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Magnetic hydrogels with supracolloidal structures prepared by suspension polymerization stabilized by Fe2O3 nanoparticles
Acta Biomaterialia 6 (2010) 275–281
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Magnetic hydrogels with supracolloidal structures prepared by suspension polymerization stabilized by Fe2O3 nanoparticles Hongxia Liu, Chaoyang Wang *, Quanxing Gao, Xinxing Liu, Zhen Tong * Research Institute of Materials Science, South China University of Technology, Guangzhou 510640, China
a r t i c l e
i n f o
Article history: Received 2 December 2008 Received in revised form 8 June 2009 Accepted 10 June 2009 Available online 14 June 2009 Keywords: Magnetic hydrogels Nanocomposite Interfacial self-assembly Suspension polymerization Thermosensitive
a b s t r a c t Magnetic hydrogels with supracolloidal structures were fabricated by suspension polymerization of N-isopropylacrylamide (NIPAm) and/or acrylamide (Am) stabilized by Fe2O3 nanoparticles. Fe2O3 nanoparticles can self-assemble at liquid–liquid interfaces to form stable water in oil Pickering emulsion droplets. Monomers dissolved in suspended aqueous droplets were subsequently polymerized at 60 °C. When NIPAm was homopolymerized the PNIPAm produced deposited from the interior water phase onto the interface to form Fe2O3/PNIPAm nanocomposite shells because of its hydrophobicity at the reaction temperature. Magnetic and thermosensitive hollow microcapsules were obtained. When Am was homopolymerized magnetic core–shell microcapsules with PAm hydrogel cores and Fe2O3 nanoparticle shells were obtained. When NIPAm and Am were co-polymerized, magnetic hydrogel microcapsules with two kinds of supracolloidal structures were obtained varying with the NIPAm/Am ratio. These microcapsule beads may find applications as delivery vehicles for biomolecules, drugs, cosmetics, food supplements and living cells. Suspension polymerization based on Pickering emulsion droplets opens up a new route to synthesize a variety of hybrid hydrogels with supracolloidal structures. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Hydrogels with a reversible volume response to external stimuli have been studied extensively as biomaterials for tissue engineering and cell encapsulation and as carriers of drugs, peptides or proteins, due to their hydrophilic character and possible biocompatibility [1–4]. Over the last few years artificial conjugation of hydrogels and inorganic components has received increasing attention due to the synergistic properties of hydrogels and inorganic components [5–11]. In particular, hydrogel-based hybrid composites incorporating colloidal inorganic particles could be designed with tailored mechanical and functional properties and have many important uses in bioseparation, biomedical and catalytic applications [6–9]. To date a number of approaches for the preparation of these hybrid hydrogels have been suggested and developed. They can basically be divided into four kinds. The first is the incorporation of preformed inorganic particles into the hydrogel matrix by mixing and subsequent in situ gelation [9,10]. The second is the deposition of inorganic particles into the preformed hydrogel matrix by in situ mineralization [6,8]. The third is coupling both preformed inorganic particles and hydrogel matrix together [11]. The possible fourth is the simultaneous formation of both hydrogel matrix and inorganic particles in the reac* Corresponding authors. Tel./fax: +86 20 87112886 (C. Wang). E-mail addresses: [email protected] (C. Wang), [email protected] (Z. Tong).
tion system. Although commonly used, these classical methods have the limitation that inorganic particles randomly distribute in the hydrogel matrix. New, better controlled synthetic routes for such hybrid materials are eagerly anticipated. Recently, self-assembly of colloidal particles at the liquid–liquid interface has been well documented and offers a straightforward pathway for the production of organized nanostructures [12]. In this approach colloidal particles spontaneously localize at the interface to minimize the Helmholtz free energy. Typically, a so-called Pickering emulsion is stabilized and novel microcapsules known as colloidosomes whose shells consist of colloidal particles have been created using this strategy [13–18]. Noble et al. [19] and Cayre et al. [20] produced colloidosome hydrogel beads with agarose gel cores and shells of polymeric colloidal particles by self-assembly of colloid particles at liquid–liquid interfaces and subsequent gelation of the aqueous cores. Duan et al. [21], continuing their work, prepared magnetic colloidosome hydrogel beads with agarose gel cores and shells of inorganic particles based on interfacial self-assembly of Fe3O4 nanoparticles. We have fabricated alginate gel beads with shells of porous CaCO3 microparticles by self-assembly of colloidal particles at liquid–liquid interfaces and subsequent in situ gelation at room temperature [22,23]. Therefore, well-defined hydrogel beads with shells of inorganic particles can be easily obtained by interfacial self-assembly of inorganic particles. Most recently, Pickering emulsion droplets have been used as polymerization vessels to fabricate hybrid polymer particles with
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.06.018
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supracolloidal structures [24–33]. Polymerization based on Pickering emulsion droplets includes Pickering emulsion polymerization, mini-emulsion polymerization, suspension polymerization and dispersion polymerization. The solid particles first self-assemble at the liquid–liquid interface and act as the effective stabilizers during the polymerization process without the need for any conventional stabilizers. After completion of polymerization the particles are captured on the surface of the resultant polymer beads, where they can be most effective for subsequent applications. Such surfactant-free emulsion polymerization is more attractive for the preparation of hybrid beads than conventional emulsion polymerization. Bon et al. [32] used poly(methyl methacrylate) microgels to generate micron sized Pickering emulsions as polymerization vessels to create supracolloidal interpenetrating polymer network reinforced microcapsules by phase separation. The polymerized network should interlock the microgel building blocks and fix the core–shell morphology. At the same time the mechanical properties of the capsule can be readily tailored by varying the chemical composition of the interpenetrating polymer network. They also prepared organic–inorganic hybrid hollow spheres by TiO2-stabilized Pickering emulsion polymerization [33]. In this work we demonstrate a simple and effective method for the fabrication of magnetic hydrogel microcapsules with supracolloidal structures by Pickering suspension polymerization stabilized by Fe2O3 nanoparticles [34], inspired by the studies of Bon’s group [33]. Fe2O3 nanoparticles spontaneously adsorbed to the liquid– liquid interfaces to stabilize water in oil emulsion droplets. The radical polymerization of N-isopropylacrylamide (NIPAm) and/or acrylamide (Am) took place in the interior water phase. Hollow microcapsules with Fe2O3/polymer nanocomposite shells and core–shell microcapsules with hydrogel cores and Fe2O3 nanoparticles shells were obtained by varying the NIPAm/Am ratio. The existence of superparamagnetic Fe2O3 nanoparticles means the microcapsules with supracolloidal structures obtained are easily manipulated by external magnetic fields.
