NANO LETTERS
Cooperative Assembly of Magnetic Nanoparticles and Block Copolypeptides in Aqueous Media
2003 Vol. 3, No. 11 1489-1493
Larken E. Euliss,†,‡,§ Stephanie G. Grancharov,†,‡,| Stephen O’Brien,| Timothy J. Deming,§ Galen D. Stucky,§ C. B. Murray,‡ and G. A. Held*,‡ IBM TJ Watson Research Center, PO Box 218, Yorktown Heights, New York 10598, Departments of Chemistry and Biochemistry and Materials, UniVersity of California, Santa Barbara, California 93106, and Department of Applied Physics, Columbia UniVersity, New York, New York 10027 Received July 3, 2003
ABSTRACT We demonstrate that highly crystalline, monodisperse maghemite (γ-Fe2O3) nanoparticles, synthesized in organic solvents, can be effectively transferred into an aqueous medium using an ammonium salt and stabilized at neutral pH. The nanoparticles remain monodisperse, as characterized by TEM and XRD, as well as superparamagnetic, as determined by SQUID magnetometry. When the aqueous maghemite is combined with the block copolypeptide poly(EG2-lys)100-b-poly(asp)30, the nanoparticles assemble into uniform clusters comprised of approximately 20 nanoparticles, resulting in a water soluble block copolypeptide−nanoparticle composite structure.
As methods of controlling the crystalline structure, magnetic properties, and interparticle ordering of magnetic nanoparticles improve,1 so also does the interest in utilizing them as building blocks for new devices2,3 and composite materials.4 In particular, there has been much interest in utilizing magnetic nanoparticles in biological applications such as magnetic resonance imaging contrast enhancement5 and drug delivery.6,7 In the case of drug delivery, magnetic fields can be utilized to direct the particles (and thus the drug) to specific locations within the body. However, before these particles can act as drug delivery agents, they must be stabilized in a physiological environment. Block copolypeptides provide one promising means of stabilizing drug carriers.8 These block copolypeptides can self-assemble into micelles that are capable of trapping drugs within a hydrophobic core, while their hydrophilic exteriors can be designed so as to be stable within a wide range of physiological environments. In this paper we demonstrate the use of block copolypeptides to stabilize clusters of magnetic nanoparticles in an aqueous environment. We first demonstrate that highly monodisperse maghemite (γ-Fe2O3) nanoparticles, which are * Corresponding author. e-mail:
[email protected]. phone: 914-9452609. † L.E.E. and S.G.G. contributed equally to this work and should be regarded as joint first authors. ‡ IBM TJ Watson Research Center. § University of California, Santa Barbara. | Columbia University. 10.1021/nl034472y CCC: $25.00 Published on Web 09/06/2003
© 2003 American Chemical Society
synthesized in organic media, can be transferred to an aqueous solution with no loss of structural or magnetic properties. We then find that combining these particles with the block copolypeptide poly(EG2-lys)100-b-poly(asp)309 results in the formation of magnetic nanoparticle clusters. As both ends of this block copolypeptide are hydrophilic, we believe that the aspartic acid residues bind electrostatically with the surface of the nanoparticles, followed by the formation of micelle-like assemblies, each containing approximately 20 nanoparticles in its core.10 The resulting micellar assemblies will have a magnetic moment 20 times that of a single nanoparticle, and have the high physiological stability provided by a poly(EG2-lys) shell. Both of these attributes increase the viability of using such particles for effective drug delivery. Experimental Section. Synthesis of Water Soluble Maghemite Nanocrystals. In preparing monodisperse iron nanocrystals, known methods11 were followed. Specifically, 28 mL of octyl ether was added to 3 mL of oleic acid. The mixture was heated to 100 °C under an inert atmosphere for 30 min. To this mixture, 0.8 mL of Fe(CO)5 was added, the temperature was then raised to 300 °C, and the solution stirred for 1 h. The mixture was then allowed to cool to room temperature. The solution was centrifuged and the precipitate removed. The remaining supernatant solution was then collected, and ethanol was added to produce reversible flocculations of the nanocrystals. The solution was centrifuged again, and the precipitate saved and subse-
Figure 1. Molecular structure of block copolypeptide poly(EG2lys)100-b-poly(asp)30 ) poly(Ne-2[2-(2-methoxyethoxy)ethoxy]acetylL-lysine)100-b-poly(L-aspartic acid, sodium salt)30.
