Article pubs.acs.org/Langmuir
In Situ Functionalization and PEO Coating of Iron Oxide Nanocrystals Using Seeded Emulsion Polymerization Hauke Kloust,† Christian Schmidtke,† Artur Feld,† Theo Schotten,‡ Robin Eggers,† Ursula E. A. Fittschen,§ Florian Schulz,† Elmar Pöselt,† Johannes Ostermann,† Neus G. Bastús,†,⊥ and Horst Weller*,†,‡,∥ †
Institute of Physical Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany Center for Applied Nanotechnology, Grindelallee 117, 20146 Hamburg, Germany § Institute of Inorganic and Applied Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany ∥ The Hamburg Center for Ultrafast Imaging, University of Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany ‡
S Supporting Information *
ABSTRACT: Herein we demonstrate that seeded emulsion polymerization is a powerful tool to produce multiply functionalized PEO coated iron oxide nanocrystals. Advantageously, by simple addition of functional surfactants, functional monomers, or functional polymerizable linkerssolely or in combinations thereofduring the seeded emulsion polymerization process, a broad range of in situ functionalized polymercoated iron oxide nanocrystals were obtained. This was demonstrated by purposeful modulation of the zeta potential of encapsulated iron oxide nanocrystals and conjugation of a dyestuff. Successful functionalization was unequivocally proven by TXRF. Furthermore, the spatial position of the functional groups can be controlled by choosing the appropriate spacers. In conclusion, this methodology is highly amenable for combinatorial strategies and will spur rapid expedited synthesis and purposeful optimization of a broad scope of nanocrystals.
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INTRODUCTION Inorganic nanocrystals are well established in biological and medical applications.1 Especially the superparamagnetic iron oxide nanocrystals are of great interest and find application as contrast agents for T2- or T2*-weighted and recently even T1weighted magnetic resonance imaging (MRI).2,3 The primary challenge for biological or medical applications is rendering initially hydrophobic nanocrystals water-soluble and biocompatible, without compromising their characteristic properties. To this end, numerous efficient phase transfer protocols, mainly based on polymeric ligand systems, have been developed to insulate the nanocrystals by a polymeric shell from all kinds of biological relevant media and to minimize unspecific protein and tissue interactions.4−11 A second challenge is the purposeful functionalization of the nanocrystals for biological targeting, e.g., adjusting the zeta potential or attaching affinity molecules or molecular probes like peptides, carbohydrates, antibodies, or nucleotides to the particles for targeting e.g. cell receptors. Conventionally, functionalization of the stabilizing ligands on the surface of the nanocrystals can be done before or after the phase transfer from organic media into water.7,12,13 Both concepts afford additional preparation steps and sophisticated and often tedious coupling strategies. Herein we propose a facile, broad scope in situ functionalization methodology by addition of functional surfactants, functional monomers, or functional polymerizable linkers in a seeded emulsion polymerization process, hence generating the © 2013 American Chemical Society
protective polymer shell and a controlled functionalization simultaneously in a single step. Emulsion polymerization is a well-established method to produce uniform polymeric particles. Therein, a mixture of monomers, carrying polymerizable group, and surfactants, having an amphiphilic character, form an oil-in-water emulsion. By means of a radical initiator, the monomer-filled micelles get converted into polymeric particles. Advantageously, this technique allows the in situ functionalization of the polymeric particles by different strategies. First, functional monomers can be copolymerized during the radical polymerization.14,15 Second, functional surfactants may be used, e.g., so-called surfmers. Surfmers bear functional groups which are prone to participate in the polymerization reaction and hence get covalently bound to the surface.16−18 In our previous publication, we succeeded in the use of emulsion polymerization for the encapsulation of hydrophobic iron oxide nanocrystals. Therein, the nanocrystals or clusters thereof acted as seeds, stabilized by polysorbate-80 as a surfactant.19 In brief, oleic acid-coated iron oxide nanocrystals, as obtained from the synthesis (Scheme 1, I) in high boiling organic solvents were dispersed in THF. After addition of a surfactant, Received: February 25, 2013 Revised: March 26, 2013 Published: March 26, 2013 4915