after 15 min continuous stirring. To speed up the precipitation of nanoparticles the resulting solution was placed in a refrigerator for 5 h. The MPs were subsequently washed three times with methanol and settled by centrifugation at 10,000 rpm for 5 min. A 10 mM oleic acid solution, in 50 ml methanol, was then added to the nanoparticles and this mixture was stirred for 3 h. After completing synthesis the maghemite nanoparticles were thoroughly washed with methanol to remove excess surfactant and finally dispersed in 50 ml hexane. This nanoparticle suspension was used as synthesized. The resulting oleic acid-stabilized maghemite nanoparticles were single crystalline and uniform with diameters of 5 nm (see Fig. 1). Partial Fe2O3 nanoparticles were labeled with FITC. FITC was dissolved in dimethyl sulfoxide at a concentration of 1 mg ml1. A 10 ll aliquot of dye solution of was added to the aqueous dispersion of nanoparticles and stirred at 40 °C for 1 h. After reaction the nanoparticles were washed with water until the fluorescence intensity of the water became minimal. Then the FITC-labeled nanoparticles were capped with oleic acid. 2.3. Suspension polymerization based on Pickering emulsion droplets Given quantities of NIPAm, AM, V-50 and BIS were added to 0.9 ml pure water. This aqueous solution was stirred for 20 min and deoxygenated by bubbling through nitrogen gas for 10 min at
a
2. Materials and methods 2.1. Materials
2.2. Preparation of c-Fe2O3 nanoparticles capped with oleic acid
700 600
b
500
Intensity (a.u.)
N-Isopropylacrylamide (NIPAm) (Kohjin Co. Ltd, Japan) and acrylamide (Am) (Ruijie Chemical Industries, China) were recrystallized before use. 2,20 -Azobis(2-methylpropionamidine)dihydrochloride (V-50) (Huaxin Chemical Industries Co., China), N,N0 -methylene diacrylamide (BIS) (Acros Organics), fluorescein isothiocynate (FITC) (Alfa) were used as received. Iron (II) chloride tetrahydrate (FeCl24H2O), iron (III) chloride hexahydrate (FeCl36H2O), 2-propanol (HPLC grade), concentrated ammonia, oleic acid, n-hexane and ethanol were bought from Guangzhou Chemical Factory (China) and were used without further purification. The water used in all experiments was purified by deionization and filtration with a Millipore purification apparatus to a resistivity higher than 18.0 M X cm.
400 300 200
Iron oxide (c-Fe2O3, maghemite) magnetic nanoparticles (MPs) were synthesized using methods similar to that used by Koo et al. [35]. A 0.5 g portion of FeCl36H2O was added to 50 ml nitrogenpurged HPLC grade 2-propanol. Then, 0.25 g FeCl24H2O was added while the solution was continuously stirred. The solution color was yellowish-orange after complete dissolution of the iron precursors. Then the solution temperature was gradually raised to 50 °C and concentrated aqueous ammonia was added to the solution in excess (<10 ml). The color of the solution changed to dark brown
100 0 20
30
40
50
60
Two theta (degree Cu Kα) Fig. 1. (a) TEM image and (b) XRD pattern of Fe2O3 magnetic nanoparticles capped with oleic acid.
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H. Liu et al. / Acta Biomaterialia 6 (2010) 275–281 Table 1 Processing parameters of Pickering emulsions for polymerization. Sample
N10B1 N10B1A2 N5B1A5 B1A10
Oil phase
Aqueous phase
Hexane (1% Fe2O3) (ml)
Water (ml)
NIPAm (g)
6 6 6 6
0.9 0.9 0.9 0.9
0.1 0.1 0.05
Am (g)
BIS (g)
V-50 (g)
0.02 0.05 0.1
0.01 0.01 0.01 0.01
0.005 0.005 0.005 0.005
room temperature in the dark. Then 6 ml hexane containing Fe2O3 nanoparticles as the oil phase was added to the aqueous phase and the system was emulsified by shaking. The stable emulsion which formed was placed in a 60 °C water bath to polymerize for 6 h. At the end of the reaction the oil phase was decanted. After complete evaporation of the remaining hexane magnetic flexible microcapsules with inorganic/polymer nanocomposite shells or core–shell hydrogel beads were obtained. In our experiments four batches with different weight ratios of NIPAm and AM in the aqueous phase were used to prepare microcapsules or hydrogel beads, as illustrated in Table 1. The samples are referred to as N10B1, N10B1A2, N5B1A5 and B1A10. Here N is for NIPAm, B for BIS and A for Am. The numbers following the letters give the weight of these substances in the aqueous phase, with one standing for 0.01 g. 2.4. Fabrication of FITC-labeled microcapsules Samples N10B1A2, N5B1A5, B1A10 were washed three times with ethanol and then transferred to 4 ml of a pH 9.9 aqueous buffer solution of NaOH/Na2CO3 and washed three times with this buffer solution. A 1 mg ml1 sample of aqueous FITC solution was added to the microcapsule dispersion and then the mixture was shaken for 48 h in the dark. Unreacted FITC was washed multiple times with pure water until the fluorescence intensity at 512 nm of the washing water was nearly 0. 2.5. Characterization A drop of c-Fe2O3 nanoparticle suspension in ethanol was spread on a carbon-coated copper grid and dried overnight at room
Fig. 3. Confocal laser scan microscope image of water in hexane Pickering emulsion stabilized FITC-labeled Fe2O3 nanoparticles.
