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Functionalization of Monodisperse Magnetic Nanoparticles Marco Lattuada† and T. Alan Hatton* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139 ReceiVed July 18, 2006. In Final Form: October 27, 2006 We report a new strategy for the preparation of monodisperse, water-soluble magnetic nanoparticles. Oleic acidstabilized magnetic nanocrystals were prepared by the organic synthesis route proposed by Sun et al. (J. Am. Chem. Soc. 2004, 126, 273.), with size control obtained via seeded-mediated growth. The oleic groups initially present on the nanoparticle surfaces were replaced via ligand exchange reactions with various capping agents bearing reactive hydroxyl moieties. These hydroxyl groups were (i) exploited to initiate ring opening polymerization (ROP) of polylactic acid from the nanoparticle surfaces and (ii) esterified by acylation to permit the addition of alkyl halide moieties to transform the nanoparticle surfaces into macroinitiators for atom transfer radical polymerization (ATRP). By appropriate selection of the ligand properties, the nanoparticle surfaces can be polymerized in various solvents, providing an opportunity for the growth of a wide variety of water-soluble polymers and polylectrolyte brushes (both cationic and anionic) from the nanoparticle surfaces. The nanoparticles were characterized by Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), electron microscopy, and light scattering. Light scattering measurements indicate that the nanoparticles are mostly present as individual nonclustered units in water. With pH-responsive polymers grown on the nanoparticle surfaces, reversible aggregation of nanoparticles could be induced by suitable swings in the pH between the stable and unstable regions.
Introduction Magnetic nanoparticles have been a research topic of great interest over the past 30 years.1-15 Since the early studies, performed on organic ferrofluids that found applications in ball bearings and magnetic sealing devices,1 a considerable amount of work has been carried out to better understand,16,17 characterize, and improve the performance of these systems.18-20 In more recent years, many novel applications of magnetic nanoparticles have been proposed and investigated in the fields of environmental * To whom correspondence should be addressed. † Present address: Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland. (1) Rosensweig, R. E. Ferrohydrodynamics; Dover Publications Inc.: Mineola, NY, 1985. (2) Wooding, A.; Kilner, M.; Lambrick, D. B. J. Colloid Interface Sci. 1991, 144 (1), 236-242. (3) Wooding, A.; Kilner, M.; Lambrick, D. B. J. Colloid Interface Sci. 1992, 149 (1), 98-104. (4) Shen, L. F.; Laibinis, P. E.; Hatton, T. A. J. Magn. Magn. Mater. 1999, 194 (1-3), 37-44. (5) Shen, L. F.; Laibinis, P. E.; Hatton, T. A. Langmuir 1999, 15 (2), 447-453. (6) Murray, C. B.; Sun, S. H.; Doyle, H.; Betley, T. MRS Bull. 2001, 26 (12), 985-991. (7) Moeser, G. D.; Roach, K. A.; Green, W. H.; Laibinis, P. E.; Hatton, T. A. Ind. Eng. Chem. Res. 2002, 41 (19), 4739-4749. (8) Sun, S. H.; Zeng, H. J. Am. Chem. Soc. 2002, 124 (28), 8204-8205. (9) Hyeon, T. Chem. Commun. 2003, 927-934. (10) Sun, S. H.; Anders, S.; Thomson, T.; Baglin, J. E. E.; Toney, M. F.; Hamann, H. F.; Murray, C. B.; Terris, B. D. J. Phys. Chem. B 2003, 107 (23), 5419-5425. (11) Sun, S. H.; Zeng, H.; Robinson, D. B.; Raoux, S.; Rice, P. M.; Wang, S. X.; Li, G. X. J. Am. Chem. Soc. 2004, 126 (1), 273-279. (12) Parvin, K.; Ma, J.; Ly, J.; Sun, X. C.; Nikles, D. E.; Sun, K.; Wang, L. M. J. Appl. Phys. 2004, 95 (11), 7121-7123. (13) Park, J.; An, K. J.; Hwang, Y. S.; Park, J. G.; Noh, H. J.; Kim, J. Y.; Park, J. H.; Hwang, N. M.; Hyeon, T. Nat. Mater. 2004, 3 (12), 891-895. (14) Kang, E.; Park, J.; Hwang, Y.; Kang, M.; Park, J. G.; Hyeon, T. J. Phys. Chem. B 2004, 108 (37), 13932-13935. (15) Jing, Z. H.; Wu, S. H. Mater. Lett. 2004, 58 (27-28), 3637-3640. (16) Degennes, P. G.; Pincus, P. A. Pair Correlations in a Ferromagnetic Colloid. Phys. Kondens. Mater. 1970, 11 (3), 189. (17) Huke, B.; Lucke, M. Rep. Prog. Phys. 2004, 67 (10), 1731-1768. (18) Ditsch, A.; Laibinis, P. E.; Wang, D. I. C.; Hatton, T. A. Langmuir 2005, 21 (13), 6006-6018. (19) Lai, J. I.; Shafi, K.; Ulman, A.; Loos, K.; Lee, Y. J.; Vogt, T.; Lee, W. L.; Ong, N. P. J. Phys. Chem. B 2005, 109 (1), 15-18.
and biomolecular separations,7,21 in magnetic resonance imaging as contrast agents,22,23 in magnetic drug targeting and delivery,24 and in cancer treatments through hyperthermia.25 It goes without saying that a very intensive investigation has been conducted to look for new strategies to prepare magnetic nanoparticles with tailor-made properties through appropriately attached functional moieties. In particular, for biomedical and separation applications, water-soluble nanoparticles are commonly required. Among the wide variety of magnetic materials that can be prepared in the form of nanoparticles, iron oxides have certainly been and still are the most intensively studied. This is due to several factors, one being the approval by the Food and Drug Administration (FDA) of the use of superparamagnetic iron oxide nanoparticles (SPION) as contrast agents in magnetic resonance imaging. FDA approval implies that iron oxides (in particular, magnetite) seem to be quite benign toward humans. Certainly, another factor is the facility with which iron oxide (magnetite) nanoparticles can be prepared through the coprecipitation of iron salts in alkaline water solutions in the presence of stabilizers.2-5,18 This method is very flexible since it allows for the preparation of magnetite nanoparticles carrying a wide variety of stabilizers including copolymers, surfactants, and mixtures thereof. Despite all these advantages, the aqueous coprecipitation method has some major drawbacks when it comes to the control of the average size and monodispersity of the produced magnetite nanocrystals. Broad distributions of magnetite nanocrystals are generally synthesized through this method, together with irregular crystallite (20) Lee, Y.; Lee, J.; Bae, C. J.; Park, J. G.; Noh, H. J.; Park, J. H.; Hyeon, T. AdV. Funct. Mater. 2005, 15 (3), 503-509. (21) Ditsch, A.; Lindenmann, S.; Laibinis, P. E.; Wang, D. I. C.; Hatton, T. A. Ind. Eng. Chem. Res. 2005, 44 (17), 6824-6836. (22) Jun, Y. W.; Huh, Y. M.; Choi, J. S.; Lee, J. H.; Song, H. T.; Kim, S.; Yoon, S.; Kim, K. S.; Shin, J. S.; Suh, J. S.; Cheon, J. J. Am. Chem. Soc. 2005, 127 (16), 5732-5733. (23) Lee, S. J.; Jeong, J. R.; Shin, S. C.; Kim, J. C.; Chang, Y. H.; Chang, Y. M.; Kim, J. D. J. Magn. Magn. Mater. 2004, 272-276, 2432-2433. (24) Neuberger, T.; Schopf, B.; Hofmann, H.; Hofmann, M.; von Rechenberg, B. J. Magn. Magn. Mater. 2005, 293 (1), 483-496. (25) Gonzales, M.; Krishnan, K. M. J. Magn. Magn. Mater. 2005, 293 (1), 265-270.