quently suspended in hexanes to produce a suspension of 6 nm iron particles. Exposing this suspension to air for 1 week resulted in the complete oxidation of the iron nanoparticles, the result being a dispersion of highly crystalline 6 nm maghemite (γ-Fe2O3) nanoparticles. The concentration of oleic acid stabilized maghemite particles in this suspension was approximately 12 mg/mL. The precipitation of the nanoparticles present in a 5 mL aliquot of this suspension was accomplished by the addition of ethanol, followed by centrifugation. The supernatant was then discarded, and the precipitate was resuspended in 15 mL of 1.0 M tetramethylammonium hydroxide (TMAOH).12,13 Deionized water was added to bring the total volume to 100 mL. The addition of 0.2 gm sodium citrate followed, and adding 0.1 M HCl dropwise to the solution brought the resulting mixture to a pH of 6.5. During this reduction in pH, a precipitate appeared and was removed by filtration (Pall Acrodisc 0.8 µm Versapor filter no. 4459). Synthesis of Block Copolypeptide Poly(EG2-lys)100-b-poly(asp)30 ) poly(Ne-2[2-(2-methoxyethoxy)ethoxy]acetyl-Llysine)100-b-poly(L-aspartic acid, sodium salt)30. The synthesis of this block copolypeptide was accomplished using previously reported methods.14,15 Stepwise polymerization of the monomer (Ne-2[2-(2-methoxyethoxy)ethoxy]acetyl-L-lysine)N-carboxyanhydride, followed by the monomer b-benzyl(L-aspartic acid)-N-carboxyanhydride using 2,2′-bipyridylNi-1,5-cyclooctadiene initiator gave the protected polymer, which was then deprotected using equimolar amounts of trifluoroacetic acid and 33% HBr in acetic acid. The structure of the resulting polymer is shown in Figure 1. Impurities and byproducts were removed by dialysis against water. The protected block copolypeptide was analyzed using gel permeation chromatography in 0.1 M LiBr/dimethylformamide (DMF) at 60 °C to determine the molecular mass. Polymer block composition was analyzed by 1H NMR of the deprotected copolypeptide in trifluoroacetic acid (TFA)-d and found to be within 10% of the predicted ratios. The polymer was then dissolved in water to produce a 2-mg/mL solution (pH 7). Preparation of Poly(EG2-lys)100-b-poly(asp)30/Maghemite Nanocrystal Composite. Deionized water (1 mL) was added to 1 mL of the aqueous suspension of maghemite nanocrystals described above (pH 6.5) and stirred. To this mixture, 50 µL of the 2-mg/mL solution of poly(EG2-lys)100-b-poly(asp)30 was added. The mixture was allowed to stand 1490
overnight and then filtered (Pall Acrodisc 0.8 µm Versapor filter no. 4459). Sample Preparation and Characterization. Samples studied with transmission electron microscopy (TEM) were prepared by placing one drop of a nanoparticle dispersion (hexanes or aqueous based) onto the amorphous carbon substrate of a TEM grid and then drying the grid under vacuum at 100 °C for several hours. TEM images were taken with a Philips CM-12 120 keV TEM. Samples studied with X-ray diffraction (XRD) were prepared by drying the nanoparticle dispersion onto a glass substrate under vacuum at 100 °C. Those substrates onto which an aqueous dispersion had been dried were then lightly washed with water; this had the effect of dissolving excess TMAOH salt, while the nanoparticles remained bound to the substrate. XRD data were collected using Co KR radiation (λ ) 1.78892 Å) with a Seimens D-500 diffractometer. Magnetometry measurements were taken with a Quantum Design MPMS SQUID magnetometer. Samples were prepared by drying the nanoparticle dispersion in a glass sample tube under vacuum at 100 °C. Results and Discussion. Water Soluble 6 nm Maghemite Nanocrystals. Previous studies have shown that the decomposition of Fe(CO)5 in hydrocarbon solvents provides a simple and effective method to prepare highly monodisperse nanocrystals of magnetic iron.11 We find that simple exposure of these particles dispersed in hexanes to the atmosphere results in their complete oxidation into monodisperse, highly crystalline 6 nm maghemite nanoparticles. Further, we have demonstrated that nanoparticles prepared by this method can be transferred into an aqueous medium with the objective of biocompatibility. TEM and X-ray diffraction (XRD) were