temperature for high resolution transmission electron microscopy (JEM-2010HR). The observations were carried out under accelerating voltages of 200 kV. X-ray diffraction of the magnetic nanoparticles was performed in transmission geometry using an X’pert PRO diffractometer (40 kV and 40 mA) equipped with a Cu-Ka source (wavelength 0.154 nm) at room temperature. The wet microcapsules were observed with an optical microscope (Carl Zeiss, German). Confocal micrographs were taken with a Leica TCS-SP2 confocal laser scanning microscope (CLSM) with a 20 objective with a numerical aperture of 1.4 at an excitation wavelength of 480 nm. The morphology of dry microcapsules was observed by scanning electron microscopy (SEM) with a Philips XL 30 at an acceleration voltage of 15 kV. Samples were prepared by dropping the microcapsule suspension onto a quartz wafer, freeze-drying it for 4 h and then sputtering it with gold. Thermo-gravimetric analysis (TGA) curves for the dry samples were collected with a thermo-analyzer (TG 209, NETZCH Co.) within the temperature range 20–800 °C with a rate of temperature increase of 10 °C min1. 3. Results and discussion Nano/microparticle interfacial self-assembly to form Pickering emulsion droplets has been well documented [12]. Herein, magnetic hollow microcapsules with inorganic/polymer composite shells were facilely and high effectively fabricated by suspension
Oil phase
NIPAm
Oil phase
Oil phase
Emulsifition Water phase
Water phase
Polymerization at 60 oC
Monomers and initiators
Oil phase
Monomers and initiators
Am
Fig. 2. Schematic illustration of the preparation of magnetic hollow microcapsules and core–shell hydrogel beads by suspension polymerization based on Pickering emulsion droplets.
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polymerization stabilized by Fe2O3 nanoparticles based on Pickering emulsion droplets. As is known, PNIPAm is a temperature-sensitive polymer which has the sharpest transition of the
thermosensitive alkylacrylamide polymers. Cross-linked PNIPAm exhibits a drastic hydrophobic–hydrophilic transition at its lower critical solution temperature (LCST) of 32 °C. At temperatures
Fig. 4. Optical microscope images of wet samples of N10B1 (a), N10B1A2 (c), N5B1A5 (e) and B1A10 (g) dispersed in ethanol and dry samples of N10B1 (b), N10B1A2 (d), N5B1A5 (f) and B1A10 (h) after the ethanol had been evaporated.
Fig. 5. Optical microscope images of dry N10B1 (a) and N10B1 redispersed in water for 5 (b) and 10 min (c).
H. Liu et al. / Acta Biomaterialia 6 (2010) 275–281
lower than the LCST PNIPAm exhibits hydrophilicity and at temperatures above the LCST it exhibits hydrophobicity [36]. So we utilized the characteristic of this hydrophobic–hydrophilic transition at the LCST of PNIPAm to fabricate magnetic hollow microcapsules with inorganic/polymer composite shells by particle interfacial self-assembly. The fabrication strategy is illustrated in Fig. 2. An aqueous solution of NIPAm monomer, initiator V-50 and cross-linker BIS was rapidly emulsified in n-hexane in the presence of Fe2O3 nanoparticles to produce water in oil Pickering emulsion by shaking and nanoparticles were self-assembled at liquid–liquid interfaces. The system was placed in 60 °C water bath and water droplets containing monomers and initiator were subsequently polymerized based on a Pickering emulsion. Because of its hydrophobicity at this temperature, the PNIPAm obtained should deposit from the interior water phase onto the interface and form Fe2O3/PNIPAm nanocomposite shells. The resulting hollow microcapsules with hybrid shells were easily collected by decanting and then evaporating off the hexane. The microcapsules obtained can be transferred to water by repeated washing with ethanol and water. If NIPAm is replaced by Am in this suspension polymerization based on Pickering emulsion droplets hybrid hydrogel beads with nanoshells should be produced, because PAM is not temperature-sensitive and is hydrophilic at the polymerization temperature of 60 °C. If NIPAM and Am are co-polymerized based on this Pickering emulsion system then hollow microcapsules or core–shell hydrogel beads may be obtained by varying the NIPAm/Am ratio. Fig. 3 is confocal laser scan microscope (CLSM) image of a water in hexane Pickering emulsion stabilized with FITC-labeled Fe2O3 nanoparticles. There are obvious green circles of emulsion droplets. No obvious color is found in the continuous phase and inside the
279
Fig. 8. Photographs of N10B1 in water under an external magnetic field (inset: N10B1 in water without an external magnetic field).