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shapes, mainly due to the uncontrolled rate of nucleation that occurs in aqueous solutions.26 In addition, the typical average size of magnetic crystals cannot readily be increased above the limit of 7-8 nm.5,27 The above problems indicate why alternative synthetic routes have been explored to provide better control over the size distribution of magnetic nanoparticles. So far, only two routes have successfully ensured good control of magnetite nanoparticle size and distribution: the use of emulsions as nanoreactors to nucleate nanoparticles and the nucleation of nanoparticles in high temperature organic solvents. The first method, pioneered by the Pileni group,28,29 is a very flexible technique for the preparation of many different types of monodisperse metallic and metal oxide nanoparticles using micelles to tailor particle size. Unfortunately, in many cases it has been reported that the crystallinity of magnetite particles produced through this method is poor and they require annealing at high temperatures after synthesis.9 This obviously limits the applicability of the method to the production of water-soluble magnetic nanoparticles since many capping agents, typically used to stabilize the nanoparticles, can undergo thermal decomposition at such high temperatures. The second class of methods that have been applied successfully in the preparation of monodisperse magnetic nanoparticles is given by organic chemical routes.8,11,30 In these methods, nanoparticles nucleate and grow in an organic solvent as the temperature is increased progressively in a controlled fashion, usually up to the boiling point of the solvent. While the mechanism leading to particle nucleation under these conditions is not wellknown, the method has proven to be effective in producing magnetite nanoparticles with good crystallinity and a relatively monodisperse, controlled size distribution. In particular, the recently proposed strategy developed by Sun et al.11 allows one to prepare monodisperse nanoparticles with a particle size ranging from 4 to 20 nm by using the concept of seeded growth. The drawback of Sun’s method is that the particles can only be prepared in the presence of oleic acid or oleyl amine as stabilizer, and they are, therefore, soluble only in nonpolar and moderately polar organic solvents. The preparation of monodisperse magnetite crystals is not the only problem faced by scientists working in nanoparticle technology, as the preparation of very small polymer-coated monodisperse particles is itself a difficult task.31-34 Usually two different approaches have been reported for coating particles with polymers: “grafting to” and “grafting from” the particle surface. The former involves the grafting of pre-existing polymer chains onto the particle by means of either electrostatic or hydrophobic interactions or simply by using the affinity of certain chemical groups for the particle surface.18,26,35 This method tends to be very flexible because many different types of polymers and copolymers can be grafted, and thus it can be effectively used with large particles. However, in the case of very small nanoparticles, polymer chains have a high probability of grafting
onto more than one particle at a time, inducing the formation of clusters. In the case of magnetic nanoparticles, this procedure has been exploited to produce clusters with defined sizes.18 In addition, the “grafting to” method usually requires particles with a bare surface and is, therefore, most efficiently adopted to stabilize particles during their nucleation phase. In the “grafting from” approach, on the other hand, a polymer brush is grown directly from the particle surface. This usually requires the presence of initiators on the particle surface that can induce the polymerization process. At present, the most widely used polymerization technique for the growth of polymer brushes from surfaces is atom transfer radical polymerization (ATRP)31,32,34,36-38 because it is a living polymerization process which can produce polymer (and copolymer) brushes with narrow molecular weight distributions and for which a broad variety of monomers can be used. In the past few years, ATRP has been employed successfully to coat both organic and inorganic nanoparticles with different types of polymers, most of which are hydrophobic. Another popular approach is ring opening polymerization (ROP), which has also been used to coat nanoparticles with layers of certain biodegradable polyesters.32,39,40 In this work, we have used Sun’s method to prepare monodisperse magnetite nanocrystals and have developed a novel strategy to functionalize their surfaces to obtain various types of monodisperse magnetic nanoparticles (mostly water-soluble) with tunable core sizes coated by a variety of stabilizers and polymers. Some of these particles bear functional groups that would make them ideal candidates for biological applications. The oleic acid and oleyl amine moieties initially present on the nanoparticle surface are replaced with other ligands that both possess high affinity for the nanoparticle surface and either provide direct solubility in water or expose functional hydroxyl groups. The hydroxyl groups either have been used to initiate catalyzed ROP from the particle surfaces and coat the particles with a layer of polylactic acid or have been reacted with a powerful acylating agent to form esters that can be used as initiators for ATRP polymerization. The remarkable feature of the proposed strategy is that, by appropriately choosing the ligand, particles that are soluble either in organic solvents (both apolar and highly polar) or in water can be prepared, giving our method great flexibility in the selection of monomers and polymerization conditions. In this work, many polyelectrolytes, both anionic and cationic, have been grown from the nanoparticle surface. With pH-responsive polymers, aggregation of the particles was induced by changing pH conditions and, in almost all cases, the aggregation behavior could be reversed by switching back to the pH conditions that ensured the presence of charges on the particle surfaces. As a final remark, it should be noted that other methods have recently been proposed to prepare monodisperse nanoparticles.13,15,20,41-44 Many of these follow the same line as Sun’s method but start from cheaper reagents and are more suitable to be scaled up for the production of larger quantities of particles.
(26) Moeser, G. D.; Green, W. H.; Laibinis, P. E.; Linse, P.; Hatton, T. A. Langmuir 2004, 20 (13), 5223-5234. (27) Kang, Y. S.; Risbud, S.; Rabolt, J. F.; Stroeve, P. Chem. Mater. 1996, 8 (9), 2209. (28) Pileni, M. P. Langmuir 1997, 13 (13), 3266-3276. (29) Pileni, M. P. AdV. Funct. Mater. 2001, 11 (5), 323-336. (30) Sun, X.; Zhang, Y. W.; Si, R.; Yan, C. H. Small 2005, 1 (11), 1081-1086. (31) von Werne, T.; Patten, T. E. J. Am. Chem. Soc. 2001, 123 (31), 74977505. (32) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. ReV. 2004, 33 (1), 14-22. (33) Advincula, R. C. J. Dispersion Sci. Technol. 2003, 24 (3-4), 343-361. (34) Pyun, J.; Kowalewski, T.; Matyjaszewski, K. Macromol. Rapid Commun. 2003, 24 (18), 1043-1059. (35) Si, S.; Kotal, A.; Mandal, T. K.; Giri, S.; Nakamura, H.; Kohara, T. Chem. Mater. 2004, 16 (18), 3489-3496.
(36) Gu, B.; Sen, A. Macromolecules 2002, 35 (23), 8913-8916. (37) Vestal, C. R.; Zhang, Z. J. J. Am. Chem. Soc. 2002, 124 (48), 1431214313. (38) Marutani, E.; Yamamoto, S.; Ninjbadgar, T.; Tsujii, Y.; Fukuda, T.; Takano, M. Polymer 2004, 45 (7), 2231-2235. (39) Choi, I. S.; Langer, R. Macromolecules 2001, 34 (16), 5361-5363. (40) Schmidt, A. M. Macromol. Rapid Commun. 2005, 26 (2), 93-97. (41) Park, J.; Lee, E.; Hwang, N. M.; Kang, M. S.; Kim, S. C.; Hwang, Y.; Park, J. G.; Noh, H. J.; Kini, J. Y.; Park, J. H.; Hyeon, T. Angew. Chem., Int. Ed. 2005, 44 (19), 2872-2877. (42) Woo, K.; Hong, J.; Choi, S.; Lee, H. W.; Ahn, J. P.; Kim, C. S.; Lee, S. W. Chem. Mater. 2004, 16 (14), 2814-2818. (43) Jana, N. R.; Chen, Y. F.; Peng, X. G. Chem. Mater. 2004, 16 (20), 39313935. (44) Caruntu, D.; Caruntu, G.; Chen, Y.; O’Connor, C. J.; Goloverda, G.; Kolesnichenko, V. L. Chem. Mater. 2004, 16 (25), 5527-5534.