used to obtain information about the maghemite nanoparticles dispersed in both water and hexanes, as well as about their interaction with poly(EG2-lys)100-b-poly(asp)30. TEM images show that the maghemite nanoparticles are crystalline and monodisperse. In addition, these images indicate that no degradation takes place upon transfer from organic to aqueous solution (Figure 2a,b). Figure 2a shows maghemite particles taken from a dispersion in hexanes in which they were stabilized against aggregation with a layer of oleic acid. The subsequent change of ligand from oleic acid in hexanes to TMAOH in water results in continued stabilization of the particles in solution; there continues to be very little aggregation (Figure 2b). Figure 3 shows XRD data taken from the maghemite nanoparticles following removal from both aqueous and organic solutions. The locations of expected, indexed Bragg reflections of the maghemite structure are also shown. It is clear from this figure that particles dispersed in both hexanes and water exhibit a maghemite crystal structure. Further, XRD confirms the high degree of crystallinity of the particles; Debye-Scherrer calculations16 predict a diameter of 5.6 nm for particles taken from both the hexanes and water dispersions, in good agreement with the average diameter of 5.7 nm obtained from TEM (again, the same value for particles from both dispersions). Thus, both XRD and TEM characterizations indicate that neither particle crystallinity nor monodispersity was Nano Lett., Vol. 3, No. 11, 2003
Figure 4. Low-field susceptibility as a function of temperature measured after zero-field cooling (0) and cooling in a 10 Oe field (9) measured for 6 nm maghemite nanoparticles deposited from dispersions in (a) hexanes and (b) water. The field-cooled and zerofield-cooled scans for a given sample overlap at temperatures where the sample is in thermal equilibrium and diverge at the blocking temperature.
Figure 2. TEM micrographs of maghemite nanoparticles deposited from dispersions in (a) hexanes and (b) water. Inset in (a) is a selected area electron diffraction pattern.
Figure 3. X-ray diffraction patterns of maghemite nanoparticles deposited onto glass substrates from dispersions in hexanes (blue) and water (red). Intensity is plotted as a function of wavevector ((4π/λ)sinθ). Background has been subtracted from the red spectrum and the intensity of the blue spectrum has been offset by 200. Expected positions of Bragg peaks corresponding to the maghemite structure are shown as black lines and labeled.
compromised by the exchange of ligand, transfer to water, and subsequent neutralization of the nanoparticle dispersion with HCl. The use of TMAOH to stabilize magnetite nanoparticles synthesized under aqueous conditions is well known.12,17 However, the synthesis of metallic nanoparticles under organic conditions followed by transfer into aqueous solutions has thus far been limited to nonmagnetic particles.18 Nano Lett., Vol. 3, No. 11, 2003
As magnetic nanoparticles synthesized in an organic environment often have better crystallinity and monodispersity than those synthesized in an aqueous environment,11,18 a method of transferring organically synthesized particles to aqueous conditions is clearly desirable. After precipitating maghemite nanoparticles out of a hexanes solution (by the addition of ethanol followed by centrifugation), we find that these particles readily dissolve in a 1:6 1.0 M TMAOH/ deionized water mixture. As these particles are insoluble in water alone, we believe that the oleic acid ligands are displaced by the ammonium salt, TMAOH. Specifically, when placed in water with TMAOH, the positively charged surface of the each maghemite nanocrystal associates electrostatically with the negative hydroxide ions, which are surrounded, in turn, by positive tetramethylammonium cations, the result being an electrostatic double layer of ions that stabilizes the particles in aqueous solution.12,19,20 We find that the nanoparticles precipitated from hexanes will not dissolve in NaOH (aq) or tetramethylammonium bromide (aq), confirming the need for both tetramethylammonium cations and hydroxide anions in producing an aqueous maghemite