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Scheme 1. Encapsulation of Nanocrystals by Seeded Emulsion Polymerization: (I) Hydrophobic Nanocrystal; (II) Nanocrystal in Micelle after Phase Transfer; (III) Nanocrystal Encapsulated in Polystyrene; (A) without Additional Functional Monomer; (B) with Copolymerized Functional Monomer; (C) with Copolymerized Functional Linker; (D) with Functional Surfmer
residue was dissolved in 10 mL of water and washed with 6 mL of diethyl ether. The aqueous phase was lyophilized yielding a light yellow oil (458 mg, 87%). 1H NMR (500 MHz, CDCl3, 25 °C): δ (ppm) = 1.43 (s, 9H, tert-butyl), 3.21−3.32 (m, 6H, 3× −N−CH2), 3.40−3.72 (m, 4H per PEO unit), 3.98 (d, 2H, −O−CH2−CH=, 3J = 5.68 Hz), 4.59−4.64 (m, 1H, −CH2−CHOH−CH2−), 5.17−5.29 (m, 2H, −CHCH2), 5.80−5.95 (m, 1H, −CHCH2). 816 mg of Boc-amino-PEO10-N-(allyl 2-hydroxypropyl ether) was dissolved in 50 mL of THF/HCl (1 M). After stirring the solution for 23 h at room temperature 15 mL of an aqueous 5 M NaOH solution was added dropwise. The solution was neutralized with hydrochloric acid and evaporated to dryness. The resulting yellowish solid was triturated with CHCl3 (15, 10, 10, and 10 mL) until the CHCl3 phase was colorless. The solid residue was dried in vacuum and redissolved in 50 mL of acetone. The solution was diluted with 120 mL of nhexane and stored at −10 °C for 1 h. The precipitate was isolated by centrifugation at −10 °C (6654g, 10 min) and dissolved in 30 mL of water. The aqueous solution was lyophilized. A brownish oil was obtained (yield: 277 mg, 39%). 1H NMR (500 MHz, CDCl3, 25 °C): δ (ppm) = 3.15−3.25 (m, 6H, 3× N−CH2), 3.42−3.87 (m, 4H per PEO unit), 3.98 (d, 2H −O−CH2−CH=, 3J = 5.56 Hz), 4.58−4.63 (m, 1H, −CH2−CHOH−CH2−), 5.16−5.26 (m, 2H, −CHCH2), 5.81−5.90 (m, 1H, −CHCH2). Synthesis of Biotin-PEO60-N-(allyl 2-hydroxypropyl ether). 53 μL (4.8 × 10−4 mol) of allyl 2,3-epoxypropyl ether, 18 mg (1.6 × 10−4 mol) of DABCO, and 475 mg (1.58 × 10−4 mol) of biotinPEO60-amine were dissolved in 20 mL of dry CHCl3. After stirring for 22.5 h at 60 °C all volatiles were evaporated. The residue was redissolved in 15 mL of acetone, diluted with 30 mL of n-hexane, and stored at −10 °C for 15 h. The precipitate was isolated by centrifugation at −10 °C (6654g, 10 min), redissolved in 20 mL of water, and lyophilized. A colorless solid was obtained (yield: 480 mg, 97%). 1H NMR (500 MHz, CDCl3, 25 °C): δ (ppm) = 0.83−2.75 (m, 8H, −CO−CH2−CH2−CH2−CH2−), 2.89−2.94 (m, 4H, −S−CH2−, −CO−N−CH2), 3.13−3.26 (m, 4H, 2× N−CH2), 3.53−3.76 (m, 4H per PEO unit), 3.98 (d, 2H, −O−CH2−CH=, 3J = 5.68 Hz), 4.30− 4.33 (m, 1H, biotin-tert-C−H), 4.49−4.52 (m, 1H, biotin-tert-C−H), 4.61−4.67 (m, 1H −CH2−CHOH−CH2−), 5.17−5.26 (m, 2H −CHCH2), 5.80−5.89 (m, 1H −CHCH2). Synthesis of Nanocrystals. Iron oxide nanocrystals were synthesized according to Yu et al.20 Gold nanocrystals were synthesized according to Bastús et al.21
the solution was injected into water. The surfactant forms a micelle (II) enclosing the nanocrystal. In the hydrophobic region of the micelle a seeded emulsion polymerization occurs that forms a polystyrene shell around the nanocrystal (III), eventually providing the product (A). Additionally, we showed that these products are suitable to act as contrast agent for T2or T2*-weighted MRI. In this paper we will report the seeded emulsion polymerization for in situ functionalization of iron oxide nanocrystals. Compared to previously described ligand systhems, our method offers major advantages with respect to the availability of precursors, stability of the nanoconstructs, convenience as well as flexibility of the process, and chemical diversity of the nanocrystal surface. The method is highly amenable for parallelization and will spur the surface modification of nanocrystals in a quasi-combinatorial fashion.