emulsion droplet. It seems that most nanoparticles are indeed at the emulsion droplet interface. Four samples with different weight ratios of NIPAm and AM (N10B1, N10B1A2, N5B1A5 and B1A10) were fabricated by Pickering suspension polymerization. Optical microscope images of samples dispersed in ethanol and dried samples are shown in Fig. 4. The four samples were spherical. N10B1 and N10B1A2 showed some black, while N5B1A5 and B1A10 were clear. In fact, when seen by eye N10B1 and N10B1A2 were white and N5B1A5 and B1A10 were transparent. The possible reasons are that N10B1 and N10B1A2 are hollow microcapsules with hybrid impact shells and N5B1A5 and B1A10 are hydrogel beads with nanoshells. After ethanol evaporation N10B1 and N10B1A2 could completely collapse into a film with a crinkly appearance, while N5B1A5 and B1A10 kept their spherical shape. This is evidence that N10B1 and N10B1A2 are hollow microcapsules and N5B1A5 and B1A10
Fig. 6. Confocal laser scan microscope images of FITC-labeled samples of N10B1A2 (a), N5B1A5 (b) and B1A10 (c).
Fig. 7. SEM images of N10B1A2 (a) and N10B1 (b). (c and d) Enlarged images of those parts of (b) indicated by arrows.
H. Liu et al. / Acta Biomaterialia 6 (2010) 275–281
are hydrogel beads. The monomer conversion can be simply calculated from TGA curves of the dry samples [37]. The extent of monomer conversion for the four samples, N10B1, N10B1A2, N5B1A5 and B1A10, were 90%. However, the extent of monomer conversion calculated from TGA is biased. One reason is that a few nanoparticles are possibly in the continuous phase and some nanoparticles may be lost in the polymerization process. Another reason is that the samples for TGA were not completely dry and contained a small amount of residual water. The four samples had a wide size distribution. During a former experiment on the fabrication of novel core–shell hybrid alginate hydrogel beads we found the bead size distribution to be independent of the number of CaCO3 colloidal particles used [23]. Kralchevsky et al. indicated that the size of the droplets was solely determined by the input of mechanical energy during emulsification for a water in oil emulsion of Bancroft type [38]. Here the nanoparticles, rather than the surfactant molecules, were used to stabilize an emulsion. As is known, the size of the emulsion drop is not related to the amount of surfactant used. Thus, we can conclude that the particle size distribution does not correlate with the number of nanoparticles used. When dry N10B1 was redispersed in water the microcapsule films adsorbed water and again became spherical, as can be seen from Fig. 5. This indicates that PNIPAm/Fe2O3 nanoparticle composite shells are flexible and permeable, so it will be difficult to break them, even if the microcapsules completely collapse, and it allows them to resume their original appearance when they are redispersed in water. To further determine the structure of microcapsules of N10B1 and N10B1A2 and hydrogel beads of N5B1A5 and B1A10, we labeled N10B1A2, N5B1A5 and B1A10 with FITC, because only PAM can react with FITC forming a covalent bond. Fig. 6 shows CLSM images of FITC-labeled N10B1A2, N5B1A5 and B1A10. There are obvious green circles for N10B1A2, which means that N10B1A2 forms hollow capsules. Whole green beads were observed for N5B1A5 and B1A10, which means that they are hydrogel beads. From the CLSM image of N10B1A2 we can deduce that N10B1 should be microcapsules with hybrid impact shells. Fig. 7 shows SEM images of freeze-dried N10B1 and N10B1A2. The microcapsules of N10B1 and N10B1A2 maintained a spherical shape, although a few microcapsules were damaged during the process of preparing the samples for SEM, as seen in Fig. 7b. From Fig. 7c we can see that the interior of these damaged microcapsules wais hollow and the shell was a little rough. This again testifies to the fact that the microcapsules obtained were hollow with a inorganic/polymer nanocomposite shell. Fig. 7d shows that the appearance of the intact microcapsules was also rough. Because of the magnetism of Fe2O3 nanoparticles and the thermosensitivity of PNIPAm, microcapsules with PNIPAm/Fe2O3 nanoparticle composite shells should be magnetic and thermosensitive. Fig. 8 shows photographs of N10B1 microcapsules in water under an external magnetic field (inset: microcapsules in water without an external magnetic field). N10B1 microcapsules exhibited a clear magnetic response. The magnetic microcapsules can readily be moved and collected using an external magnetic field. The change in diameter of N10B1 and N10B1A2 with temperature relative to that at 26 °C is presented in Fig. 9. With increasing temperature the microcapsules shrank. The microcapsules were slightly thermosensitive. However, no temperature at which sharp shrinkage occurred was found. The inset photographs are of N10B1A2 at 26 and 50 °C, and obvious shrinkage can be observed. In summary, magnetic hydrogels with supracolloidal structures were fabricated by suspension polymerization of NIPAm and/or Am based on Pickering emulsion droplets. These microcapsule beads may find application as delivery vehicles for biomolecules, drugs, cosmetics, food supplements and living cells. Suspension
1.01 1.00
N10B1 N10B1A2
0.99 0.98
relative diameter
280
0.97
26 ºC
0.96 0.95 0.94 0.93 0.92 0.91
50º C
0.90 0.89
25
30
35
40
45
50
T/ ºC Fig. 9. Relationship between relative mean diameter of N10B1 and N10B1A2 dispersed in water and temperature (insets: optical microscope images of N10B1A2 dispersed in water at 26 and 50 °C).
