution followed by in site reduction of the uptaken cations using NaBH4. The obtained magnetic composite particles were observed by TEM. As shown in Figure 1f, Au nanoparticles of about 6 nm in diameter are indeed attached on the outer surface of the Fe3O4@PAA/CS particles. The XRD patterns of the Fe3O4@PAA/CS−Au particles as well as the MCNCs are shown in Figure 4. Apart from the characteristic peaks of Fe3O4 encapsulated in the MCNCs, the Fe3O4@PAA/ CS−Au particles show four new peaks at 38.1°, 44.4°, 64.6°, and 77.6° attributed to (111), (200), (220), and (311) crystal planes of cubic gold, matching well with the standard PDF card 65-2870. As-prepared hybrid particles demonstrated significant catalytic activity for the complete reduction of 4-nitrobenzene with NaBH4 within 17.4 min at room temperature; meanwhile, the reaction did not proceed in the absence of the catalyst. The typical UV−vis spectra showing the gradual catalytic reduction of 4-nitrobenzene are provided in Figure S1 of the Supporting Information. Moreover, these catalyst particles could be reused for up to six times without noticeable decrease in the catalytic activity (data not shown), indicating that the polymer layers

and the weight loss increases with elevating the AA/CS ratio in the synthesis recipes. The hydrodynamic diameters (Dh) and size distribution of these particles were also determined by DLS in ethanol (ethanol can give more stable dispersion than water), as shown in Figure 2B. The average values of Dh, as listed in Table 1, illustrate the same increasing trend as the weigh loss reflected in corresponding TG curves. The thickness of the polymer shell of different Fe3O4@PAA/CS particles can be estimated according to their Dh and that of the MCNCs, as shown in Table 1. The thickness thus obtained is significantly larger than estimated from the TEM images; e.g., the ∼16 nm thickness from the TEM image (Figure 1d) increases to a thickness of 33.6 nm based on DLS (entry 3 in Table 1). This is due to the swelling of the PAA/CS shell in ethanol. Particularly, the polydispersity index (PDI) values based on DLS of these samples are all less than 0.1, indicating rather uniform size distribution of both the original MCNCs and the resulting Fe3O4@PAA/CS core−shell particles. The zeta potentials of these particles were measured by DLS in water from pH 4 to 8. As can be seen from Figure 3, the

Figure 3. Effect of pH on the zeta potentials of MCNCs (■) and Fe3O4@PAA/CS particles with AA/CS molar ratios 0.6:1(●), 0.8:1 (▲), and 1:1 (▼).

MCNCs show minus zeta potentials, decreasing from −6.2 to −47.3 mV with the increase of the medium pH values from 4 to 8, due to the ionized carboxyl groups on the surface. In comparison with the MCNCs, all the Fe3O4@PAA/CS particles exhibit significantly higher zeta potentials, however, and show the similar decreasing profile from about 25 to −35 mV within 11774