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Since most of these methods produce oleic acid-stabilized particles, the functionalization strategy proposed here can be applied immediately to such particles. Experimental Section Materials. Benzyl ether (99%), 2-bromo-2-methyl propionic acid (BMPA) (98%), 2-bromo-2-methyl propionyl bromide (BMPB; 98%), (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione (L-lactide; 98%), citric acid (CA; 99+%), copper(I) bromide (CuBr; 98%), 1,2dichlorobenzene (DCB; 99%), dimethylaminoethyl methacrylate (DMAEMA; 98%), N,N′-dimethylformamide (DMF; 99.8%), dimethyl sulfoxide (DMSO; 99.6%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 97%), galactaric acid (97%), hydroxyethylmethacrylate (HEMA; 97%), iron tri(acetylacetonate) (97%), N-hydroxysuccinimide (NHS; 98%), N-isopropylacrylamide (NIPAm; 97%), N,N,N,N′,N′,N′-hexamethyltriethyltetramine (HMTETA; 97%), N,N,N′,N′,N′-pentamethyldiethyltrimine (PMDETA; 99%), oleic acid (OA; 90%), oleyl amine (OAm; 70%), potassium bromide (g99%, IR grade), pyridine (99%), ricinoleic acid (RA; 80%), 4-styrenesulfonic acid sodium salt hydrate (SSNa; 98%), succinic anhydride (SA; 99%), 1,2-tetradecanediol (90%), tin(II) ethylhexanoate (95%), triethylamine (99.9%), trimethylsilyl acrylate (TMSA; 98%), and trimethylsilyl methacrylate (TMSMA; 98%) were purchased from Sigma Aldrich. Dichloromethane (DCM; 99.97%), diethyl ether (99.9%), hexane (99.9%), and tetrahydrofuran (THF; 99.94%) were purchased from Omisolv. Acetone (99.5%), ethanol (98%), and methanol (99.8%) were purchased from Mellinkrod. Amino end-functionalized polyethylene glycol (NH2PEG, 10 kDa) was purchased from Nektar Therapeutics. All chemicals were used as received. All water utilized in the experiments was Milli-Q (Millipore) deionized water. Nanoparticle Preparation. (1a) Preparation of Oleic Acid-Coated Magnetic Nanoparticles. The procedure followed for the preparation of monodisperse magnetic nanoparticles can be found in ref 11 (Sun et al.). Iron tri(acetylacetonate) (2 mmol), 1,2-tetradecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol), and benzyl ether (20 mL) were mixed and stirred magnetically under a constant flow of nitrogen. The mixture was heated gradually to 100 °C and kept at 100 °C for an overall period of 45 min. Depending on the heating rate used from room temperature to 100 °C, different particle sizes were obtained. The mixture was then heated to 200 °C for a period of 40 min and kept at 200 °C for 2 h. Finally, under a blanket of nitrogen, the mixture was heated to reflux (∼300 °C) for 1 h. The black-colored mixture was cooled to room temperature by removing the heat source. Methanol (∼40 mL) was added to the mixture, and a black material was precipitated and separated via centrifugation (7000 rpm, 10 min). The black precipitate was dissolved in hexane and centrifuged once more (7000 rpm, 10 min) to remove any undispersed residue. The nanoparticles were stored in hexane. Before undergoing any ligand exchange reaction, all particles were precipitated through the addition of a large excess of ethanol or methanol, separated magnetically by means of a powerful electromagnet, and dried in the oven at 80 °C for 20 min to evaporate all alcohol. (1b) Preparation of Oleic Acid-Coated Magnetic Nanoparticles Using Seeded Growth. The seeded-growth procedure followed for the preparation of monodisperse magnetic nanoparticles was as described by Sun et al.11 Briefly, the same mixture prepared as described in step 1a was seeded by the addition of 80 mg of 11-nm particles. The mixture was heated gradually to 200 °C over a period of 1.5 h and kept at 200 °C for 1 h. At that point, under a blanket of nitrogen, the mixture was heated to reflux (∼300 °C) for 0.5 h. The remainder of the procedure was identical to that described in step 1a. (2) Preparation of Ricinoleic Acid (RA)-Coated Magnetic Nanoparticles. Oleic acid- and oleyl amine-coated nanoparticles (120 mg) prepared as described in step 1a or 1b were dispersed in 1,2-dichlorobenzene (15 mL), to which 1 g of ricinoleic acid was added. The mixture was then stirred at 80 °C for ∼24 h. The particles were subsequently precipitated by the addition of methanol
Lattuada and Hatton (∼40 mL) and 2 mL of water and recovered by means of an electromagnet. The particles were then dried in a vacuum oven for 20 min at 80 °C. (3) Preparation of Polylactic-Coated Magnetic Nanoparticles. Ricinoleic acid- and oleyl amine-coated nanoparticles (120 mg) prepared as described in step 2 were dispersed in THF (15 mL), to which 1.44 g of L-lactide and 0.1 mL of tin(II) ethylhexanoate were added. The mixture was then stirred at 60 °C for ∼72h. The particles were then precipitated by the addition of hexane (∼40 mL) and recovered by means of an electromagnet. After three redispersions in THF followed by reprecipitations with hexane, the particles were dried in a vacuum oven for 1 h at 80 °C for TGA and FTIR analysis. (4a) Preparation of Citric Acid (CA)-Coated Magnetic Nanoparticles. Oleic acid- and oleyl amine-coated nanoparticles (120 mg) prepared as described in steps 1a or 1b were dispersed in a 50/50 mixture of 1,2-dichlorobenzene and N,N′-dimethylformamide (15 mL of total volume), to which 0.1 g of citric acid was added. The mixture was then stirred at 100 °C for ∼24 h. The particles were subsequently precipitated by the addition of ethyl ether (∼40 mL) and recovered by means of an electromagnet. The particles were redispersed in acetone and reprecipitated by means of an electromagnet 3 to 4 times to remove all traces of free citric acid. The particles were then dried in a vacuum oven for 20 min at 80 °C. (4b) Preparation of CA/PEG-Coated Magnetic Nanoparticles. CA-coated nanoparticles (50 mg) prepared as described in step 4a were dispersed in 10 mL of water solution at pH 9. To this solution, 0.1 g of NH2-PEG (10 kDa) was added, followed by 0.05 g of EDC and 0.06 g of NHS. The mixture was stirred for 24 h and subsequently dialyzed against an aqueous solution at pH 9 for 2 days using a cellulose membrane with pores having a cut-off size of 50 kDa. (5) Preparation of Citric Acid/2-Bromo-2-methylpropionic Acid (CA/BMPA)-Coated Magnetic Nanoparticles. The procedure used here is identical to that described in step 4a, with the exception that, a mixture of 0.025 g of citric acid and 0.4 g of 2-bromo-2methylpropionic acid was simultaneously used for the ligand exchange reaction. (6) Preparation of Galactaric Acid (GA)-Coated Magnetic Nanoparticles. Oleic acid- and oleyl amine-coated nanoparticles (120 mg) prepared as described in step 1a or 1b were dispersed in a 50/50 mixture of 1,2-dichlorobenzene and dimethylsulfoxide (15 mL of total volume), to which 0.1 g of galactaric acid was added. The mixture was then stirred at 100 °C for ∼24 h. The particles were subsequently precipitated by the addition of ethyl ether (∼40 mL) and recovered by means of an electromagnet. The particles were redispersed in acetone and reprecipitated by means of an electromagnet 3 to 4 times to remove all traces of free galactaric acid. The particles were then dried in a vacuum oven for 20 min at 80 °C. (7) Preparation of Magnetic Nanoparticle Macroinitiators (RA/ BMPB, GA/BMPB, CA-BMPA/BMPB). Nanoparticles (120 mg), which had previously undergone a ligand exchange reaction according to one of the procedures described in step 2, 5, or 6, were redispersed either in dichloromethane (as in the case of RA-coated particles) or in N,N′-dimethylformamide (15 mL) and then vigorously stirred. To this mixture, 1 mL of triethylamine was added, followed by the dropwise addition of 0.5 mL of 2-bromo-2-methylpropionyl bromide. The acylation reaction was allowed to proceed for 3-4 h at room temperature. Particles were then precipitated through the addition of acetone and recovered by means of an electromagnet. The particles were subsequently redispersed in acetone and reprecipitated by means of an electromagnet 3 to 4 times to remove all traces of reagents, followed by 20 min of drying in a vacuum oven at 80 °C. (8) Preparation of Poly(methacrylic acid) (PAA/PMAA)-Coated Nanoparticles. RA/BMPB magnetic nanoparticles (120 mg) were dissolved in 7.5 mL of DCB and 7.5 mL of DMF inside a three-neck, round-bottom flask. To this mixture, 5 mL of TMSA (TMSMA)and 0.11 g of CuBr were added. After 30 min of nitrogen bubbling under vigorous magnetic stirring, 0.4 mL of HMTETA was injected and the temperature was raised to 90 °C. The reaction was allowed to proceed for 24 h. Finally, the particles were precipitated by adding ∼30 mL of a hexane/diethyl ether mixture and then recovered by means of an electromagnet. The nanoparticles were thoroughly