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Magnetic hydrogels with supracolloidal structures prepared by suspension polymerization stabilized by Fe2O3 nanoparticles Hongxia Liu, Chaoyang Wang *, Quanxing Gao, Xinxing Liu, Zhen Tong * Research Institute of Materials Science, South China University of Technology, Guangzhou 510640, China
a r t i c l e
i n f o
Article history: Received 2 December 2008 Received in revised form 8 June 2009 Accepted 10 June 2009 Available online 14 June 2009 Keywords: Magnetic hydrogels Nanocomposite Interfacial self-assembly Suspension polymerization Thermosensitive
a b s t r a c t Magnetic hydrogels with supracolloidal structures were fabricated by suspension polymerization of N-isopropylacrylamide (NIPAm) and/or acrylamide (Am) stabilized by Fe2O3 nanoparticles. Fe2O3 nanoparticles can self-assemble at liquid–liquid interfaces to form stable water in oil Pickering emulsion droplets. Monomers dissolved in suspended aqueous droplets were subsequently polymerized at 60 °C. When NIPAm was homopolymerized the PNIPAm produced deposited from the interior water phase onto the interface to form Fe2O3/PNIPAm nanocomposite shells because of its hydrophobicity at the reaction temperature. Magnetic and thermosensitive hollow microcapsules were obtained. When Am was homopolymerized magnetic core–shell microcapsules with PAm hydrogel cores and Fe2O3 nanoparticle shells were obtained. When NIPAm and Am were co-polymerized, magnetic hydrogel microcapsules with two kinds of supracolloidal structures were obtained varying with the NIPAm/Am ratio. These microcapsule beads may find applications as delivery vehicles for biomolecules, drugs, cosmetics, food supplements and living cells. Suspension polymerization based on Pickering emulsion droplets opens up a new route to synthesize a variety of hybrid hydrogels with supracolloidal structures. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Hydrogels with a reversible volume response to external stimuli have been studied extensively as biomaterials for tissue engineering and cell encapsulation and as carriers of drugs, peptides or proteins, due to their hydrophilic character and possible biocompatibility [1–4]. Over the last few years artificial conjugation of hydrogels and inorganic components has received increasing attention due to the synergistic properties of hydrogels and inorganic components [5–11]. In particular, hydrogel-based hybrid composites incorporating colloidal inorganic particles could be designed with tailored mechanical and functional properties and have many important uses in bioseparation, biomedical and catalytic applications [6–9]. To date a number of approaches for the preparation of these hybrid hydrogels have been suggested and developed. They can basically be divided into four kinds. The first is the incorporation of preformed inorganic particles into the hydrogel matrix by mixing and subsequent in situ gelation [9,10]. The second is the deposition of inorganic particles into the preformed hydrogel matrix by in situ mineralization [6,8]. The third is coupling both preformed inorganic particles and hydrogel matrix together [11]. The possible fourth is the simultaneous formation of both hydrogel matrix and inorganic particles in the reac* Corresponding authors. Tel./fax: +86 20 87112886 (C. Wang). E-mail addresses: [email protected] (C. Wang), [email protected] (Z. Tong).
tion system. Although commonly used, these classical methods have the limitation that inorganic particles randomly distribute in the hydrogel matrix. New, better controlled synthetic routes for such hybrid materials are eagerly anticipated. Recently, self-assembly of colloidal particles at the liquid–liquid interface has been well documented and offers a straightforward pathway for the production of organized nanostructures [12]. In this approach colloidal particles spontaneously localize at the interface to minimize the Helmholtz free energy. Typically, a so-called Pickering emulsion is stabilized and novel microcapsules known as colloidosomes whose shells consist of colloidal particles have been created using this strategy [13–18]. Noble et al. [19] and Cayre et al. [20] produced colloidosome hydrogel beads with agarose gel cores and shells of polymeric colloidal particles by self-assembly of colloid particles at liquid–liquid interfaces and subsequent gelation of the aqueous cores. Duan et al. [21], continuing their work, prepared magnetic colloidosome hydrogel beads with agarose gel cores and shells of inorganic particles based on interfacial self-assembly of Fe3O4 nanoparticles. We have fabricated alginate gel beads with shells of porous CaCO3 microparticles by self-assembly of colloidal particles at liquid–liquid interfaces and subsequent in situ gelation at room temperature [22,23]. Therefore, well-defined hydrogel beads with shells of inorganic particles can be easily obtained by interfacial self-assembly of inorganic particles. Most recently, Pickering emulsion droplets have been used as polymerization vessels to fabricate hybrid polymer particles with
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.06.018
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supracolloidal structures [24–33]. Polymerization based on Pickering emulsion droplets includes Pickering emulsion polymerization, mini-emulsion polymerization, suspension polymerization and dispersion polymerization. The solid particles first self-assemble at the liquid–liquid interface and act as the effective stabilizers during the polymerization process without the need for any conventional stabilizers. After completion of polymerization the particles are captured on the surface of the resultant polymer beads, where they can be most effective for subsequent applications. Such surfactant-free emulsion polymerization is more attractive for the preparation of hybrid beads than conventional emulsion polymerization. Bon et al. [32] used poly(methyl methacrylate) microgels to generate micron sized Pickering emulsions as polymerization vessels to create supracolloidal interpenetrating polymer network reinforced microcapsules by phase separation. The polymerized network should interlock the microgel building blocks and fix the core–shell morphology. At the same time the mechanical properties of the capsule can be readily tailored by varying the chemical composition of the interpenetrating polymer network. They also prepared organic–inorganic hybrid hollow spheres by TiO2-stabilized Pickering emulsion polymerization [33]. In this work we demonstrate a simple and effective method for the fabrication of magnetic hydrogel microcapsules with supracolloidal structures by Pickering suspension polymerization stabilized by Fe2O3 nanoparticles [34], inspired by the studies of Bon’s group [33]. Fe2O3 nanoparticles spontaneously adsorbed to the liquid– liquid interfaces to stabilize water in oil emulsion droplets. The radical polymerization of N-isopropylacrylamide (NIPAm) and/or acrylamide (Am) took place in the interior water phase. Hollow microcapsules with Fe2O3/polymer nanocomposite shells and core–shell microcapsules with hydrogel cores and Fe2O3 nanoparticles shells were obtained by varying the NIPAm/Am ratio. The existence of superparamagnetic Fe2O3 nanoparticles means the microcapsules with supracolloidal structures obtained are easily manipulated by external magnetic fields.