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bearing particles. Figure 6A shows FT-IR spectra of these modified particles. For the spectrum of MCNCs, the bands at 1626 and 1398 cm−1 are associated with the carboxylate groups due to chelated sodium citrate.14 After coating with the PAA/ CS layer onto the bare MCNCs, the spectrum of the resulting Fe3O4@PAA/CS particles shows a new absorption band at 1628 cm−1, which can be assigned to the NH3+ absorption of CS.29 For the Fe3O4@PAA/CS-COOH particles, a new peak at 1727 cm−1 is observed, which is assigned to the carboxyl group of immobilized succinic acid. In the spectrum of Fe3O4@PAA/ CS-N(CH3)3+ particles, a new absorption band appeared at 1482 cm−1, which corresponds to an asymmetric angular bending of methyl groups of quaternary hydrogen.39 The infrared bands of the Fe3O4@PAA/CS−Br particles appear in close proximity to those of the Fe3O4@PAA/CS samples, and the absorption band of the carbonyl groups introduced from 2bromo-2-methylpropionyl bromide may overlap with those of other groups of the Fe3O4@PAA/CS particles. The successful modification was also proved by the significant zeta potential transformation of the differently modified particles. Figure 6B shows the zeta potential variation profiles of these particles. The original MCNCs exhibit the most negative due to a plenty of ionized carboxylic groups on the surface, and the Fe3O4@ PAA/CS particles show greatly less negatively charged. It is reasonable to note that Fe3O4@PAA/CS−COOH particles become more negative, and the Fe3O4@PAA/CS-N(CH3)2+ turn even oppositely charged in comparison with the Fe3O4@ PAA/CS particles. The Fe3O4@PAA/CS-Br particles show somewhat less negative, indicating that part of the amine together with hydroxyl groups on the surface of the Fe3O4@ PAA/CS particles has been reacted with 2-bromo-2-methylpropionyl bromide. It indicates that the zeta potentials of the Fe3O4@PAA/CS particles can be readily tuned by introducing desired functional groups on the surface. This is advantageous for their potential bioapplications such as bioseparation and enzyme immobilization. These particles were also studied by TGA, and Figure 7 shows their TG curves. Compared with the original Fe3O4@PAA/CS particles, all the modified samples exhibit significantly larger mass loss, hence further verifying successful immobilization of the corresponding functional moieties. According to the residual weight percentage at 900 °C, the contents of grafted carboxyl, quaternary ammonium, and bromine were estimated to be 2.44, 0.29, and 1.52 mmol/g particles. The smaller amount of grafted quaternary ammonium groups may be due to the fact that the modification was carried out in basic aqueous solution where the surface CS chains is collapsed, hence not favoring the reaction of CS with glycidyltrimethylammonium chloride. The charged Fe3O4@PAA/CS-COOH and Fe3O4@PAA/ CS-N(CH3)3+ particles can be used as ion exchangers for adsorptive separation. With abundant carboxyl groups on the surface for ligand coupling, the Fe3O4@PAA/CS-COOH particles may also be a versatile magnetic matrix for biotechnological and biomedical applications. The Fe3O4@ PAA/CS-N(CH3)3+ particles may be employed as a magnetically recyclable antibacterial agent.40 The Fe3O4@PAA/CS-Br particles can be applied as an ATRP maroinitiator for endow the Fe3O4@PAA/CS particles with other desired functionalities via surface-initiated ATRP of corresponding functional monomers, which is demonstrated as follows. Surface-Initiated ATRP of DMAEMA from Fe3O4@PAA/ C-Br Particles. PDMAEMA, a pH and temperature dualresponsive polymer,41 was grafted from the surface of the

Figure 4. XRD patterns of (a) MCNCs and (b) Fe3O4@PAA/CS−Au particles (asterisk represents the diffraction peaks of Fe3O4).

could effectively stabilize the in site generated Au nanoparticles. These results also suggest that there exist CS chains tethered on the surface of the Fe3O4@PAA/CS. The systematic study on the synthesis and application of the Fe3O4@PAA/CS−Au particles will be reported in the future. Surface Modification of Fe3O4@PAA/CS Particles. If there were free CS tethered on the surface of the Fe3O4@PAA/ CS particles, they would share the same reactivity with CS due to its plentiful amino and hydroxyl groups. This hypothesis was further confirmed by successful coupling an amine-reactive fluorescent reagent FITC to the particles’ surface. The photoluminescence spectrum of the FITC modified Fe3O4@ PAA/CS particles shows a strong emission peak at 520 nm, while the unmodified controls show negligible fluorescence (see Figure 5). Also, the modified particle visualized by using a fluorescent microscope shows obvious green fluorescent signal (data not shown).

Figure 5. Fluorescent emission spectra of (a) Fe3O4@PAA/CS particles and (b) FITC modified Fe3O4@PAA/CS particles dispersed in 1% NH4OH aqueous solution (λex = 480 nm).

