Journal of Colloid and Interface Science 424 (2014) 98–103
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Fluorescent-magnetic Janus particles prepared via seed emulsion polymerization Chariya Kaewsaneha a,b, Ahmad Bitar a, Pramuan Tangboriboonrat b,⇑, Duangporn Polpanich c, Abdelhamid Elaissari a,⇑ a b c
University of Lyon, F-69622 Lyon, France, University Lyon 1, Villeurbanne, CNRS, UMR 5007, LAGEP-CPE, 43 Bd. 11 Novembre 1918, F-69622 Villeurbanne, France Department of Chemistry, Faculty of Science, Mahidol University, Phyathai, Bangkok 10400, Thailand National Nanotechnology Center (NANOTEC), Thailand Science Park, PathumThani 12120, Thailand
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Article history: Received 27 November 2013 Accepted 1 March 2014 Available online 15 March 2014 Keywords: Janus particle Anisotropic hybrid particle Fluorescent-magnetic particle
a b s t r a c t Anisotropic polymeric colloidal or Janus particles possessing simultaneous magnetic and fluorescent properties were successfully prepared via the swelling-diffusion or the in situ emulsion polymerization method. In the swelling-diffusion process, magnetic emulsions (an organic ferrofluid dispersed in aqueous medium) were synthesized and used for seeds of submicron magnetic Janus particles. After swelling the anisotropic particles obtained by 1-pyrene-carboxaldehyde fluorescent dye dissolved in tetrahydrofuran, well-defined fluorescent-magnetic Janus particles were produced. In the in situ emulsion polymerization, styrene monomer mixed with fluorescent dye monomers, i.e., 1-pyrenylmethyl methacrylate (PyMMA) or fluorescein dimethacrylate (FDMA), and an oil-soluble initiator (2,20 -azobis(2-isobutyronitrile)) were emulsified in the presence of magnetic seed emulsions. The confocal microscopic images showed the fluorescent-magnetic Janus particles with high fluorescent intensity when a fluorescent crosslinker monomer FDMA was employed. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction In the last decade, the preparation of magnetic nanoparticles (MNPs) containing active compounds has been of great interest especially in biological and medical applications [1–3]. In order to rapidly separate via an external magnet and to easily surface functionalize with biomolecules, MNPs in the form of clusters having high magnetization are embedded in polymer matrices [4–9]. Several techniques, e.g., miniemulsion [10–12], inverse miniemulsion [13,14] and seed emulsion polymerizations [15–18], have been used for preparing uniform magnetic polymeric particles (MPPs). By the miniemulsion polymerization, the desired core– shell MPPs (a cluster of MNPs engulfed in poly(styrene/divinyl benzene/acrylic acid); P(St/DVB/AA)) were achieved when a water-soluble initiator, i.e., potassium persulfate (KPS), and an appropriate St/DVB) ratio were employed [10]. It was due to a large amount of sulfate groups at the surface of MPPs and the high internal viscosity of PS matrix crosslinked by DVB which ⇑ Corresponding authors. Addresses: Department of Chemistry, Faculty of Science, Mahidol University, Phyathai, Bangkok 10400, Thailand (P. Tangboriboonrat), University Lyon-1, Villeurbanne, CNRS, UMR 5007, LAGEP-CPE, 43 bd 11 Novembre 1918, F-69622 Villeurbanne, France. Fax: +33 4 72 43 16 82 (A. Elaissari). E-mail addresses: [email protected] (P. Tangboriboonrat), elaissari@ lagep.univ-lyon1.fr (A. Elaissari). http://dx.doi.org/10.1016/j.jcis.2014.03.011 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
significantly increased the compatibility between polymer shell and water phase and prevented the phase separation inside each particle. When using an oil-soluble initiator, i.e., 2,20 -azobis (2-isobutyronitrile) (AIBN), the MPPs having Janus morphology, i.e., a cluster of MNPs located on one side of PS particle, were obtained [11,12]. Since the oil-soluble initiator initiated and preformed polymerization inside each monomer droplet, the phase separation between polymer matrix and MNPs stabilized by oleic acid (OA) took place during polymerization and, hence, MNPs were pushed aside of the droplet providing Janus MPPs. However, the magnetic content inside the Janus MPPs was low (ca.19 wt%) due to the limitation of compatibility between St monomer and hydrophobic OA backbone on the surface of MNPs [12]. In order to generate Janus MPPs with high magnetic content, the seed emulsion polymerization using magnetic emulsion (an organic ferrofluid of MNPs coated with OA emulsified in aqueous medium by Triton X-405) as seed was introduced [15–18]. In the process, St. monomer containing AIBN was diffused inside magnetic seed emulsion before being polymerized. The phase separation between PS matrix and encapsulated MNPs, having OA and Triton X-405 at the surface, took place. Consequently, hemispherical or anisotropic particles with high magnetic content (>60 wt%) were produced. Besides being controlled by an external magnet, hydrophobic ingredients, e.g., drugs and/or molecular probes, can be