nanoparticle dispersion. Neutralization of this dispersion by the addition of HCl removes some of the hydroxide ions from the maghemite surface, resulting in a small amount of precipitation. This is consistent with earlier reports on colloidal nickel ferrite spheres.20 However, we find that the addition of sodium citrate ensures that the nanoparticles remain suspended in solution, presumably because the citrate anions are able to associate with the nanoparticle surface and, thus, replace the neutralized hydroxide anions. SQUID magnetrometry measurements demonstrate that the magnetic properties of the maghemite nanoparticles are not significantly affected by the change from an organic to an aqueous solvent. In Figures 4a and b are shown field-cooled/ zero-field-cooled measurements of the magnetic susceptibility of nanoparticles that had been dispersed in organic and aqueous media, respectively. The superparamagnetic blocking temperature is identified as the temperature below which the field-cooled and zero-field-cooled susceptibilities diverge.21 From Figure 4, we conclude that the blocking temperatures of the nanoparticles dispersed in hexanes and 1491
Figure 5. Magnetic moment as a function of applied magnetic field measured for 6 nm maghemite nanoparticles deposited from dispersions in (a) hexanes and (b) water. Data were collected at 300 K and (inset to a) 10 K. Solid lines are best fits of the data to the Langevin paramagnetic function (see text).
water are 75 and 60 K, respectively. This relatively small shift in blocking temperature suggests a modest reduction in the magnetic volume of the nanoparticles as a result of their transfer to an aqueous environment. In addition, we find that measurements of magnetic moment as a function of applied field taken at 300 K show little change between nanoparticles taken from hexanes and aqueous dispersions (Figures 5a and b). Neither measurement shows evidence of hysteresis, and thus both may be modeled by the Langevin paramagnetic function, which is known to describe the magnetization of superparamagnetic particles:22 M(x) ) Nµ(coth x - (1/x)), where x ) µH/kBT, N is the number of nanoparticles, µ is the magnetic moment of an individual nanoparticle, H is the applied field, kB is Boltzmann’s constant, and T is the absolute temperature. Fits of the data in Figure 5a and b to this function (shown as solid lines in the figures) yield values of µ equal to 2.57 × 10-17 emu and 1.56 × 10-17 emu, respectively. Assuming a saturation magnetization of 363 G for the maghemite nanoparticles,23 these results correspond to magnetic particles with diameters of 6.3 and 5.3 nm for particles taken from hexanes and aqueous solution, respectively. Again, the data are consistent with a modest reduction in the effective magnetic radius as a result of the transfer to an aqueous environment. Note that at 10 K (inset, Figure 5a) the nanoparticles exhibit the hysteretic behavior expected for particles below their blocking temperature. Block Copolypeptide-Maghemite Nanoparticle Assembly. When either the block copolypeptide poly(EG2-lys)100-b-poly(asp)30 or the homopolymer poly(asp)30 is added to an aqueous dispersion of 6 nm maghemite nanoparticles, the nanoparticles assemble into soluble clusters. Transmission electron micrographs of these clusters, taken following their removal by drying from aqueous dispersions of nanoparticle/ poly(EG2-lys)100-b-poly(asp)30 (Figures 6a and 6b) and nanoparticle/ homopolymer poly(asp)30 (Figure 6c) illustrate the structure of these assemblies. We note that no similar clustering phenomenon is observed in the presence of the homopolymer poly(EG2-lys)100. This suggests that the assembly of the nanoparticles is facilitated by favorable electrostatic interactions between the negatively charged carboxylic acid side groups of the poly(asp)30 and the positively charged surfaces of the maghemite nanocrystals. 1492
Figure 6. TEM micrographs of clusters of maghemite nanoparticles deposited from dispersions in water to which (a), (b) poly(EG2lys)100-b-poly(asp)30, and (c) poly(asp)30 have been added. Note that the homopolymer-nanoparticle mixture was not filtered prior to preparation of the TEM grid.