By utilization of the amine and hydroxyl functionalities of the surface CS, the Fe3O4@PAA/CS particles were modified to introduce a variety of functional groups. The tethered CS chains were allowed to react with succinic anhydride in DMF to introduce negatively charged carboxylic groups, with glycidyltrimethylammonium chloride via in aqueous solution for the coupling of positively charged quaternary ammonium and with 2-bromo-2-methylpropionyl bromide in DMF to give bromine 11775

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Figure 6. FT-IR spectra (A) and zeta potentials measured in phosphate buffer (50 mM, pH 7.4) (B) of differently modified Fe3O4@PAA/CS particles: (a) MCNCs, (b) Fe3O4@PAA/CS particles, (c) Fe3O4@PAA/CS-COOH, (d) Fe3O4@PAA/CS-N(CH3)3+, (e) Fe3O4@PAA/CS-Br, and (f) Fe3O4@PAA/CS-PDMAEMA.

and temperature responsive (see Figure S3 in the Supporting Information). The synthesis, characterization, and application of the Fe3O4@PAA/CS-PDMAEMA particles will be reported elsewhere in detail. Magnetic Properties. The magnetic properties of the typical magnetic particles were determined by VSM at room temperature. The magnetic hysteresis curves are shown in Figure 7. The saturation magnetization (Ms) of the carboxylcapped MCNCs was 49.1 emu/g. After coating with a layer pf CS/AA (CA/AA = 1:1), the Ms of the resulting Fe3O4@PAA/ CS particles decreased to 34.9 emu/g. With grafting alkyl bromide and PDMAEMA onto the surface of the Fe3O4@ PAA/CS particles sequentially, the corresponding values of Ms decreased in turn to 32.1 and 17.6 emu/g, respectively. All these samples showed near superparamagnetism (coercive force less than 20 Oe); that is, minor remanence would exist when the magnetic field were removed, and hence the magnetic materials would be easily redispersed in the medium. A water dispersion of Fe3O4@PAA/CS particles (1 mg/mL) could be separated by a magnet (4000 G) within 1 min and could be readily dispersed again by shaking after the magnet was removed (see the inset photographs in Figure 8). Aqueous Dispersion Stability. Stable dispersion of magnetic polymer microspheres in aqueous media is partic-

Figure 7. TG curves of differently modified Fe3O4@PAA/CS particles: (a) unmodified, (b) Fe3O4@PAA/CS-N(CH3)3+, (c) Fe3O4@PAA/ CS-COOH, (d) Fe3O4@PAA/CS-Br, and (e) Fe3O4@PAA/CSPDMAEMA.

Fe3O4@PAA/CS-Br particles by surface-initiated ATRP of DMAEMA. For the FT-IR spectrum of thus obtained Fe3O4@ PAA/CS-PDMAEMA in Figure 6A,f, a peak at 1732 cm−1 appears which is attributable to CO stretching vibrations of ester, showing that DMAEMA is linked. In contrast to the negatively charged Fe3O4@PAA/CS-Br, the positive zeta potential of the Fe3O4@PAA/CS-PDMAEMA particles (see Figure 6B,f) also confirms the grafting of PDMAEMA. As seen from the TG curve Figure 7e, the PDMAEMA grafted particles exhibit greatly larger mass loss than the initiator particles, and the mass ratio of the grafted PDMAEMA relative to the PDMAEMA modified particles was estimated to be 51.6%. EBriB as a co-initiator was added to the polymerization system to synthesize the PDMAEMA homopolymers for estimating the molecular weight and PDI of the grafted PDMAEMA, which were characterized by GPC. The total conversion of DMAEMA reached 18.3%. According to the GPC trace of the free PDMAEMA generated from the co-initiator (see Figure S2 in the Supporting Information), its number-averaged molecular weight of was calculated to be ∼37 900 with a narrow PDI of 1.086, indicative of good controllability over the graft polymerization of DMAEMA from the initiator particles’ surface. The Fe3O4@PAA/CS-PDMAEMA particles were studied by measuring their hydrodynamic diameters via DLS at different pH values and temperatures, exhibiting both pH