Monodisperse Magnetic Nanoparticle Functionalization washed using methanol and then precipitated several times by means of an electromagnet. Subsequently, the nanoparticles were dispersed in methanol and sonicated in a sonicating bath to deprotect the polymer and give PAA- or PMAA-coated particles. Finally, the particles were redispersed in 15 mL of water with the aid of 0.1 mL of PMDETA. A similar procedure was also used with GA/BMPB magnetic nanoparticles, with the difference being that these particles were dissolved in 15 mL of dimethyl sulfoxide and the polymerization reaction was carried out at room temperature. All subsequent steps were identical. (9) Preparation of Poly(hydroxyethylmethacrylate) (PHEMA)Coated Nanoparticles. RA/BMPB nanoparticles (120 mg) were dissolved in 10 mL of DCB inside a three-neck, round-bottom flask. To this mixture, the desired amount of HEMA and 0.11 g of CuBr were added. After 30 min of nitrogen bubbling under vigorous magnetic stirring, 0.4 mL of HMTETA was injected and then the solution was kept at room temperature. The reaction was allowed to proceed for 24 h. Finally, the particles were precipitated by adding ∼30 mL of hexane and recovered by means of an electromagnet. The nanoparticles were then thoroughly washed using methanol, precipitated several times by means of hexane, and recovered using an electromagnet. (10) Preparation of PHEMA/SA-Coated Nanoparticles. All PHEMA-coated magnetic nanoparticles prepared as described in step 10 were dissolved in 15 mL of pyridine. Subsequently, an amount of succinic anhydride that was twice as large as the amount of HEMA monomer used in step 9 was added to the mixture. This mixture was then stirred for 48 h. Subsequently, the particles were precipitated using a mixture of acetone and diethyl ether and recovered by means of an electromagnet. The particles were then washed several times using acetone and recovered by means of an electromagnet. Finally, the particles were dissolved in water (15 mL), where 0.1 mL of PMDETA was added to speed up the dissolution in water, followed by dialysis of the particle solution against a pH 9 water solution. (11) Preparation of Poly(dimethylaminoethyl methacrylate) (PMDETA)-Coated Nanoparticles. GA/BMPB magnetic nanoparticle macroinitiators (120 mg) were dissolved in 15 mL of DMSO inside a three-neck, round-bottom flask. To this mixture, 5 mL of DMAEMA and 0.11 g of CuBr were added. After 30 min of nitrogen bubbling under vigorous magnetic stirring, 0.4 mL of HMTETA was injected. The reaction was allowed to proceed for at least 24 h at room temperature. Finally, the particles were precipitated by adding ∼30 mL of diethyl ether and recovered by means of an electromagnet. The nanoparticles were then thoroughly washed using acetone and precipitated several times by means of an electromagnet. Finally, the particles were redispersed in 15 mL of phosphate buffer at pH 5 (20 mmol solution in water) and sonicated for 0.5 h. (12) Preparation of Polystyrene Sulfonate (PSSNa)-Coated Nanoparticles. CA-BMPA/BMPB magnetic nanoparticles (120 mg) were dissolved in 15 mL of a 3/1 (v/v) mixture of water and methanol. To this dispersion, 4.125 g of sodium styrene sulfonate and 0.11 g of CuBr were added. After 30 min of nitrogen bubbling under vigorous magnetic stirring, 0.4 mL of HMTETA was injected. The reaction was then allowed to proceed for 24 h at room temperature. Finally, the particles were precipitated by adding ∼30 mL of acetone and recovered by means of an electromagnet. The nanoparticles were then thoroughly washed using acetone and precipitated several times. Finally, the particles were redispersed in 15 mL of water. (13) Preparation of Poly(N-isopropylacrylamide) (PNIPAm)Coated Nanoparticles. The procedure is identical to that described in step 12, except that 2.5 g of N-isopropylacrylamide was used as the monomer. Magnetic Filtration of Polymer-Coated Nanoparticles. A 10cm-long column with a diameter of ∼0.5 cm was packed with type 430 fine-grade stainless steel wool (40-66 µm diameter), supplied by S. G. Frantz Co., Inc. (Trenton, NJ), up to a 16% packing fraction. The column was placed between the two poles of an electromagnet. The outlet of the column was connected via silicon rubber tubes to a peristaltic pump. Magnetic filtration was performed by letting the aqueous solution containing the polymer-coated nanoparticles pass
Langmuir, Vol. 23, No. 4, 2007 2161 through the column with the electromagnet on (which is capable of creating a magnetic flux of 1.3 T). In the presence of a magnetic field, the isolated magnetic particles are not trapped by the column, while clusters containing several particles are retained by the column. Additional water was added until the liquid coming out of the column was clear. At this point, with the electromagnet turned off, additional water was passed through the column to remove the retained clusters. Zeta Potential Measurements. All zeta potential measurements were performed using a Brookhaven ZetaPALS zeta potential analyzer (Brookhaven Instruments Corporation). The particles were diluted to 0.1-0.5 wt % of magnetite. Measurements were taken in buffer solutions at given pH. 10 mM phosphate buffer was used to tune the pH from 5 to 9, while 10 mM acetate buffer was used to set the pH to 4. The Smoluchowski equation was used to extract the zeta potential ζ from the measured particle electrophoretic mobility µe: η ζ ) µe
(1)
where η and are the viscosity and the dielectric constant of the dispersion medium, respectively. The reported zeta potential values are an average over six measurements, each of which was obtained over 20 electrode cycles. The Smoluchowski equation is only applicable when the particle size is much larger than the Debye length of the electrical double layer in the solution, a condition that was always satisfied in all measurements. Dynamic Light Scattering (DLS) Measurements. DLS experiments were performed using a Brookhaven BI-200SM light scattering system (Brookhaven Instruments Corporation) at a measurement angle of 90°. The autocorrelation function was fit with various fitting algorithms (cumulant method and CONTIN) to extract the diffusion coefficient, and the Stokes-Einstein equation was used to convert the diffusion coefficient to the hydrodynamic diameter. Intensityaveraged size distributions were converted to number-averaged size distributions for further analysis. Samples were measured for 5 min, and measurements were repeated three times to verify the reproducibility of the results. Transmission Electron Microscopy (TEM) Measurements. TEM experiments were performed on a JEOL 200CX (200 kV) microscope. All samples were prepared by evaporating dilute suspensions on a carbon-coated film. Fourier Transform Infrared (FTIR) Spectroscopy Experiments. FTIR experiments were performed on a Perkin Elmer 2000 FTIR instrument. Spectra were recorded in the wavenumber interval between 4000 and 400 nm-1. All samples were ground and mixed with KBr and then pressed to form pellets. The background spectrum was subtracted from the sample spectrum. Each spectrum was acquired twice, and an average of the two measurements was taken and analyzed. Thermogravimetric Analysis (TGA). TGA measurements were performed on a Perkin Elmer TGA7 instrument. All measurements were taken under a constant flow of nitrogen of 50 mL/min. The temperature was increased at a pace of 15 °C/min, starting from room temperature up to 960 °C, and then held constant at maximum temperature for 45 min. All samples were dried in a vacuum oven at 60-80 °C prior to each TGA measurement to remove most of the water or solvent. The initial weight of each sample was between 4 and 20 mg. All reported TGA curves were normalized with respect to the weight at 100 °C to make sure that only the solid fraction was measured. All nanocrystals that underwent TGA analysis had an average size of 11 nm unless specified otherwise.
Results and Discussion Preparation of Monodisperse Magnetite Particles (and Seeded-Growth Procedure). We prepared OA/OAm-coated particles following the procedure proposed by Sun et al.,11 using 1,2-tetradecane diol instead of the much more expensive 1,2hexadecane diol. The rate at which the solution was heated, particularly in the first phase of the reaction, when the temperature
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Figure 1. TEM micrographs of freshly prepared OA/OAm-coated magnetic nanoparticles: (a) average diameter 6.5 nm (low heating rate) and (b) average diameter 11 nm (high heating rate).