after 15 min continuous stirring. To speed up the precipitation of nanoparticles the resulting solution was placed in a refrigerator for 5 h. The MPs were subsequently washed three times with methanol and settled by centrifugation at 10,000 rpm for 5 min. A 10 mM oleic acid solution, in 50 ml methanol, was then added to the nanoparticles and this mixture was stirred for 3 h. After completing synthesis the maghemite nanoparticles were thoroughly washed with methanol to remove excess surfactant and finally dispersed in 50 ml hexane. This nanoparticle suspension was used as synthesized. The resulting oleic acid-stabilized maghemite nanoparticles were single crystalline and uniform with diameters of 5 nm (see Fig. 1). Partial Fe2O3 nanoparticles were labeled with FITC. FITC was dissolved in dimethyl sulfoxide at a concentration of 1 mg ml1. A 10 ll aliquot of dye solution of was added to the aqueous dispersion of nanoparticles and stirred at 40 °C for 1 h. After reaction the nanoparticles were washed with water until the fluorescence intensity of the water became minimal. Then the FITC-labeled nanoparticles were capped with oleic acid. 2.3. Suspension polymerization based on Pickering emulsion droplets Given quantities of NIPAm, AM, V-50 and BIS were added to 0.9 ml pure water. This aqueous solution was stirred for 20 min and deoxygenated by bubbling through nitrogen gas for 10 min at
a
2. Materials and methods 2.1. Materials
2.2. Preparation of c-Fe2O3 nanoparticles capped with oleic acid
700 600
b
500
Intensity (a.u.)
N-Isopropylacrylamide (NIPAm) (Kohjin Co. Ltd, Japan) and acrylamide (Am) (Ruijie Chemical Industries, China) were recrystallized before use. 2,20 -Azobis(2-methylpropionamidine)dihydrochloride (V-50) (Huaxin Chemical Industries Co., China), N,N0 -methylene diacrylamide (BIS) (Acros Organics), fluorescein isothiocynate (FITC) (Alfa) were used as received. Iron (II) chloride tetrahydrate (FeCl24H2O), iron (III) chloride hexahydrate (FeCl36H2O), 2-propanol (HPLC grade), concentrated ammonia, oleic acid, n-hexane and ethanol were bought from Guangzhou Chemical Factory (China) and were used without further purification. The water used in all experiments was purified by deionization and filtration with a Millipore purification apparatus to a resistivity higher than 18.0 M X cm.
400 300 200
Iron oxide (c-Fe2O3, maghemite) magnetic nanoparticles (MPs) were synthesized using methods similar to that used by Koo et al. [35]. A 0.5 g portion of FeCl36H2O was added to 50 ml nitrogenpurged HPLC grade 2-propanol. Then, 0.25 g FeCl24H2O was added while the solution was continuously stirred. The solution color was yellowish-orange after complete dissolution of the iron precursors. Then the solution temperature was gradually raised to 50 °C and concentrated aqueous ammonia was added to the solution in excess (<10 ml). The color of the solution changed to dark brown
100 0 20
30
40
50
60
Two theta (degree Cu Kα) Fig. 1. (a) TEM image and (b) XRD pattern of Fe2O3 magnetic nanoparticles capped with oleic acid.
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H. Liu et al. / Acta Biomaterialia 6 (2010) 275–281 Table 1 Processing parameters of Pickering emulsions for polymerization. Sample
N10B1 N10B1A2 N5B1A5 B1A10
Oil phase
Aqueous phase
Hexane (1% Fe2O3) (ml)
Water (ml)
NIPAm (g)
6 6 6 6
0.9 0.9 0.9 0.9
0.1 0.1 0.05
Am (g)
BIS (g)
V-50 (g)
0.02 0.05 0.1
0.01 0.01 0.01 0.01
0.005 0.005 0.005 0.005
room temperature in the dark. Then 6 ml hexane containing Fe2O3 nanoparticles as the oil phase was added to the aqueous phase and the system was emulsified by shaking. The stable emulsion which formed was placed in a 60 °C water bath to polymerize for 6 h. At the end of the reaction the oil phase was decanted. After complete evaporation of the remaining hexane magnetic flexible microcapsules with inorganic/polymer nanocomposite shells or core–shell hydrogel beads were obtained. In our experiments four batches with different weight ratios of NIPAm and AM in the aqueous phase were used to prepare microcapsules or hydrogel beads, as illustrated in Table 1. The samples are referred to as N10B1, N10B1A2, N5B1A5 and B1A10. Here N is for NIPAm, B for BIS and A for Am. The numbers following the letters give the weight of these substances in the aqueous phase, with one standing for 0.01 g. 2.4. Fabrication of FITC-labeled microcapsules Samples N10B1A2, N5B1A5, B1A10 were washed three times with ethanol and then transferred to 4 ml of a pH 9.9 aqueous buffer solution of NaOH/Na2CO3 and washed three times with this buffer solution. A 1 mg ml1 sample of aqueous FITC solution was added to the microcapsule dispersion and then the mixture was shaken for 48 h in the dark. Unreacted FITC was washed multiple times with pure water until the fluorescence intensity at 512 nm of the washing water was nearly 0. 2.5. Characterization A drop of c-Fe2O3 nanoparticle suspension in ethanol was spread on a carbon-coated copper grid and dried overnight at room
Fig. 3. Confocal laser scan microscope image of water in hexane Pickering emulsion stabilized FITC-labeled Fe2O3 nanoparticles.