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encapsulated in the polymer matrix part of Janus particles without magnetic interference. Moreover, this morphology offers unique feature and provides advanced multifunctionality, e.g., composition, hydrophobicity and hydrophilicity, combined and modular functionalities, which enable important applications unavailable to their symmetrical counterparts [19–22]. The colloidal hybrid particles that possess simultaneous inorganic material [23,24] and principally magnetic and optical properties are of particular interest in various fields, including materials science and biomedical applications i.e., separation, detection, sensing and diagnosis [7,25–29]. A variety of fluorescent-magnetic Janus particles (F-MJPs) with complex shapes, anisotropic nature, and diverse functionalities, have been developed by several methods, e.g., microfluidic device and solvent evaporation methods. Although the microfluid device produces a large amount of F-MJPs in a continuous fashion, the size of resultant particles is typically large (1–100 lm) [30,31]. Therefore, submicron F-MJPs were prepared via the solvent evaporation based method [32]. MNPs and pyrene labeled poly(styrene-block-allyl alcohol) were dissolved in chloroform and then emulsified in water phase having poly(vinyl alcohol) as stabilizing agent. After solvent evaporation, the phase separation between MNPs and polymer labeled fluorescent matrix occurred and spherical F-MJPs having MNPs located on one side and polymer labeled with fluorescent dye on the other side were generated. Based on this technique, various hydrophobic molecular probes, i.e., rhodamine perchlorate, Nile Red and fluorescein isothiocyanate (FITC), could be simply incorporated into hydrophobic polymer matrix of F-MJP [33–35]. For example, a small amount of hydrophobic fluorescent dye, i.e., FITC, dissolved in CH2Cl2 was diffused into poly(methyl methacrylate) particle with gentle stirring. After partial removal of CH2Cl2, monodisperse fluorescent polymeric particles with high dye loading were obtained. Another method to prepare fluorescent-magnetic particles was the in situ emulsion and/or miniemulsion polymerization. A novel poly[(Nvinylimidazole)-co-(1-pyrenylmethyl methacrylate)] (VI-co-PyMMA) ferric complex was synthesized through a convenient free-radical copolymerization using AIBN as initiator [36]. Due to an excellent quantum yield of 0.65 in ethanol at 293 K with a long fluorescent time of 410 ns, pyrene and/or its derivatives commonly used as fluorescent compounds were incorporated into polymer chain. However, the F-MJPs prepared by the in situ emulsion polymerization have not yet been reported. The aim of this present study was to adapt and to improve the seed emulsion polymerization method for the preparation of F-MJPs. Submicron Janus magnetic particles were synthesized via emulsion polymerization using magnetic emulsions as seeds [17]. Fluorescent dye (1-pyrenecarboxaldehyde) dissolved in chloroform (CHCl3), dimethylformamide (DMF) or tetrahydrofuran (THF) was then allowed to diffuse into the particles. In parallel, in situ seed emulsion polymerization was used for the preparation of F-MJPs by polymerizing fluorescent dye monomers, i.e., 1-pyrenylmethyl methacrylate or fluorescein dimethacrylate dissolved in St monomer with AIBN initiator. The effects of types of organic solvents and fluorescent dye monomers on the particle morphology, polymerization conversion, particle size, magnetic and fluorescent properties, were investigated by transmission electron microscopy, gravimetric method, zeta sizer, thermal gravimetric analyzer and confocal microscopy, respectively.
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containing OA dispersed in octane, prepared by the coprecipitation method, was emulsified in water with aid of nonionic surfactant (t-octylphenoxypolyethoxyethanol (Triton X-405)) [15–18]. Styrene (St) monomer (Sigma–Aldrich, Purum) was purified by washing with 5 M NaOH thrice. 2,20 -azobis(2-isobutyronitrile) (AIBN) (Sigma–Aldrich, Purum), oleic acid (OA) (Sigma–Aldrich, Technical), 1-pyrenecarboxaldehyde (PyCHO) (Polysciences, Inc., R&D), 1-pyrenylmethyl methacrylate (PyMMA) (Polysciences, Inc., R&D), fluorescein dimethacrylate (FDMA) (Polysciences, Inc., R&D), chloroform (CHCl3) (Labscan, AR), dimethylformamide (DMF) (Sigma–Aldrich, A.C.S. reagent), tetrahydrofuran (THF) (Fisher Chemical, Analysis) were used as received. 2.2. Preparation of magnetic Janus particles The method for preparation of anisotropic magnetic Janus particles (MJPs) via emulsion polymerization using magnetic emulsion as seeds was reported elsewhere [17]. Briefly, the magnetic emulsion was washed with 1.5 g l1 Triton X-405 solution thrice and purged with N2 for 2 h. Then, St monomer (900 mg) mixed with AIBN initiator (2 wt% of St monomer) was poured into the magnetic emulsion (50 mg, 4.1% solid content) and stirred at 300 rpm for 1 h. The polymerization was carried out at 70 °C for 20 h. 2.3. Preparation of fluorescent-magnetic Janus particles 2.3.1. Swelling-diffusion method 5 103 M of fluorescent dye, i.e., PyCHO, in CHCl3, DMF or THF (100 ll) was poured into Janus magnetic seeds latex (1 ml, 0.1% solid content). The mixture was gently stirred overnight to allow the solvent diffusion inside seed particles. The organic solvent was then removed by evaporating in fume hood at room temperature. 2.3.2. In situ seed emulsion polymerization St monomer (900 mg) containing AIBN (2 wt% of St monomer) and fluorescent monomer, i.e., PyMMA or FDMA (3 wt% of St monomer), was poured into the magnetic emulsion (50 mg, 4.1% solid content) in the reactor. After stirring for 1 h, the polymerization was started at 70 °C in dark cabinet for 20 h. 2.4. Characterizations of hybrid particles Hydrodynamic diameter (Dh) of the prepared colloidal particles was measured by using zeta sizer (Malvern, Nano ZS) at 25 °C thrice. Theirs chemical composition and magnetic content were investigated by applying thermogravimetric analyzer (TGA; NETZSCH TG 209 F1) varying from 25 to 600 °C under N2 at heating rate of 10 °C/min. Transmission electron microscope (TEM; Philips CM120) was used for morphological study of the prepared hybrid colloidal particles. A drop of highly diluted sample was deposited onto a carbon-coated copper grid and allowed to dry at room temperature overnight before TEM imaging. Fluorescent property of the hybrid F-MJPs was examined by using confocal microscopy (OLYMPUS, FLUOVIEW/FV1000). Before being deposited onto a glass slide, the sample containing PyCHO fluorescent dye was highly diluted with ethanol, whereas the other one containing FDMA was diluted with 1% NH4OH.