Figure 8. Magnetization curves of (a) carboxyl-capped MCNCs, (b) Fe3O4@PAA/CS (AA/CS = 1:1), (c) Fe3O4@PAA/CS-Br, and (d) Fe3O4@PAA/CS-PDMAEMA. 11776

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magnetic matrix for biotechnological and biomedical applications.

ularly important for their potential applications in bio-related fields such as bioseparation and bioassay. Different magnetic particles were suspended in a phosphate buffer (pH 7.4, 50 mM) with a concentration of 1 mg/mL for examining their dispersion stability by turbidity method using a UV/vis spectrophotometer at 600 nm. As shown in Figure 9, the

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures including UV−vis spectra showing the gradual reduction of 4-nitrobenzene with NaBH4 in water using Fe3O4@PAA/CS−Au composite particles as catalyst, GPC trace of fee PDMAEMA chains, and hydrodynamic diameters of Fe3O4@PAA/CS−PDMAEMA particles at different temperatures and pH values. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel +86 22 23501443 (G.F.). Notes

The authors declare no competing financial interest. Figure 9. Plots of transmittance as a function of time measured for phosphate buffered dispersions (pH 7.4, 50 mM) of (a) MCNCs, (b) Fe3O4@PAA/CS, (c) Fe3O4@PAA/CS-COOH, (d) Fe3O4@PAA/ CS-N(CH3)3+, (e) Fe3O4@PAA/CS-Br, and (f) Fe3O4@PAA/CSPDMAEMA. Insets are the photographs of the corresponding dispersions: (A) initially dispersed by ultrasound and (B) after 3.5 h of setting.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the National Natural Science Foundation of China (No. 21074061), PCSIRT (IRT1257), and the Natural Science Foundation of Tianjin (No. 09JCYBJC02900).

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transmittance of the dispersion for MCNCs, Fe3O4@PAA/CSCOOH, Fe3O4@PAA/CS-N(CH3)3+, and Fe3O4@PAA/CSPDMAEMA maintained unchanged after 3 h of setting, indicative of sustained dispersion stability. The Fe3O4@PAA/ CS-Br particles showed much less stable dispersion, and the unmodified Fe3O4@PAA/CS particles exhibited the worst aqueous stability. The corresponding photographs reflected the similar results (see insets in Figure 9). Among these particles, however, both Fe3O4@PAA/CS-Br and Fe3O4@ PAA/CS show much higher surface charged than the stably dispersed Fe3O4@PAA/CS-N(CH3)3+ and PAA/CS-PDMAEMA in spite of the opposite charge signs (see Figure 6B). This can be explained as follows. Both electrostatic and steric repulsion contribute to the stabilization of magnetic particles against aggregation and setting.42 At pH 7.4, the CS chains on the surface of Fe3O4@PAA/CS or Fe3O4@PAA/CS-Br are deprotonated and hence collapsed. On the contrary, both Fe3O4@PAA/CS-N(CH3)3+ and Fe3O4@PAA/CS-PDMAEMA particles bear stretching protonated polymer chains and hence showed better stability, though less charged.

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CONCLUSIONS With submicrometer MCNCs bearing carboxylic groups as the magnetic cores suspended in the aqueous solution of CS, AA, and MBA, we have demonstrated a simple and green method for the synthesis of highly magnetic and uniform-sized Fe3O4@ PAA/CS core−shell particles by cross-linking copolymerization of AA and MBA. The thickness of the polymer shell can be tuned by varying the polymerization parameters. Desired functionality can be readily introduced on the surface of the core−shell particles by reaction with the amino and/or hydroxyl groups of the CS chains in the outer shell. Therefore, as-prepared Fe3O4@PAA/CS particles may be a versatile 11777

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