Each nanoparticle may associate with more than one strand of the polyaspartic acid and, likewise, each strand of polyaspartic acid may bind to more than one nanoparticle, the result being a bridging aggregation leading to controlled organization. For the case of the nanoparticle/homopolymer poly(asp)30 mixture, these clusters typically have dimensions of order 100 nm and, as such, are only observed if the nanoparticlehomopolymer mixture is not filtered. Many, but not all of these clusters, appear to be comprised of an aggregation of smaller nanoparticle clusters (as in the upper cluster shown in Figure 6c). The fact that these clusters are soluble suggests that they may be stabilized by portions of the poly(asp)30 not directly attached to nanoparticles. When the block copolypeptide poly(EG2-lys)100-b-poly(asp)30 is introduced to an aqueous dispersion of maghemite nanoparticles, the nanoparticles cooperatively assemble into clusters that are significantly more uniform than those observed when the homopolymer poly(asp)30 is added to the dispersion (Figure 6). Again, we assume that favorable electrostatic interactions between the aspartic acid residues and the nanoparticles occur. However, in the block copolypeptide case, the aggregates that form are limited in size by the poly(EG2-lys)100 block. As both ends of poly(EG2lys)100-b-poly(asp)30 are hydrophilic, one would expect the block copolypeptide to be soluble in water at physiological pH. However, it is entirely plausible that the association of the polyaspartic acid segment of the copolypeptide with a maghemite nanoparticle would shift the critical point of micellar formation,24 the result being micelles with cores comprised of clusters of maghemite nanopartcles electrostatically bound to polyaspartic acid and outer shells comprised of the highly hydrophilic poly(EG2-lys) segments of the block copolypeptide. A schematic illustration of such a micellar assembly is shown in Figure 7. In summary, we have investigated the transfer of magnetic nanoparticles synthesized in organic media into an aqueous Nano Lett., Vol. 3, No. 11, 2003
manuscript, and Peter Allen for the artful rendering of Figure 7. L.E.E. and S.G.G. acknowledge financial support from a NSF IGERT Fellowship and a NSF GK-12 Teaching Fellowship, respectively. Part of this work was carried out at the UCSB-MRL Central Facilities, supported by the NSF under Award No. DMR-0080034. This work was also supported in part by the MRSEC Program of the NSF at Columbia University under award number DMR-0213574. References
Figure 7. Schematic illustration of the proposed structure of the micellar assemblies formed following the addition of the block copolypeptide poly(EG2-lys)100-b-poly(asp)30 to an aqueous dispersion of 6 nm maghemite nanoparticles. The shell of the micelle is comprised of poly(EG2-lys)100, which is known to form a stable R-helical conformation in water,15 while the core is comprised of maghemite nanoparticles electrostatically bound to the poly(asp)30 segments of the block copolypeptide.
environment, finding that this transfer can be accomplished with no loss in the crystallinity, monodispersity, or magnetic properties of the particles. In addition, we have studied the cooperative assembly of magnetic nanoparticles in the presence of amino acid based polymers, demonstrating that electrostatic interactions between block copolypeptides and nanoparticles can control the organization of these components. The addition of polyaspartic acid initiates the aggregation of maghemite nanoparticles into clusters, without the formation of a precipitate. The addition of the block copolypeptide poly(EG2-lys)100-b-poly(asp)30 initiates