temperature for high resolution transmission electron microscopy (JEM-2010HR). The observations were carried out under accelerating voltages of 200 kV. X-ray diffraction of the magnetic nanoparticles was performed in transmission geometry using an X’pert PRO diffractometer (40 kV and 40 mA) equipped with a Cu-Ka source (wavelength 0.154 nm) at room temperature. The wet microcapsules were observed with an optical microscope (Carl Zeiss, German). Confocal micrographs were taken with a Leica TCS-SP2 confocal laser scanning microscope (CLSM) with a 20 objective with a numerical aperture of 1.4 at an excitation wavelength of 480 nm. The morphology of dry microcapsules was observed by scanning electron microscopy (SEM) with a Philips XL 30 at an acceleration voltage of 15 kV. Samples were prepared by dropping the microcapsule suspension onto a quartz wafer, freeze-drying it for 4 h and then sputtering it with gold. Thermo-gravimetric analysis (TGA) curves for the dry samples were collected with a thermo-analyzer (TG 209, NETZCH Co.) within the temperature range 20–800 °C with a rate of temperature increase of 10 °C min1. 3. Results and discussion Nano/microparticle interfacial self-assembly to form Pickering emulsion droplets has been well documented [12]. Herein, magnetic hollow microcapsules with inorganic/polymer composite shells were facilely and high effectively fabricated by suspension
Oil phase
NIPAm
Oil phase
Oil phase
Emulsifition Water phase
Water phase
Polymerization at 60 oC
Monomers and initiators
Oil phase
Monomers and initiators
Am
Fig. 2. Schematic illustration of the preparation of magnetic hollow microcapsules and core–shell hydrogel beads by suspension polymerization based on Pickering emulsion droplets.
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polymerization stabilized by Fe2O3 nanoparticles based on Pickering emulsion droplets. As is known, PNIPAm is a temperature-sensitive polymer which has the sharpest transition of the
thermosensitive alkylacrylamide polymers. Cross-linked PNIPAm exhibits a drastic hydrophobic–hydrophilic transition at its lower critical solution temperature (LCST) of 32 °C. At temperatures
Fig. 4. Optical microscope images of wet samples of N10B1 (a), N10B1A2 (c), N5B1A5 (e) and B1A10 (g) dispersed in ethanol and dry samples of N10B1 (b), N10B1A2 (d), N5B1A5 (f) and B1A10 (h) after the ethanol had been evaporated.
Fig. 5. Optical microscope images of dry N10B1 (a) and N10B1 redispersed in water for 5 (b) and 10 min (c).
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lower than the LCST PNIPAm exhibits hydrophilicity and at temperatures above the LCST it exhibits hydrophobicity [36]. So we utilized the characteristic of this hydrophobic–hydrophilic transition at the LCST of PNIPAm to fabricate magnetic hollow microcapsules with inorganic/polymer composite shells by particle interfacial self-assembly. The fabrication strategy is illustrated in Fig. 2. An aqueous solution of NIPAm monomer, initiator V-50 and cross-linker BIS was rapidly emulsified in n-hexane in the presence of Fe2O3 nanoparticles to produce water in oil Pickering emulsion by shaking and nanoparticles were self-assembled at liquid–liquid interfaces. The system was placed in 60 °C water bath and water droplets containing monomers and initiator were subsequently polymerized based on a Pickering emulsion. Because of its hydrophobicity at this temperature, the PNIPAm obtained should deposit from the interior water phase onto the interface and form Fe2O3/PNIPAm nanocomposite shells. The resulting hollow microcapsules with hybrid shells were easily collected by decanting and then evaporating off the hexane. The microcapsules obtained can be transferred to water by repeated washing with ethanol and water. If NIPAm is replaced by Am in this suspension polymerization based on Pickering emulsion droplets hybrid hydrogel beads with nanoshells should be produced, because PAM is not temperature-sensitive and is hydrophilic at the polymerization temperature of 60 °C. If NIPAM and Am are co-polymerized based on this Pickering emulsion system then hollow microcapsules or core–shell hydrogel beads may be obtained by varying the NIPAm/Am ratio. Fig. 3 is confocal laser scan microscope (CLSM) image of a water in hexane Pickering emulsion stabilized with FITC-labeled Fe2O3 nanoparticles. There are obvious green circles of emulsion droplets. No obvious color is found in the continuous phase and inside the
279
Fig. 8. Photographs of N10B1 in water under an external magnetic field (inset: N10B1 in water without an external magnetic field).