2. Materials and methods
3. Results and discussion
2.1. Material
3.1. Magnetic emulsion and magnetic Janus particles
Magnetic emulsions were prepared by following the process of Ademtech, SA Pesac, Bordeaux, France. Briefly, ferrofluid, i.e., MNPs
The sizes, morphologies and magnetic content of the synthesized magnetic emulsions and MJPs were determined after
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washing with deionized (DI) water under magnetic field to remove all non-magnetic particles. 3.1.1. Morphology and size distribution TEM images of magnetic emulsions and MJPs are displayed in Fig. 1A and B, respectively, whereas the size distribution curve of MJPs is shown in Fig. 1C. Fig. 1A shows the raspberry-like morphology with uniform magnetic (black) distribution in each magnetic emulsion particle. Since the particles were not monodisperse, the elaborated magnetic emulsions were fractionated under magnetic field by varying intensities (size-sorting process). In Fig. 1B, Janus or hemisphere morphology consisting of magnetic part (black) located on one side and PS (white) on the other side of particle was clearly noticed. This morphology was resulted from thermodynamic incompatibility between PS and magnetic core of the droplet [15,17]. In particular, a large amount of OA in the oil droplets could act as poor solvent of PS enhancing the phase separation process. The narrow size distribution with an average size of 165 nm of MJPs is displayed in Fig. 1C. 3.1.2. Magnetic content The TGA thermograms of magnetic emulsions and MJPs are shown in Fig. 2. Curve A revealed the thermal degradation at 25–500 °C of 17 wt% relating to the weight loss of OA and Triton X-405 presented at the surface of magnetic emulsions [17]. The degradation of polymer matrix of MJPs in curve B with the weight loss of 33 wt% took place at 400 °C. This indicated the high magnetic
Fig. 2. TGA thermograms of magnetic emulsions (curve A) and MJPs (curve B).
content of 67 wt% which permitted rapid separation of the prepared hybrid MJPs from the medium after applying an external magnet for a few minutes. 3.2. Fluorescent-magnetic Janus particles To obtain F-MJPs, the two methods, i.e., swelling-diffusion and in situ seed emulsion polymerization, were applied. The morphologies, polymerization conversion, magnetic content and fluorescent properties of the prepared F-MJPs were determined by TEM, gravimetric method, TGA and confocal microscopy.
Fig. 1. TEM images of (A) magnetic emulsions and (B) MJPs; (C) size distribution of MJPs.
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3.2.1. Swelling-diffusion method In the swelling-diffusion technique, the solvent plays many roles, e.g., in the swelling of polymer particle, diffusion of dye from solution into particle and entrapment of dye after solvent removal. Both types and amount of organic solvents are important parameters not only to produce the particles with high fluorescent intensity but also to maintain Janus morphology. Therefore, DMF, CHCl3 and THF (100 ll) were used in the preparation of MJPs (1 ml; 0.1% solid content) containing 5 103 M PyCHO and the morphologies of F-MJPs after solvent removal, investigated by TEM, are shown in Fig. 3A–C. By using DMF, Fig. 3A revealed broken particles while CHCl3 caused the spread of MNPs (black) around PS particles as observed in Fig. 3B. On contrary, the well-defined Janus morphology in Fig. 3C was obtained when THF was applied. The explanation concerned the physicochemical properties, i.e., boiling point, vapor pressure, solubility parameter and density, of these solvents [37,38]. Since the MJPs could be easily swelled by these solvents, the miscibility between hydrophobic fluorescent dye (PyCHO) and PS matrix was increased. The two solvents with low boiling points, i.e., CHCl3 (61 °C) and THF (65–67 °C), vaporized at much higher rate than DMF (135–155 °C) and, hence, the particle separation or broken particle was unable to take place before completely vaporization. However, the well-defined Janus morphology was formed only when using THF as solvent. It was because of the lower density of THF (0.889 g/cm3) compared to that of CHCl3 (1.492 g/ cm3) which also caused faster evaporation. 3.2.2. In situ seed emulsion polymerization When the seed emulsion polymerization was used for the synthesis of F-MJPs, the maximum percent conversion of hybrid particles prepared by using FDMA as fluorescent monomer (61.8%) was higher than that of particles containing PyMMA (57.3%). It was explained that the presence of FDMA comonomer acting as crosslinking agent resulted in high viscosity inside PS particle and, consequently, the polymerization rate was increased due to the auto-acceleration [39,40]. The comparable polymerization conversion of both types of hybrid particles was confirmed by TGA and the thermograms of particles containing PyMMA and FDMA are shown in Fig. 4. It was observed that the degradation of polymer matrix in F-MJPs having PyMMA (curve A) and FDMA (curve B) took place at ca. 400 °C with the weight loss of 23 and 30 wt%, respectively. This confirmed that the polymer content of particles having FDMA was higher than that containing PyMMA. Both types of hybrid particles had high magnetic content (>70%) which could be sufficiently manipulated by applying an external magnet [41]. The morphology of F-MJPs containing PyMMA and FDMA was examined by TEM and the micrographs are shown in Fig. 5A and B, respectively. For the F-MJPs having PyMMA, Fig. 5A shows that magnetic substances (dark) were uniformly distributed in polymer matrix
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Fig. 4. TGA thermograms of F-MJPs containing PyMMA (curve A) and FDMA (curve B).