Figure 2. Measured particle size distribution and corresponding log-normal fitting of freshly prepared OA/OAm-coated magnetic nanoparticles with an average size of 6.5 nm (low heating rate), 11 nm (high heating rate), and 13.5 nm (first seeded growth particles starting from 11 nm particles).
was raised from room temperature to 100 °C, was a crucial factor in determining the average size of the particles. At the slower heating rate of ∼2.5 °C/min, the size distribution was significantly smaller than that obtained when the rate was ∼5 °C/min, as is evident from the TEM micrographs shown in Figure 1. The particle size distributions appear to be quite narrow in both cases, and the particle shape is reasonably spherical. The size distributions have been quantified through a visual analysis of ∼250 particles in the TEM micrographs, with the rather narrow distributions shown in Figure 2 confirming that the organic route is an effective method for the preparation of monodisperse magnetic nanoparticles. Both distributions can be fit by a lognormal function with mean values of 6.41 and 11.08, and polydispersity indexes of 0.238 and 0.152 for the slow and fast heating cases, respectively. DLS measurements of freshly prepared particles diluted in hexane provided an average hydrodynamic diameter of particles in solution equal to ∼10 and 15 nm for the 6.5 and 11 nm particles, respectively, with the differences between the two sets of measurements being due to the thicknesses of the OA/OAm layers on the particle surfaces. Thus, the OA/OAm nanoparticles are mostly isolated and not clustered in organic solution. The first seeded growth proceeded smoothly, with an average size increase from 11 to 13.5 nm and a small polydispersity change from 0.152 to 0.162, as shown in Figure 2. Seed-grown
particles increased in size as expected (about 2 to 2.5 nm for each growth), but this occurred at the expense of deterioration in the sphericity of the particles, which took on more polyhedral-like shapes (cubic, prismatic, etc.), and of a progressively broader size distribution. This is consistent with the concept that larger seeds tend to grow faster than smaller ones as a consequence of Ostwald ripening during synthesis. To obtain a narrower distribution of large particles, it might be necessary to apply a size sorting technique (for example, magnetic chromatography). Only nanoparticles obtained through up to one seeded growth were employed in the subsequent functionalization studies. Nanoparticle Functionalization. The general procedure followed for the surface functionalization of OA/OAm-coated magnetite nanoparticles is illustrated schematically in Figure 3. It can be observed that the first step in our functionalization procedure always consists of a ligand exchange reaction, followed either by ring opening polymerization (ROP) or by another reaction step aimed at preparing the particle for a surface-initiated radical polymerization (ATRP). All steps are discussed individually in the subsequent paragraphs. Ligand Exchange Reactions. The first step in our functionalization procedure consisted of a ligand exchange (or surface exchange) reaction, which is a flexible route for attaching to a particle molecules that have a high affinity for its surface. The Whitesides group45 was the first to show that molecules carrying thiol groups can adsorb very efficiently onto gold surfaces, with the thiol moiety binding to the gold surface. It has since been shown that it is relatively easy to replace one molecule with another by using an excess of the new molecule while providing enough heat and/or sonication.46 The same principle applies to iron oxide surfaces and molecules containing carboxyl acid groups, the interactions between which have been investigated by means of IR spectroscopy.47,48 It was suggested that the carboxyl group interacts with a trivalent iron atom located on the surface by forming a bridge with the two oxygen atoms. (45) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111 (1), 321-335. (46) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121 (30), 7081-7089. (47) Rocchicciolideltcheff, C.; Franck, R.; Cabuil, V.; Massart, R. J. Chem. Res., Synop. 1987, 126-126. (48) Lesnikovich, A. I.; Shunkevich, T. M.; Naumenko, V. N.; Vorobyova, S. A.; Baykov, M. V. J. Magn. Magn. Mater. 1990, 85 (1-3), 14-16.
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Figure 3. Scheme for the magnetic nanoparticles functionalization procedure described in this work. Steps 1A and 1B: ligand exchange reactions. Step 2: acylation of hydroxyl groups to prepare ATRP surface initiators. Step 3A: surface-initiated ring opening polymerization of L-lactide. Step 3B: surface-initiated ATRP. Step 4: deprotection or additional reaction after polymerization. Step 5: grafting of endfunctionalized PEG chains onto the nanoparticle surface using amidation chemistry.
Bourlinos et al.49 have exploited this method to prepare both water-soluble particles (with betaines on their surfaces) and particles with phenyl groups by starting from oleic acid-coated nanoparticles. The ligand exchange reaction shown in steps 1A and 1B in Figure 3 proved to be a very effective method for independently changing both the particle reactivity, by attaching molecules bearing functional groups to the particle surface, and the particle solubility, a feature which proved to be of great advantage in our preparation of water-soluble, polymer-coated nanoparticles. The grafting of a previously prepared polymer chain onto a single particle surface after its synthesis is very difficult, since there is a high probability that a macromolecule can bind to the surface of two particles at the same time and, consequently, bridge them. It is, therefore, preferable to adopt a surface-initiated polymerization method, where polymer chains are grown directly from the nanoparticle surface. ATRP is the most commonly used method of the many available polymerization methods that enable a surface-initiated growth;34 the ability to use solvents of varying polarity is a particularly important factor in our decision to use this method because it enormously increases the variety of monomers that can be polymerized. The selection of ligands that allow for the preparation of particles with the desired solubility was, therefore, a priority in this work. We have investigated four different ligands that proved to be particularly flexible: ricinoleic acid (RA), citric acid (CA), galactaric acid (GA), and 2-bromo-2-methyl propionic acid (BMPA), whose structures are shown in Figure 4. The first three
possess hydroxyl groups which allow for enhanced reactivity relative to oleic acid, while the fourth is a well-known initiator for ATRP.50 Hydroxyl groups are effective as functional moieties mainly because they can undergo acylation reactions under mild conditions, and in contrast to other more reactive groups, such as aminos, they do not have a high affinity for iron oxide surfaces. The hydroxyl groups on the nanoparticle surface can also be used to initiate ROP, a useful method for the preparation of certain biodegradable polymers that cannot be prepared by other methods and are potentially very useful in biological and biomedical applications.39,40 The presence of hydroxyl groups does not permit the direct utilization of these molecules during the synthesis of magnetite nanoparticles, which proceeds via the reduction of iron acetylacetonate by a diol. Under these conditions, hydroxyl groups oxidize to ketones, rendering them chemically nonreactive. In addition, not many molecules are known to provide as good a stabilization of newly formed particles as does oleic acid at the high temperatures reached during the synthesis, which is one of the reasons that oleic acid is used for this step. Ricinoleic acid was found to be as efficient a ligand as oleic acid for providing particle stability against aggregation in nonpolar (or mildly polar) organic solvents. It has essentially the same structure as oleic acid, with the only difference being the presence of a hydroxyl group in position 12, as shown in Figure 4. The ligand exchange reaction to replace oleic acid with ricinoleic acid could not be performed at temperatures higher than 80 °C, since, at higher temperatures, ricinoleic acid seemed to undergo
(49) Bourlinos, A. B.; Bakandritsos, A.; Georgakilas, V.; Petridis, D. Chem. Mater. 2002, 14 (8), 3226.
(50) Coessens, V.; Pintauer, T.; Matyjaszewski, K. Prog. Polym. Sci. 2001, 26 (3), 337-377.