emulsion droplet. It seems that most nanoparticles are indeed at the emulsion droplet interface. Four samples with different weight ratios of NIPAm and AM (N10B1, N10B1A2, N5B1A5 and B1A10) were fabricated by Pickering suspension polymerization. Optical microscope images of samples dispersed in ethanol and dried samples are shown in Fig. 4. The four samples were spherical. N10B1 and N10B1A2 showed some black, while N5B1A5 and B1A10 were clear. In fact, when seen by eye N10B1 and N10B1A2 were white and N5B1A5 and B1A10 were transparent. The possible reasons are that N10B1 and N10B1A2 are hollow microcapsules with hybrid impact shells and N5B1A5 and B1A10 are hydrogel beads with nanoshells. After ethanol evaporation N10B1 and N10B1A2 could completely collapse into a film with a crinkly appearance, while N5B1A5 and B1A10 kept their spherical shape. This is evidence that N10B1 and N10B1A2 are hollow microcapsules and N5B1A5 and B1A10
Fig. 6. Confocal laser scan microscope images of FITC-labeled samples of N10B1A2 (a), N5B1A5 (b) and B1A10 (c).
Fig. 7. SEM images of N10B1A2 (a) and N10B1 (b). (c and d) Enlarged images of those parts of (b) indicated by arrows.
H. Liu et al. / Acta Biomaterialia 6 (2010) 275–281
are hydrogel beads. The monomer conversion can be simply calculated from TGA curves of the dry samples [37]. The extent of monomer conversion for the four samples, N10B1, N10B1A2, N5B1A5 and B1A10, were 90%. However, the extent of monomer conversion calculated from TGA is biased. One reason is that a few nanoparticles are possibly in the continuous phase and some nanoparticles may be lost in the polymerization process. Another reason is that the samples for TGA were not completely dry and contained a small amount of residual water. The four samples had a wide size distribution. During a former experiment on the fabrication of novel core–shell hybrid alginate hydrogel beads we found the bead size distribution to be independent of the number of CaCO3 colloidal particles used [23]. Kralchevsky et al. indicated that the size of the droplets was solely determined by the input of mechanical energy during emulsification for a water in oil emulsion of Bancroft type [38]. Here the nanoparticles, rather than the surfactant molecules, were used to stabilize an emulsion. As is known, the size of the emulsion drop is not related to the amount of surfactant used. Thus, we can conclude that the particle size distribution does not correlate with the number of nanoparticles used. When dry N10B1 was redispersed in water the microcapsule films adsorbed water and again became spherical, as can be seen from Fig. 5. This indicates that PNIPAm/Fe2O3 nanoparticle composite shells are flexible and permeable, so it will be difficult to break them, even if the microcapsules completely collapse, and it allows them to resume their original appearance when they are redispersed in water. To further determine the structure of microcapsules of N10B1 and N10B1A2 and hydrogel beads of N5B1A5 and B1A10, we labeled N10B1A2, N5B1A5 and B1A10 with FITC, because only PAM can react with FITC forming a covalent bond. Fig. 6 shows CLSM images of FITC-labeled N10B1A2, N5B1A5 and B1A10. There are obvious green circles for N10B1A2, which means that N10B1A2 forms hollow capsules. Whole green beads were observed for N5B1A5 and B1A10, which means that they are hydrogel beads. From the CLSM image of N10B1A2 we can deduce that N10B1 should be microcapsules with hybrid impact shells. Fig. 7 shows SEM images of freeze-dried N10B1 and N10B1A2. The microcapsules of N10B1 and N10B1A2 maintained a spherical shape, although a few microcapsules were damaged during the process of preparing the samples for SEM, as seen in Fig. 7b. From Fig. 7c we can see that the interior of these damaged microcapsules wais hollow and the shell was a little rough. This again testifies to the fact that the microcapsules obtained were hollow with a inorganic/polymer nanocomposite shell. Fig. 7d shows that the appearance of the intact microcapsules was also rough. Because of the magnetism of Fe2O3 nanoparticles and the thermosensitivity of PNIPAm, microcapsules with PNIPAm/Fe2O3 nanoparticle composite shells should be magnetic and thermosensitive. Fig. 8 shows photographs of N10B1 microcapsules in water under an external magnetic field (inset: microcapsules in water without an external magnetic field). N10B1 microcapsules exhibited a clear magnetic response. The magnetic microcapsules can readily be moved and collected using an external magnetic field. The change in diameter of N10B1 and N10B1A2 with temperature relative to that at 26 °C is presented in Fig. 9. With increasing temperature the microcapsules shrank. The microcapsules were slightly thermosensitive. However, no temperature at which sharp shrinkage occurred was found. The inset photographs are of N10B1A2 at 26 and 50 °C, and obvious shrinkage can be observed. In summary, magnetic hydrogels with supracolloidal structures were fabricated by suspension polymerization of NIPAm and/or Am based on Pickering emulsion droplets. These microcapsule beads may find application as delivery vehicles for biomolecules, drugs, cosmetics, food supplements and living cells. Suspension
1.01 1.00
N10B1 N10B1A2
0.99 0.98
relative diameter
280
0.97
26 ºC
0.96 0.95 0.94 0.93 0.92 0.91
50º C
0.90 0.89
25
30
35
40
45
50
T/ ºC Fig. 9. Relationship between relative mean diameter of N10B1 and N10B1A2 dispersed in water and temperature (insets: optical microscope images of N10B1A2 dispersed in water at 26 and 50 °C).
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