(white) which were surrounded by polymer shell. It was believed that the polymerization of PyMMA fluorescent comonomer might be occurred at the surface of magnetic emulsion due to the more hydrophilic characteristic of methyl methacrylate (MMA) group of PyMMA compared with PS. This morphology was similar to that of PS-poly(methyl methacrylate) (PMMA) prepared by the suspension-emulsion combined polymerization [42]. In that case, the second MMA monomer was added at the mid of suspension polymerization where the viscosity of PS (dispersed phase) rapidly increased. A part of MMA monomer would diffuse to the surface of PS particles and polymerize at the outer layer leading to the formation of PS-PMMA core–shell structure. On contrary, with using FDMA, the F-MJPs with Janus morphology having narrow size distribution (ca. 164 nm) were obtained as shown in Fig. 5B and in the inserted image. As already mentioned, the phase separation between PS and magnetic part in Janus particle took place due to the incompatibility between PS and OA coated MNPs having Triton X-405 at the surface of magnetic seeds emulsion [15]. Although DVB as crosslinking agent reduced the mobility of PS chains and the phase separation, the asymmetric particles were still generated. Since the concentration of FDMA crosslinking comonomer (3 wt% of St. monomer) used was too low to reduce the mobility of PS, the phase separation between magnetic seed and polymer part could be occurred resulting in Janus particles. 3.2.3. Fluorescent properties of hybrid F-MJPs The presence of fluorescent dyes inside F-MJPs or the fluorescent property of the hybrid particles was investigated under confocal microscopy. The fluorescent images of MJPs and F-MJPs prepared via (1) the solvent-diffusion method using THF and PyCHO (F-MJPs@PyCHO) and (2) the in situ seed emulsion polymerization using FDMA as fluorescent comonomer (F-MJPs@FDMA) are shown in Fig. 6A–C.
Fig. 3. TEM images of F-MJPs prepared by using 5 103 M PyCHO, MJPs (1 ml, 0.1% solid content) with (A) DMF, (B) CHCl3, and (C) THF (100 ll).
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Fig. 5. TEM images of hybrid F-MJPs prepared by using (A) PyMMA and (B) FDMA (the size distribution curve was also inserted).
Fig. 6. Fluorescent photographs of (A) MJPs, (B) F-MJPs@PyCHO, and (C) F-MJPs@FDMA.
Under confocal microscopy, the fluorescence signal of MJPs could not be observed in Fig. 6A, whereas uniform F-MJPs@PyCHO and F-MJPs@FDMA in Fig. 6B and C displayed high fluorescent signal with blue and green colors, respectively. This confirmed that the two methods are effective to produce F-MJPs in spite of the fact that the solvent-diffusion technique was facile and fluorescent dye might be leaked from the particles due to physical interaction with polymer matrix. It was also undoubtful that the in situ seed emulsion polymerization provided the uniform distribution of dye along the polymer backbone via chemical bonding [43,44]. The prepared F-MJPs not only demonstrated strongly fluorescent signal under
confocal microscopy but also possessed magnetic property under an external magnetic field. Therefore, these particles would be efficiently used as imaging probe in biomedical applications.
4. Conclusion Novel hybrid particles possessing fluorescent and magnetic properties with well-defined Janus morphologies were accomplished by using the two methods based on seed emulsion radical polymerization which have not yet been reported [17,18,36].
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Controllable phase separation between inorganic (magnetic part) and organic (fluorescent polymeric) part is a key factor to produce the hybrid fluorescent-magnetic particles with Janus morphology (F-MJPs). Such anisotropic morphology was claimed to exhibit interesting optical properties (for in vitro detection) and not interfered by magnetic domains [32,45]. By using the developed technique based on the swelling-diffusion method, the anisotropic magnetic particles were firstly prepared via seed emulsion polymerization and then swollen by an organic fluorescent dye solution. The swelling process via solvent diffusion leads to the transfer of fluorescent dyes inside polymer part. After solvent evaporation, the fluorescent anisotropic magnetic particles (F-MJPs) were then obtained. Due to its good polystyrene solubilization, low boiling point and low density, THF was an appropriate solvent in the solvent-diffusion technique for transporting the PyCHO fluorescent dye from the continuous phase to the MJPs. This facile technique has numerous advantages such as rapid process, stability of the fluorescent dye (PyCHO), homogeneous fluorescence distribution in the polymer part, rapid diffusion of solvent carrier and solvent evaporation [34,35] compared to batch labeling processes [43,44]. In the in situ seed emulsion polymerization process, the F-MJPs were simply obtained when using the proper St monomer, oil-soluble AIBN initiator and a low amount of FDMA fluorescent dye crosslinkable comonomer. The chemical linkage of fluorescent dye along the polymer chain is indicated ensuring no fluorescent dye leakage from the hybrid particles after polymerization [36,46]. Confocal microscopy confirmed the strong fluorescent properties of the hybrid particles prepared via both techniques although the PyCHO was physically entrapped into the particles. The obtained F-MJPs could be, therefore, efficiently used as imaging probes in in vitro biomedical applications. Acknowledgments The authors thank and appreciate the research grant (RTA5480007) from The Thailand Research Fund (TRF)/Commission on Higher Education to P.T., and the scholarship from TRF, Mahidol University and French Government through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0174/2552) to the Ph.D. student C.K. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.03.011. References [1] H. Ahmad, J. Colloid Sci. Biotechnol. 2 (2013) 155. [2] H. Macková, D. Horák, Š. Trachtová, B. Rittich, A. Španová, J. Colloid Sci. Biotechnol. 1 (2012) 235.