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Figure 4. Chemical structures and corresponding abbreviations of ligands and chemicals used for functionalization reactions used in this work.
some undesired reaction resulting in loss of reactivity of the hydroxyl group. Particles coated with ricinoleic acid are soluble in mildly polar solvents like THF, DCB, DCM, chloroform, and toluene. Galactaric acid has two carboxyl groups, which make it a very efficient ligand for complexing with the nanoparticle surfaces, and four hydroxyl groups that can be further functionalized to impart the desired properties to these surfaces. Good particle stability in polar solvents, such as DMF and DMSO, was observed when oleic acid was replaced by galactaric acid via ligand exchange reactions. This selection of a ligand that provides good particle solubility in DMSO is particularly beneficial to ATRP since it has been demonstrated that many polar monomers can be polymerized in DMSO under very mild conditions.51 Citric acid was selected to provide stability in water and wateralcohol mixtures, in which many polyelectrolytes can be successfully polymerized. Citric acid has three carboxyl groups and is, therefore, a very powerful ligand. Even though the presence of one hydroxyl group on the citric acid molecule, in principle, allows for further functionalization, it turns out that it does not provide sufficient reactivity toward acylation reactions, and poor results were obtained. Therefore, we used it in combination with BMPA, a very efficient ATRP initiator. BMPA alone was found to be incapable of providing sufficient stabilization to particles in any solvent. The combination of CA and BMPA in a 1:5 molar ratio was beneficial for both particle stability and reactivity, since the stability in aqueous media provided by citric acid was combined with the ATRP initiator functionality provided by BMPA. It is not well-known how the ligand exchange reaction proceeds, and only recently have a limited number of studies on the mechanism of adsorption and replacement of thiols on gold surfaces been carried out.52-54 It is generally believed that ligand exchange reactions follow second-order kinetics,52 but more complex mechanisms have been proposed.53 To our knowledge, no kinetics studies on ligand exchange on iron oxide surfaces have been published. Our experiments seem to indicate that (51) Monge, S.; Darcos, V.; Haddleton, D. M. J.Polym. Sci., Part A: Polym. Chem. 2004, 42 (24), 6299-6308. (52) Guo, R.; Song, Y.; Wang, G. L.; Murray, R. W. J. Am. Chem. Soc. 2005, 127 (8), 2752-2757. (53) Woehrle, G. H.; Brown, L. O.; Hutchison, J. E. J. Am. Chem. Soc. 2005, 127 (7), 2172-2183. (54) Ionita, P.; Caragheorgheopol, A.; Gilbert, B. C.; Chechik, V. Langmuir 2004, 20 (26), 11536-11544.
Figure 5. FTIR spectra of freshly prepared magnetic nanoparticles and of nanoparticles subjected to ligand exchange reactions with three different ligands followed by acylation.
when ligands are displaced from the surface, they do not come off by themselves, and they also remove some iron atoms from the surface. In fact, we found that, after completion of a ligand exchange reaction, when particles were recovered through precipitation, the supernatant solution was slightly red in color, indicative of the presence of iron atoms. This observation was particularly evident when CA and GA were used as ligands, probably because the ligand exchange reaction was performed at a higher temperature (100 °C) compared to the case in which RA was used, and also because both GA and CA have multiple carboxyl acid groups and, thus, stronger binding capabilities. As shown in step 2 of Figure 3, when ATRP was used to grow polymer brushes, the hydroxyl groups were functionalized by acylation using BMPB, a very reactive acyl bromide, which, in the presence of triethylamine, can form ester bonds and transform hydroxyl groups into ester moieties bearing one of the most efficient ATRP initiators currently known. Verification that the selected ligands efficiently replaced OA/ OAm was provided by the FTIR spectra of the nanoparticles that had undergone ligand exchange reactions followed by acylation. The spectra of freshly prepared nanoparticles are shown in Figure 5, together with those of the nanoparticles with RA/BMPB, with CA, and with GA/BMPB. As a first observation, it can be noted that the IR spectrum of freshly prepared nanoparticles does not have any clear “signature” that indicates which molecules are on the surface. It can certainly be claimed that alkyl chains are present, as well carboxyl acid groups, given the broad and not very well defined absorption regions at wavenumbers between
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Figure 6. Zeta potential as a function of pH for various watersoluble magnetic nanoparticles. All nanocrystals have an average size of 11 nm.
Figure 7. Z-weighted hydrodynamic diameter as a function of pH for various water-soluble magnetic nanoparticles. All nanocrystals have an average size of 11 nm.
1400 and 1800 cm-1, around 2900 cm-1, and around 3400 cm-1.55 The first region is probably due to vibrations of both the alkyl groups and the carboxyl (and amino) groups as they interact with the particle surface, the second region is due to CH2 stretching, and the third absorption peak is due to OH and NH2 vibrations. Particles that underwent ligand exchange with ricinoleic acid followed by reaction with BMPB are not significantly different from freshly prepared nanoparticles, except for a few better defined peaks in the wavenumber range between 1200 and 1700 cm-1, which could be attributed to the C-Br stretching frequency (in the 1200 cm-1 range) as well as to the ester groups (around 1600 cm-1). On the other hand, the spectra of the citric acidcoated particles show enormous differences, particularly with strong peaks at 1640 cm-1 and at 1370 cm-1, which are typical of carboxyl groups, and at 1050 cm-1, which is probably due to the hydroxyl group of citric acid. In the case of GA/BMPBcoated particles, a large number of peaks at 1180 cm-1 and at 1035 cm-1 can be observed, which are attributed to the various hydroxyl groups, as well as a strong peak around 1200 cm-1, which is due to both C-Br stretching and ester bands.55 Additional carboxyl group absorption bands can be observed at higher frequencies, in the range between 2400 and 2700 cm-1. A more quantitative analysis of the results of ligand exchange reactions based both on chemical analysis of the elements and on TGA measurements is reported in the Supporting Information. Organic Solvent-Soluble Nanoparticles. ROP of Polylactic Acid. The flexibility of our strategy in functionalizing nanoparticles has been demonstrated using ring opening polymerization (ROP) to grow polylactic acid (PLA) successfully from RAcoated nanoparticles in THF, as pictured schematically in step 3A of Figure 3. The hydroxyl group of ricinoleic acid opens up the ring of lactide in the presence of stannous dioctanoate as the catalyst and initiates the polymerization, according to the recipe by Choi and Langer.39 It was shown, using TGA and FTIR measurements, that particles coated with polylactic acid had been prepared. The TGA curves of both RA- and PLA-coated particles are reported in the Supporting Information. The profiles for RAand PLA-coated nanoparticles are different: PLA-coated particles lost more mass (∼45% compared to ∼35% for RA-coated particles), with a substantial loss of mass between 200 and 350 °C that is attributed to thermal degradation of the PLA backbone.56 In addition, the FTIR spectrum of the PLA-coated nanoparticles (again, reported in the Supporting Information) shows that welldefined ester absorption bands are present in PLA-coated particles at wavenumbers close to 1750 cm-1 and between 1100 and 1200
cm-1, indicative of the strong presence of ester groups on the nanoparticle surface. The same procedure can be easily extended to the preparation of PLGA-coated nanoparticles, which could be very useful in magnetic drug release devices, since PLGA is biocompatible and has been approved by the FDA for use. Furthermore, PLGA chains are terminated by hydroxyl groups, which allow for additional chemical modification. Water-Soluble Nanoparticles. Citric Acid-Coated Nanoparticles. A facile preparation procedure for water-soluble monodisperse magnetic nanoparticles is through a ligand exchange reaction with pure CA (step 1B in Figure 3). The nanoparticles so obtained are extremely stable and highly negatively charged at neutral to basic pH values, as demonstrated by their zeta potential, which equals -75 mV when measured in a pH 9 phosphate buffer solution. Figure 6 shows that the zeta potential is almost constant for pH g 4, while destabilization is observed for pH 3. The particle hydrodynamic diameters shown in Figure 7 follow a similar trend, being equal to ∼15 nm for pH g 4, but significantly higher at lower pH. As an example of the versatility and the type of reaction that can be performed on these magnetic nanoparticles stabilized by carboxyl acid groups, we have used EDC amidation chemistry to bind amino-mono-end-functionalized poly(ethylene glycol) (PEG) chains onto the CA-coated nanoparticle surfaces (step 5 in Figure 3). The success of the reaction can be seen by comparing the size of the nanoparticles before and after the reaction: the average hydrodynamic diameter before the reaction was ∼15 nm, and it grew to ∼25 nm after the reaction. This is consistent with the presence of a ∼5-nm PEG shell around the particles, since the estimated radius of gyration in water of a single 10K molecular weight PEG chain is about 4 to 5 nm. Such “stealth” nanoparticles could have relevant biological applications, given that PEG chains are capable of rendering molecules and small particles almost invisible to the immune system. Furthermore, NH2-PEG chains were chosen as the prototypes of large biocompatible molecules that can be effectively bound on the nanoparticles surfaces using EDC-mediated chemistry. ATRP Functionalization of Nanoparticles. Most of the experiments we have performed to prepare polymer-coated nanoparticles used ATRP because of its versatility and the facility with which an ATRP initiator can be anchored onto a surface. This crucial step of our nanoparticles functionalization procedure is sketched in Figure 3 as step 3B. In addition, ATRP is a living free-radical polymerization, which, in principle, enables controlled and monodisperse polymer chains to be obtained and offers the opportunity to grow block copolymers, if desired.50 In the following, several strategies for the preparation of water-soluble polymer-coated magnetic nanoparticles through ATRP are
(55) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: New York, 1991. (56) Nalbandi, A. Iran. Polym. J. 2001, 10 (6), 371-376.