a more controlled organization of nanocrystals, possibly through the formation of micelles with cores comprised of magnetic nanoparticles electrostatically bound to the polyaspartic acid end of the block copolypeptide. In this case, the poly(EG2lys) ends of the copolymers would form the micelle shell and, as such, both stabilize the clusters and control their size. By altering the composition of the block copolypeptide, it should be possible to control both the size and stability of the resulting dispersed nanoparticle clusters, thereby greatly expanding the potential applications and usefulness of these cooperatively assembled nanocomposites. Acknowledgment. The authors thank Franz X. Redl, Jacek C. Ostrowski, and Jennifer N. Cha for helpful discussions, Geoffrey Grinstein for his critical reading of the
Nano Lett., Vol. 3, No. 11, 2003
(1) Hyeon, T. Chem. Commun. 2003, 927-934. (2) Anders, S.; Sun, S.; Murray, C. B.; Rettner, C. T.; Best, M. E.; Thomson, T.; Albrecht, M.; Thiele, J. U.; Fullerton, E. E.; Terris, B. D. Microelectron. Eng. 2002, 61-2, 569-575. (3) Sun, S. H.; Anders, S.; Hamann, H. F.; Thiele, J. U.; Baglin, J. E. E.; Thomson, T.; Fullerton, E. E.; Murray, C. B.; Terris, B. D. J. Am. Chem. Soc. 2002, 124, 2884-2885. (4) Strable, E.; Bulte, J. W. M.; Moskowitz, B.; Vivekanandan, K.; Allen, M.; Douglas, T. Chem. Mater. 2001, 13, 2201-2209. (5) Babes, L.; Denizot, B.; Tanguy, G.; Le Jeune, J. J.; Jallet, P. J. Colloid Interface Sci. 1999, 212, 474-482. (6) Scientific and Clinical Applications of Magnetic Carriers; Hafeli, U., Schutt, W., Teller, J., Zborowski, M., Eds.; Plenum Press: New York, 1997. (7) Safarik, I.; Safarikova, M. Monatsh. Chem. 2002, 133, 737-759. (8) Deming, T. J. AdV. Drug DeliV. ReV. 2002, 54, 1145-1155. (9) EG2-lys is a notation for lysine modified by the addition of two ethylene glycol groups at the terminus of the lysine residue. Specifically, EG2 ) Ne-2[2-(2-methoxyethoxy)ethoxy]acetyl-L-lysine. (10) The type of assembly described here-a core of nanoparticles bound to one functional end of many block copolypeptide molecules and surrounded by a shell comprised of the hydrophilic other ends of these molecules-is, strictly speaking, not a micelle. Nonetheless, for simplicity we shall refer to it as such throughout this paper. (11) Hyeon, T.; Lee, S. S.; Park, J.; Chung, Y.; Bin Na, H. J. Am. Chem. Soc. 2001, 123, 12798-12801. (12) Berger, P.; Adelman, N. B.; Beckman, K. J.; Campbell, D. J.; Ellis, A. B.; Lisensky, G. C. J. Chem. Edu. 1999, 76, 943-948. (13) Correa-Duarte, M. A.; Giersig, M.; Kotov, N. A.; Liz-Marzan, L. M. Langmuir 1998, 14, 6430-6435. (14) Deming, T. J. Nature 1997, 390, 386-389. (15) Yu, M.; Nowak, A. P.; Deming, T. J.; Pochan, D. J. J. Am. Chem. Soc. 1999, 121, 12210-12211. (16) Cullity, B. D. Elements of X-ray Diffraction, 2nd ed.; AddisonWesley: Reading, MA, 1978. (17) Dresco, P. A.; Zaitsev, V. S.; Gambino, R. J.; Chu, B. Langmuir 1999, 15, 1945-1951. (18) Gittins, D. I.; Caruso, F. Angew. Chem., Int. Ed. 2001, 40, 30013004. (19) Hunter, R. J. Foundations of Colloid Science; Clarendon: Oxford, 1987. (20) Plaza, R. C.; de Vicente, J.; Gomez-Lopera, S.; Delgado, A. V. J. Colloid Interface Sci. 2001, 242, 306-313. (21) Dormann, J. L.; Fiorani, D.; Tronc, E. AdV. Chem. Phys. 1997, 98, 283-494. (22) Bean, C. P.; Livingston, J. D. J. Appl. Phys. 1959, 30, 120S-129S. (23) Berkowitz, A. E.; Schuele, W. J.; Flanders, P. J. J. Appl. Phys. 1968, 39, 1261-1263. (24) Firouzi, A.; Kumar, D.; Bull, L. M.; Besier, T.; Sieger, P.; Huo, Q.; Walker, S. A.; Zasadzinski, J. A.; Glinka, C.; Nicol, J.; Margolese, D.; Stucky, G. D.; Chmelka, B. F. Science 1995, 267, 1138-1143.
NL034472Y
1493