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Fluorescent-magnetic Janus particles prepared via seed emulsion polymerization Chariya Kaewsaneha a,b, Ahmad Bitar a, Pramuan Tangboriboonrat b,⇑, Duangporn Polpanich c, Abdelhamid Elaissari a,⇑ a b c
University of Lyon, F-69622 Lyon, France, University Lyon 1, Villeurbanne, CNRS, UMR 5007, LAGEP-CPE, 43 Bd. 11 Novembre 1918, F-69622 Villeurbanne, France Department of Chemistry, Faculty of Science, Mahidol University, Phyathai, Bangkok 10400, Thailand National Nanotechnology Center (NANOTEC), Thailand Science Park, PathumThani 12120, Thailand
a r t i c l e
i n f o
Article history: Received 27 November 2013 Accepted 1 March 2014 Available online 15 March 2014 Keywords: Janus particle Anisotropic hybrid particle Fluorescent-magnetic particle
a b s t r a c t Anisotropic polymeric colloidal or Janus particles possessing simultaneous magnetic and fluorescent properties were successfully prepared via the swelling-diffusion or the in situ emulsion polymerization method. In the swelling-diffusion process, magnetic emulsions (an organic ferrofluid dispersed in aqueous medium) were synthesized and used for seeds of submicron magnetic Janus particles. After swelling the anisotropic particles obtained by 1-pyrene-carboxaldehyde fluorescent dye dissolved in tetrahydrofuran, well-defined fluorescent-magnetic Janus particles were produced. In the in situ emulsion polymerization, styrene monomer mixed with fluorescent dye monomers, i.e., 1-pyrenylmethyl methacrylate (PyMMA) or fluorescein dimethacrylate (FDMA), and an oil-soluble initiator (2,20 -azobis(2-isobutyronitrile)) were emulsified in the presence of magnetic seed emulsions. The confocal microscopic images showed the fluorescent-magnetic Janus particles with high fluorescent intensity when a fluorescent crosslinker monomer FDMA was employed. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction In the last decade, the preparation of magnetic nanoparticles (MNPs) containing active compounds has been of great interest especially in biological and medical applications [1–3]. In order to rapidly separate via an external magnet and to easily surface functionalize with biomolecules, MNPs in the form of clusters having high magnetization are embedded in polymer matrices [4–9]. Several techniques, e.g., miniemulsion [10–12], inverse miniemulsion [13,14] and seed emulsion polymerizations [15–18], have been used for preparing uniform magnetic polymeric particles (MPPs). By the miniemulsion polymerization, the desired core– shell MPPs (a cluster of MNPs engulfed in poly(styrene/divinyl benzene/acrylic acid); P(St/DVB/AA)) were achieved when a water-soluble initiator, i.e., potassium persulfate (KPS), and an appropriate St/DVB) ratio were employed [10]. It was due to a large amount of sulfate groups at the surface of MPPs and the high internal viscosity of PS matrix crosslinked by DVB which ⇑ Corresponding authors. Addresses: Department of Chemistry, Faculty of Science, Mahidol University, Phyathai, Bangkok 10400, Thailand (P. Tangboriboonrat), University Lyon-1, Villeurbanne, CNRS, UMR 5007, LAGEP-CPE, 43 bd 11 Novembre 1918, F-69622 Villeurbanne, France. Fax: +33 4 72 43 16 82 (A. Elaissari). E-mail addresses: [email protected] (P. Tangboriboonrat), elaissari@ lagep.univ-lyon1.fr (A. Elaissari). http://dx.doi.org/10.1016/j.jcis.2014.03.011 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
significantly increased the compatibility between polymer shell and water phase and prevented the phase separation inside each particle. When using an oil-soluble initiator, i.e., 2,20 -azobis (2-isobutyronitrile) (AIBN), the MPPs having Janus morphology, i.e., a cluster of MNPs located on one side of PS particle, were obtained [11,12]. Since the oil-soluble initiator initiated and preformed polymerization inside each monomer droplet, the phase separation between polymer matrix and MNPs stabilized by oleic acid (OA) took place during polymerization and, hence, MNPs were pushed aside of the droplet providing Janus MPPs. However, the magnetic content inside the Janus MPPs was low (ca.19 wt%) due to the limitation of compatibility between St monomer and hydrophobic OA backbone on the surface of MNPs [12]. In order to generate Janus MPPs with high magnetic content, the seed emulsion polymerization using magnetic emulsion (an organic ferrofluid of MNPs coated with OA emulsified in aqueous medium by Triton X-405) as seed was introduced [15–18]. In the process, St. monomer containing AIBN was diffused inside magnetic seed emulsion before being polymerized. The phase separation between PS matrix and encapsulated MNPs, having OA and Triton X-405 at the surface, took place. Consequently, hemispherical or anisotropic particles with high magnetic content (>60 wt%) were produced. Besides being controlled by an external magnet, hydrophobic ingredients, e.g., drugs and/or molecular probes, can be
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encapsulated in the polymer matrix part of Janus particles without magnetic interference. Moreover, this morphology offers unique feature and provides advanced multifunctionality, e.g., composition, hydrophobicity and hydrophilicity, combined and modular functionalities, which enable important applications unavailable to their symmetrical counterparts [19–22]. The colloidal hybrid particles that possess simultaneous inorganic material [23,24] and principally magnetic and optical properties are of particular interest in various fields, including materials science and biomedical applications i.e., separation, detection, sensing and diagnosis [7,25–29]. A variety of fluorescent-magnetic Janus particles (F-MJPs) with complex shapes, anisotropic nature, and diverse functionalities, have been developed by several methods, e.g., microfluidic