Cooperative Assembly of Magnetic Nanoparticles and Block Copolypeptides in Aqueous Media

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 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)30 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* 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. NANO LETTERS 2003 Vol. 3, No. 11 1489-1493 10.1021/nl034472y CCC: $25.00 © 2003 American Chemical Society Published on Web 09/06/2003 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