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Figure 9. TGA curves for three polymer-coated magnetic nanoparticles: PAA nanoparticles polymerized from RA/BMPB, PAA nanoparticles polymerized from GA/BMPB, and PMAA nanoparticles polymerized from RA/BMPB. Figure 8. Chemical structures and corresponding abbreviations of monomers used in this work.
discussed, together with the corresponding nanoparticle properties. All monomers used for the ATRP polymerization, together with their protected or further functionalized forms, are shown in Figure 8. P(M)AA-Coated Particles. The main drawback to ATRP is that it does not permit direct polymerization of certain monomers, such as those that have primary amino groups or unprotected carboxyl groups, because these moieties interfere in the activation of the catalyst needed to perform ATRP and form intermediate species which deactivate the catalysis. While polymerization of certain monomers carrying amino groups can be achieved by using sufficiently strong multidentate amino ligands, monomers carrying carboxyl groups usually require protection through suitable protecting groups.50 After the polymerization is accomplished, a deprotection reaction is required to recover the carboxyl groups, as depicted in step 4 of Figure 3. Since one of the most interesting monomers to be polymerized from magnetic nanoparticle surfaces for our applications was (meth)acrylic acid, it was necessary to select a suitable protecting group. This was not an easy choice. In fact, the most common protective group for (meth)acrylic acid is the tert-butyl group,50 which can be easily removed either through the addition of acids (usually hydrochloric acid or trifluoroacetic acid) or through reaction with trimethyl silyl iodide,57 which displaces the tert-butyl group. Unfortunately, while polymerization of tert-butyl (meth)acrylate runs very smoothly, both of these deprotection reactions lead to the severe corrosion of magnetite nanoparticles. While it is wellknown that magnetite is sensitive to the presence of strong acids (and trifluoroacetic acid is strong enough to damage the particles severely), it is not understood why deprotection via trimethyl silyl iodide leads to almost complete dissolution of magnetite particles as well. The solution to this problem entailed the direct polymerization of TMSA (TMSMA) monomers, whose deprotection is straightforward and simply requires the use of an excess of methanol.58 This choice had some drawbacks too. TMSA and TMSMA monomers are much more difficult to polymerize than are tertbutyl (meth)acrylate monomers and give much lower yields. We increased the yield by operating the polymerization reaction at an experimentally determined optimum temperature of 90 °C. The quality of the polymer-coated particles, as determined by their water solubility, decreases above and below this temperature; (57) Southard, G. E.; Woo, J. T. K.; Massingill, J. L. Prog. Org. Coat. 2004, 49 (2), 160-164. (58) Zhang, J. X.; Varshney, S. K. Des. Monomers Polym. 2002, 5 (1), 79-95.
low temperatures lead to insufficient polymerization (and increasing the polymerization time does not seem to improve the result), while higher temperatures probably lead to a competition between faster polymerization and progressive loss of the initiators attached to the particle surface. In fact, 90 °C is close to the temperature used for the ligand exchange reaction. The choice of the solvent (DCB, toluene, or a mixture of DCB and DMF) did not improve the polymerization yield significantly, although a mixture of DCB and DMF seemed to be optimal since the polymer is soluble in DMF but not in dichlorobenzene or toluene, while the RA/BMPB nanoparticles are not very soluble in DMF but are soluble in DCB. Using the above procedure, we prepared PAA particles of different sizes. All water-soluble, polymer-coated particles were analyzed by TGA, FTIR, DLS, and zeta potential measurements. The TGA profile of polyacrylic acid-coated particles obtained from deprotection of TMSA, shown in Figure 9, indicates a very limited mass loss (∼15%) distributed uniformly over the entire temperature range. It can be observed that decomposition of PAA occurs in two steps: the carboxyl groups first condense with each other to give anhydrides, which occurs between 200 and 300 °C, followed by breakage of the PAA backbone, which begins at temperatures higher than 300 °C.59 In addition, another mass loss is observed around 650-750 °C, similar to the one noted for the RA/BMPB particles (see Figure 1s in the Supporting Information) but to a smaller degree, suggesting that many of the RA/BMPB initiators had disappeared, as can be judged by comparing the TGA profile of PAA-coated particles with that of the RA/BMPB particles shown in Figure 1s of the Supporting Information. Despite being coated only by a small amount of polymer, these particles exhibited excellent solubility in water, as demonstrated by both their size (measured through DLS) and their zeta potential. The zeta potential is negative at large pH values (Figure 6) and becomes progressively smaller as the pH decreases, reaching almost zero at pH 4. This is consistent with the typical values for polyacrylic acid, which has a pK value of ∼4.5. In addition, the effect of pH on particle size, shown in Figure 7, indicates that PAA-coated particles have an average size of less than 15 nm (number average ∼8 nm), which is consistent with the presence of monodisperse particles in solution. DLS provides a rigorous test for the presence (or otherwise) of aggregates because the intensity scattered by an object is proportional to the square of its volume, which implies that even if only a small fraction of particles were clustered, the measured average size would be significantly larger than the size of an (59) Ho, B. C.; Lee, Y. D.; Chin, W. K. J. Polym. Sci., Part A: Polym. Chem. 1992, 30 (11), 2389-2397.
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individual isolated particle (a small fraction of such clustered material was removed prior to these experiments by filtration). Figure 7 shows that the average size increases with decreasing pH, until complete particle instability is reached at pH values less than 4. However, if the aggregation is not permitted to proceed for too long, the aggregation process is essentially completely reversible and monodisperse particles can be recovered by adjusting the pH back to high values. The polymer layer provides a steric barrier at short separation distances so that a pair of aggregating particles is in a secondary energy minimum, and the particles are relatively easily separated and do not aggregate irreversibly. Similar considerations apply to PMAA-coated nanoparticles, with the exception that TGA analysis, shown in Figure 9, indicates a larger amount of polymer on their surfaces (∼ 20%). Also, their stability and pH-responsive behavior closely follow those of PAA-coated nanoparticles. It is to be noted that the amount of polymer on the nanoparticle surface could be tuned by changing the conditions under which polymerization was carried out. For instance, we polymerized TMSA at room temperature in DMSO starting with GA/BMBPcoated nanoparticles, to create water-soluble PAA-coated nanoparticles, with a pH response similar to those obtained for particle coatings formed at higher temperatures. The TGA analysis reported in Figure 9, however, showed that the wt % of polymer on the particle surfaces was substantially higher than that present on the RA/BMPB particles (>30% versus 15%). This is just one example of the beneficial effects of running ATRP polymerizations in DMSO, as already mentioned in the literature.51 PHEMA/SA-Coated Nanoparticles. A second strategy for the preparation of polymer-coated magnetic nanoparticles with carboxyl acid groups on their surfaces is to first polymerize HEMA, followed by reacting the product polymer with SA in pyridine. This strategy has already been utilized by others to prepare controlled-block copolymers with carboxyl acid moieties60 and can be considered as a valid alternative to the polymerization of monomers with protective groups, when acidic groups cannot be directly polymerized. In the case of nanoparticles, we began with RA/BMPB macroinitiators and carried out the HEMA polymerization in DCB, the solvent in which the polymerization is extremely fast even at room temperature. The role played by the solvent is unclear, but certainly crucial, since the same reaction takes place much more slowly in THF, even at temperatures as high as 60 °C. The other attractive feature of this strategy is the high polymerization yield, with the possibility of tuning the amount of polymer on the surface of the nanoparticles by simply changing the amount of monomer to be polymerized. The subsequent step of the process is the reaction with SA in pyridine, which also functions as a catalyst. This step converts PHEMA into a polyelectrolyte, according to the structure shown in Figure 8. The TGA analysis of PHEMA/SA-coated nanoparticles, which is shown in the Supporting Information, indicates that the weight fraction of polymer can be tuned from 52 to 81% through a 4-fold increase in the amount of monomer used. The pH-responsive behavior of PHEMA-SA particles, shown in Figures 6 and 7, is not too different from that of PAA particles, except that their estimated zeta potentials (and consequently their surface charges) are smaller than those of the PAA-coated particles, and the zeta potential is almost independent of pH down to a pH of 5. Conversely, instability is observed at the isoelectric point near a pH of 4. The size increases measurably as the polymer layers coating the particles increase in thickness, from 20 nm for 0.5 mL of HEMA particles to 45 nm for 1 mL (60) Bories-Azeau, X.; Merian, T.; Weaver, J. V. M.; Armes, S. P.; van den Haak, H. J. W. Macromolecules 2004, 37 (24), 8903-8910.