device and solvent evaporation methods. Although the microfluid device produces a large amount of F-MJPs in a continuous fashion, the size of resultant particles is typically large (1–100 lm) [30,31]. Therefore, submicron F-MJPs were prepared via the solvent evaporation based method [32]. MNPs and pyrene labeled poly(styrene-block-allyl alcohol) were dissolved in chloroform and then emulsified in water phase having poly(vinyl alcohol) as stabilizing agent. After solvent evaporation, the phase separation between MNPs and polymer labeled fluorescent matrix occurred and spherical F-MJPs having MNPs located on one side and polymer labeled with fluorescent dye on the other side were generated. Based on this technique, various hydrophobic molecular probes, i.e., rhodamine perchlorate, Nile Red and fluorescein isothiocyanate (FITC), could be simply incorporated into hydrophobic polymer matrix of F-MJP [33–35]. For example, a small amount of hydrophobic fluorescent dye, i.e., FITC, dissolved in CH2Cl2 was diffused into poly(methyl methacrylate) particle with gentle stirring. After partial removal of CH2Cl2, monodisperse fluorescent polymeric particles with high dye loading were obtained. Another method to prepare fluorescent-magnetic particles was the in situ emulsion and/or miniemulsion polymerization. A novel poly[(Nvinylimidazole)-co-(1-pyrenylmethyl methacrylate)] (VI-co-PyMMA) ferric complex was synthesized through a convenient free-radical copolymerization using AIBN as initiator [36]. Due to an excellent quantum yield of 0.65 in ethanol at 293 K with a long fluorescent time of 410 ns, pyrene and/or its derivatives commonly used as fluorescent compounds were incorporated into polymer chain. However, the F-MJPs prepared by the in situ emulsion polymerization have not yet been reported. The aim of this present study was to adapt and to improve the seed emulsion polymerization method for the preparation of F-MJPs. Submicron Janus magnetic particles were synthesized via emulsion polymerization using magnetic emulsions as seeds [17]. Fluorescent dye (1-pyrenecarboxaldehyde) dissolved in chloroform (CHCl3), dimethylformamide (DMF) or tetrahydrofuran (THF) was then allowed to diffuse into the particles. In parallel, in situ seed emulsion polymerization was used for the preparation of F-MJPs by polymerizing fluorescent dye monomers, i.e., 1-pyrenylmethyl methacrylate or fluorescein dimethacrylate dissolved in St monomer with AIBN initiator. The effects of types of organic solvents and fluorescent dye monomers on the particle morphology, polymerization conversion, particle size, magnetic and fluorescent properties, were investigated by transmission electron microscopy, gravimetric method, zeta sizer, thermal gravimetric analyzer and confocal microscopy, respectively.
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containing OA dispersed in octane, prepared by the coprecipitation method, was emulsified in water with aid of nonionic surfactant (t-octylphenoxypolyethoxyethanol (Triton X-405)) [15–18]. Styrene (St) monomer (Sigma–Aldrich, Purum) was purified by washing with 5 M NaOH thrice. 2,20 -azobis(2-isobutyronitrile) (AIBN) (Sigma–Aldrich, Purum), oleic acid (OA) (Sigma–Aldrich, Technical), 1-pyrenecarboxaldehyde (PyCHO) (Polysciences, Inc., R&D), 1-pyrenylmethyl methacrylate (PyMMA) (Polysciences, Inc., R&D), fluorescein dimethacrylate (FDMA) (Polysciences, Inc., R&D), chloroform (CHCl3) (Labscan, AR), dimethylformamide (DMF) (Sigma–Aldrich, A.C.S. reagent), tetrahydrofuran (THF) (Fisher Chemical, Analysis) were used as received. 2.2. Preparation of magnetic Janus particles The method for preparation of anisotropic magnetic Janus particles (MJPs) via emulsion polymerization using magnetic emulsion as seeds was reported elsewhere [17]. Briefly, the magnetic emulsion was washed with 1.5 g l1 Triton X-405 solution thrice and purged with N2 for 2 h. Then, St monomer (900 mg) mixed with AIBN initiator (2 wt% of St monomer) was poured into the magnetic emulsion (50 mg, 4.1% solid content) and stirred at 300 rpm for 1 h. The polymerization was carried out at 70 °C for 20 h. 2.3. Preparation of fluorescent-magnetic Janus particles 2.3.1. Swelling-diffusion method 5 103 M of fluorescent dye, i.e., PyCHO, in CHCl3, DMF or THF (100 ll) was poured into Janus magnetic seeds latex (1 ml, 0.1% solid content). The mixture was gently stirred overnight to allow the solvent diffusion inside seed particles. The organic solvent was then removed by evaporating in fume hood at room temperature. 2.3.2. In situ seed emulsion polymerization St monomer (900 mg) containing AIBN (2 wt% of St monomer) and fluorescent monomer, i.e., PyMMA or FDMA (3 wt% of St monomer), was poured into the magnetic emulsion (50 mg, 4.1% solid content) in the reactor. After stirring for 1 h, the polymerization was started at 70 °C in dark cabinet for 20 h. 2.4. Characterizations of hybrid particles Hydrodynamic diameter (Dh) of the prepared colloidal particles was measured by using zeta sizer (Malvern, Nano ZS) at 25 °C thrice. Theirs chemical composition and magnetic content were investigated by applying thermogravimetric analyzer (TGA; NETZSCH TG 209 F1) varying from 25 to 600 °C under N2 at heating rate of 10 °C/min. Transmission electron microscope (TEM; Philips CM120) was used for morphological study of the prepared hybrid colloidal particles. A drop of highly diluted sample was deposited onto a carbon-coated copper grid and allowed to dry at room temperature overnight before TEM imaging. Fluorescent property of the hybrid F-MJPs was examined by using confocal microscopy (OLYMPUS, FLUOVIEW/FV1000). Before being deposited onto a glass slide, the sample containing PyCHO fluorescent dye was highly diluted with ethanol, whereas the other one containing FDMA was diluted with 1% NH4OH.
2. Materials and methods
3. Results and discussion
2.1. Material
3.1. Magnetic emulsion and magnetic Janus particles
Magnetic emulsions were prepared by following the process of Ademtech, SA Pesac, Bordeaux, France. Briefly, ferrofluid, i.e., MNPs
The sizes, morphologies and magnetic content of the synthesized magnetic emulsions and MJPs were determined after
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washing with deionized (DI) water under magnetic field to remove all non-magnetic particles. 3.1.1. Morphology and size distribution TEM images of magnetic emulsions and MJPs are displayed in Fig. 1A and B, respectively, whereas the size distribution curve of MJPs is shown in Fig. 1C. Fig. 1A shows the raspberry-like morphology with uniform magnetic (black) distribution in each magnetic emulsion particle. Since the particles were not monodisperse, the elaborated magnetic emulsions were fractionated under magnetic field by varying intensities (size-sorting process). In Fig. 1B, Janus or hemisphere morphology consisting of magnetic part (black) located on one side and PS (white) on the other side of particle was clearly noticed. This morphology was resulted from thermodynamic incompatibility between PS and magnetic core of the droplet [15,17]. In particular, a large amount of OA in the oil droplets could act as poor solvent of PS enhancing the phase separation process. The narrow size distribution with an average size of 165 nm of MJPs is displayed in Fig. 1C. 3.1.2. Magnetic content The TGA thermograms of magnetic emulsions and MJPs are shown in Fig. 2. Curve A revealed the thermal degradation at 25–500 °C of 17 wt% relating to the weight loss of OA and Triton X-405 presented at the surface of magnetic emulsions [17]. The degradation of polymer matrix of MJPs in curve B with the weight loss of 33 wt% took place at 400 °C. This indicated the high magnetic
Fig. 2. TGA thermograms of magnetic emulsions (curve A) and MJPs (curve B).
content of 67 wt% which permitted rapid separation of the prepared hybrid MJPs from the medium after applying an external magnet for a few minutes. 3.2. Fluorescent-magnetic Janus particles To obtain F-MJPs, the two methods, i.e., swelling-diffusion and in situ seed emulsion polymerization, were applied. The morphologies, polymerization conversion, magnetic content and fluorescent properties of the prepared F-MJPs were determined by TEM, gravimetric method, TGA and confocal microscopy.
Fig. 1. TEM images of (A) magnetic emulsions and (B) MJPs; (C) size distribution of MJPs.
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3.2.1. Swelling-diffusion method In the swelling-diffusion technique, the solvent plays many roles, e.g., in the swelling of polymer particle, diffusion of dye from solution into particle and entrapment of dye after solvent removal. Both types and amount of organic solvents are important parameters not only to produce the particles with high fluorescent intensity but also to maintain Janus morphology. Therefore, DMF, CHCl3 and THF (100 ll) were used in the preparation of MJPs (1 ml; 0.1% solid content) containing 5 103 M PyCHO and the morphologies of F-MJPs after solvent removal, investigated by TEM, are shown in Fig. 3A–C. By using DMF, Fig. 3A revealed broken particles while CHCl3 caused the spread of MNPs (black) around PS particles as observed in Fig. 3B. On contrary, the well-defined Janus morphology in Fig. 3C was obtained when THF was applied. The explanation concerned the physicochemical properties, i.e., boiling point, vapor pressure, solubility parameter and density, of these solvents [37,38]. Since the MJPs could be easily swelled by these solvents, the miscibility between hydrophobic fluorescent dye (PyCHO) and PS matrix was increased. The two solvents with low boiling points, i.e., CHCl3 (61 °C) and THF (65–67 °C), vaporized at much higher rate than DMF (135–155 °C) and, hence, the particle separation or broken particle was unable to take place before completely vaporization. However, the well-defined Janus morphology was formed only when using THF as solvent. It was because of the lower density of THF (0.889 g/cm3) compared to that of CHCl3 (1.492 g/ cm3) which also caused faster evaporation. 3.2.2. In situ seed emulsion polymerization When the seed emulsion polymerization was used for the synthesis of F-MJPs, the maximum percent conversion of hybrid particles prepared by using FDMA as fluorescent monomer (61.8%) was higher than that of particles containing PyMMA (57.3%). It was explained that the presence of FDMA comonomer acting as crosslinking agent resulted in high viscosity inside PS particle and, consequently, the polymerization rate was increased due to the auto-acceleration [39,40]. The comparable polymerization conversion of both types of hybrid particles was confirmed by TGA and the thermograms of particles containing PyMMA and FDMA are shown in Fig. 4. It was observed that the degradation of polymer matrix in F-MJPs having PyMMA (curve A) and FDMA (curve B) took place at ca. 400 °C with the weight loss of 23 and 30 wt%, respectively. This confirmed that the polymer content of particles having FDMA was higher than that containing PyMMA. Both types of hybrid particles had high magnetic content (>70%) which could be sufficiently manipulated by applying an external magnet [41]. The morphology of F-MJPs containing PyMMA and FDMA was examined by TEM and the micrographs are shown in Fig. 5A and B, respectively. For the F-MJPs having PyMMA, Fig. 5A shows that magnetic substances (dark) were uniformly distributed in polymer matrix
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Fig. 4. TGA thermograms of F-MJPs containing PyMMA (curve A) and FDMA (curve B).