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of HEMA, and up to 85 nm for 2 mL of HEMA at pH 10. The very low zeta potential values can be ascribed to the type of model (eq 1) used in its estimation, which is not suitable for particles with charges distributed over a very thick layer of polymer chains, since the assumption behind Smoluchowski’s equation is that the charges are concentrated on the particle surface. Indeed, the estimated zeta potential values decrease as the thickness of the polymer layer increases for PHEMA/SA-coated particles, while their stability improves. In Figures 6 and 7, it can be observed clearly that the particle sizes for PHEMA/SAcoated nanoparticles decrease significantly with decreasing pH, due to compression and progressive loss of charge of the polymer layer. Conversely, the zeta potential is almost unchanged, because the two effects (reduction of the charge and compression of the polymer layer) balance each other. PDMAEMA-Coated Nanoparticles. To polymerize other types of water-soluble monomers using ATRP, it was necessary to change solvents. In particular, we were interested in preparing water-soluble, positively charged nanoparticles, for which we used the monomer DMAEMA. Negligible yields of almost insoluble particles were obtained in nonpolar organic solvents. With GA/BMPB particles in DMSO at room temperature, however, positively charged magnetic nanoparticles could be obtained readily. A significant increase in the polymerization yield was observed upon increasing the polymerization time, and particles with different layer thicknesses could be prepared, as confirmed by TGA analysis. IR spectra reported in the Supporting Information confirm the presence of PDMAEMA on the nanoparticles. The zeta potential of the particles polymerized for 1 day, reported in Figure 6, takes on more positive values as the pH decreases, which is consistent with the tertiary amino groups of the polymer acquiring progressively more charge. The size evolution shown in Figure 7 is, however, contrary to expectations. With decreasing pH down to a pH of 4, the average hydrodynamic diameter increases substantially, indicating that, as the polymer layer acquires more and more charge, the polymer brush tends to swell. Below a pH of 4, the size decreases with decreasing pH, possibly because the increase in ionic strength screens the charges on the polyelectrolyte brush. These mechanisms are not sufficient to explain the observed peculiar size evolution, however, and aggregation seems to be involved to some extent. Indeed, the intensity of light scattered decreased consistently with decreasing pH. Since scattered intensity is proportional to the mass squared of particles present in the solution, if some clusters were present at high pH values (which is likely because a size equal to 60 nm at pH 9 is not consistent with a monodisperse particle population), they break up progressively as the particles acquire a charge. One of the features of PDMAEMA is its solubility in water at high pH values at room temperature, while at high temperatures a progressive insolubility develops. At pH 9, an increase in solution temperature up to 45 °C leads to a progressive increase in particle (aggregate) size. This aggregation could not be reversed by just lowering the temperature, but it could be reversed by lowering the pH. This indicates that PDMAEMA is not capable of providing good stability when uncharged. PSSNa and PNIPAm-Coated Nanoparticles. Finally, we also developed a strategy to prepare particles coated by polymers that require an aqueous solution as the polymerization medium, in which we used a mixture of ligands during the ligand exchange reaction, with CA as the water-soluble ligand and BMPA as the ATRP initiator. Reaction with BMPB allowed for further functionalization of at least some of the hydroxyl groups present
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on the citric acid molecules to produce ATRP initiators that were soluble in water and water-methanol mixtures. We exploited this feature to prepare PSSNa-coated nanoparticles,61 obtaining highly soluble and almost pH-insensitive, water-soluble nanoparticles. The zeta potential of these particles is almost independent of pH, as expected, because of the strong sulfonic acid group, while the particle size tends to decrease slightly as the pH values decrease. Once again, this is caused by the screening of the charges on the polymer chains when there is a sufficient ionic strength increase. The rather large size of these particles can be explained in terms of a highly swollen polymer brush, which is also the reason why the estimated zeta potential values are rather low. As the last example of a polymer that can be grown from the nanoparticle surface in aqueous media, we have prepared magnetic nanoparticles coated with PNIPAm, which is a very well-known polymer that is hydrophilic at temperatures lower than 32 °C, defined as the lower critical solubility temperature (LCST).62 Above this threshold, PNIPAm becomes hydrophobic and tends to precipitate out of solution. Despite the thickness of this polymer layer, the temperature-responsive behavior of these nanoparticles is not really evident due to the presence of charged groups on the surface that are provided by citric acid moieties. For pH values less than 3, when almost all charges are screened, the particles flocculate and are unstable, indicating that the layer of PNIPAm grown from the nanoparticle surface is, by itself, not sufficient to stabilize nanoparticles in water.
Conclusions We have proposed a flexible methodology for the preparation of various types of monodisperse, water-soluble magnetic nanoparticles coated by different polymer brushes. The method takes advantage of a recently developed synthesis for OA/OAmcoated, monodisperse magnetite nanoparticles, which allows the size of the magnetite core to be tuned through a seeded-growth process. First, we found that particle sizes in the range of 6 to 11 nm can be tuned without using the seeded-growth process by simply varying the heating rate of the solution containing the nucleating particles. Subsequently, once the particles with the (61) Chen, X. Y.; Randall, D. P.; Perruchot, C.; Watts, J. F.; Patten, T. E.; von Werne, T.; Armes, S. P. J. Colloid Interface Sci. 2003, 257 (1), 56-64. (62) Schild, H. G. Prog. Polym. Sci. 1992, 17 (2), 163-249.
Lattuada and Hatton
desired size are obtained, they are first subjected to a ligand exchange reaction, which serves the purpose of replacing the (weakly reactive) oleic acid groups initially present on the nanoparticle surface with more reactive and useful moieties. We have tested three different compounds that allow us to tailor the particle solubility in solvents with different polarities. When RA is used as the ligand, particles that are soluble in nonpolar and weakly polar solvents are obtained that either can undergo surface-initiated ROP, through which polylactic acid brushes were grown on the particles, or could be further functionalized with BMPB to give ATRP macroinitiators. In this case, by using TMSA or TMSMA, either poly(acrylic acid) or poly(methacrylic acid) brushes could be grown on the particle surfaces. In addition, these ATRP initiators can be used to polymerize HEMA in DCB with very high yields, and PHEMA brushes can be further reacted with SA to give a poly acid on the particle surface. All of these particles are water-soluble and rather monodisperse, and a decrease in the pH of the solution induces flocculation, which can be reversed by increasing the pH again. With galactaric acid as the ligand, and with it being further acylated with BMPB, particles coated with PDMAEMA can be produced by ATRP in DMSO at room temperature; these particles are positively charged and pH-responsive. Finally, when a mixture of CA and BMPA was used as the ligand, water-soluble (and water-methanol-soluble) particles could be obtained, on which both pH-nonresponsive PSSNa and PNIPAm brushes could be grown through ATRP. To show the possible applications of magnetic nanoparticles with carboxyl groups on their surfaces, and how other molecules can be covalently bound to these surfaces, we have attached amino-functionalized PEG chains to CA-coated nanoparticles using EDC-promoted amidation chemistry, resulting in the preparation of “stealth particles”. Acknowledgment. The assistance of Szymon Leszczynski in performing some of the experiments is gratefully acknowledged. This work was supported by the DuPont-MIT Alliance. Supporting Information Available: Chemical analysis of the elements and TGA profiles for ligand-capped nanoparticles and TGA profiles and FTIR spectra for polymer-coated nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org. LA062092X