(white) which were surrounded by polymer shell. It was believed that the polymerization of PyMMA fluorescent comonomer might be occurred at the surface of magnetic emulsion due to the more hydrophilic characteristic of methyl methacrylate (MMA) group of PyMMA compared with PS. This morphology was similar to that of PS-poly(methyl methacrylate) (PMMA) prepared by the suspension-emulsion combined polymerization [42]. In that case, the second MMA monomer was added at the mid of suspension polymerization where the viscosity of PS (dispersed phase) rapidly increased. A part of MMA monomer would diffuse to the surface of PS particles and polymerize at the outer layer leading to the formation of PS-PMMA core–shell structure. On contrary, with using FDMA, the F-MJPs with Janus morphology having narrow size distribution (ca. 164 nm) were obtained as shown in Fig. 5B and in the inserted image. As already mentioned, the phase separation between PS and magnetic part in Janus particle took place due to the incompatibility between PS and OA coated MNPs having Triton X-405 at the surface of magnetic seeds emulsion [15]. Although DVB as crosslinking agent reduced the mobility of PS chains and the phase separation, the asymmetric particles were still generated. Since the concentration of FDMA crosslinking comonomer (3 wt% of St. monomer) used was too low to reduce the mobility of PS, the phase separation between magnetic seed and polymer part could be occurred resulting in Janus particles. 3.2.3. Fluorescent properties of hybrid F-MJPs The presence of fluorescent dyes inside F-MJPs or the fluorescent property of the hybrid particles was investigated under confocal microscopy. The fluorescent images of MJPs and F-MJPs prepared via (1) the solvent-diffusion method using THF and PyCHO (F-MJPs@PyCHO) and (2) the in situ seed emulsion polymerization using FDMA as fluorescent comonomer (F-MJPs@FDMA) are shown in Fig. 6A–C.
Fig. 3. TEM images of F-MJPs prepared by using 5 103 M PyCHO, MJPs (1 ml, 0.1% solid content) with (A) DMF, (B) CHCl3, and (C) THF (100 ll).
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Fig. 5. TEM images of hybrid F-MJPs prepared by using (A) PyMMA and (B) FDMA (the size distribution curve was also inserted).
Fig. 6. Fluorescent photographs of (A) MJPs, (B) F-MJPs@PyCHO, and (C) F-MJPs@FDMA.
Under confocal microscopy, the fluorescence signal of MJPs could not be observed in Fig. 6A, whereas uniform F-MJPs@PyCHO and F-MJPs@FDMA in Fig. 6B and C displayed high fluorescent signal with blue and green colors, respectively. This confirmed that the two methods are effective to produce F-MJPs in spite of the fact that the solvent-diffusion technique was facile and fluorescent dye might be leaked from the particles due to physical interaction with polymer matrix. It was also undoubtful that the in situ seed emulsion polymerization provided the uniform distribution of dye along the polymer backbone via chemical bonding [43,44]. The prepared F-MJPs not only demonstrated strongly fluorescent signal under
confocal microscopy but also possessed magnetic property under an external magnetic field. Therefore, these particles would be efficiently used as imaging probe in biomedical applications.
4. Conclusion Novel hybrid particles possessing fluorescent and magnetic properties with well-defined Janus morphologies were accomplished by using the two methods based on seed emulsion radical polymerization which have not yet been reported [17,18,36].
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Controllable phase separation between inorganic (magnetic part) and organic (fluorescent polymeric) part is a key factor to produce the hybrid fluorescent-magnetic particles with Janus morphology (F-MJPs). Such anisotropic morphology was claimed to exhibit interesting optical properties (for in vitro detection) and not interfered by magnetic domains [32,45]. By using the developed technique based on the swelling-diffusion method, the anisotropic magnetic particles were firstly prepared via seed emulsion polymerization and then swollen by an organic fluorescent dye solution. The swelling process via solvent diffusion leads to the transfer of fluorescent dyes inside polymer part. After solvent evaporation, the fluorescent anisotropic magnetic particles (F-MJPs) were then obtained. Due to its good polystyrene solubilization, low boiling point and low density, THF was an appropriate solvent in the solvent-diffusion technique for transporting the PyCHO fluorescent dye from the continuous phase to the MJPs. This facile technique has numerous advantages such as rapid process, stability of the fluorescent dye (PyCHO), homogeneous fluorescence distribution in the polymer part, rapid diffusion of solvent carrier and solvent evaporation [34,35] compared to batch labeling processes [43,44]. In the in situ seed emulsion polymerization process, the F-MJPs were simply obtained when using the proper St monomer, oil-soluble AIBN initiator and a low amount of FDMA fluorescent dye crosslinkable comonomer. The chemical linkage of fluorescent dye along the polymer chain is indicated ensuring no fluorescent dye leakage from the hybrid particles after polymerization [36,46]. Confocal microscopy confirmed the strong fluorescent properties of the hybrid particles prepared via both techniques although the PyCHO was physically entrapped into the particles. The obtained F-MJPs could be, therefore, efficiently used as imaging probes in in vitro biomedical applications. Acknowledgments The authors thank and appreciate the research grant (RTA5480007) from The Thailand Research Fund (TRF)/Commission on Higher Education to P.T., and the scholarship from TRF, Mahidol University and French Government through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0174/2552) to the Ph.D. student C.K. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.03.011. References [1] H. Ahmad, J. Colloid Sci. Biotechnol. 2 (2013) 155. [2] H. Macková, D. Horák, Š. Trachtová, B. Rittich, A. Španová, J. Colloid Sci. Biotechnol. 1 (2012) 235.
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