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EXPERIMENTAL SECTION
Materials. Rose Bengal, 4-vinylbenzyl chloride, allyl 2,3-epoxypropyl ether, 1,4-diazabicyclo[2.2.2]octane, CHCl3, diethyl ether, nhexane, 1-pentanol, TWEEN 20, BSA, and avidin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Boc-amino-PEO10amin, azido-PEO10-amine, and biotin-PEO60-amin were purchased from Polypure AS (Oslo, Norway). Alkyne-PEO70-amine was purchased from Rapp Polymere GmbH (Tübingen, Germany). Hydrochloric acid, acetone, and tetrahydrofuran (THF) were purchased from Grüssing GmbH (Filsum, Gremany). Styrene, divinylbenzene, and polysorbate-80 were purchased from Merck KGaA (Darmstadt, Germany). 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] was purchased from Wako Chemicals GmbH (Neuss, Germany). 3-Propyldisulfanylprop-1-ene was purchased from TCI Europe N.V. (Zwijndrecht, Belgium). Magnetic columns were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). All chemicals were used as received unless otherwise stated. Styrene and divinylbenzene were distilled prior to use. Synthesis of Amino-PEO10-N-(allyl 2-hydroxypropyl ether). 450 μL (3.81 × 10−3 mol) of allyl 2,3-epoxypropyl ether, 88 mg (7.84 × 10−4 mol) 1,4-diazabicyclo[2.2.2]octane (DABCO), and 450 mg (6.98 × 10−4 mol) of Boc-amino-PEO10-amine were dissolved in 15 mL of dry CHCl3. The solution was stirred for 23 h at 60 °C, and CHCl3 was evaporated after cooling to ambient temperature. The 4916
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Synthesis of PI-b-PEO−OH and PI-b-PEO−COOH. Polyisoprene-block-poly(ethylene oxide) diblock copolymers (PI-b-PEO−OH and PI-b-PEO−COOH) were synthesized according to Pöselt et al.8 The prepared polymers had a Mn = 12 700 g/mol. Synthesis of PI-b-PEO-NH2. To a vigorously stirred solution of 516 mg (3.18 mmol) 1,1′-carbonyldiimidazole (CDI) in 10 mL of dry CH2Cl2 a solution of 2.15 g (0.159 mmol) PI-b-PEO−OH in 15 mL of dry CH2Cl2 was added dropwise. After stirring for 40 h at room temperature the solution was quickly washed with distilled water (4 × 40 mL) at 4 °C and dried immediately over Na2SO4. Filtration and evaporation provided the crude activated ester as a yellow gel, which was immediately used in the next step without further purification. To a vigorously stirred solution of 383 mg (6.36 mmol) of ethylenediamine in 5 mL of dry CH2Cl2 a solution of the foregoing CDI-activated PI-b-PEO diblock copolymer (0.159 mmol) in 10 mL of dry CH2Cl2 was added dropwise within 10 min. After adding 10 mL of anhydrous CHCl3 the reaction mixture was stirred for 31 h at 55 °C. Stirring was continued for a further 7 days at room temperature. After filtration, the solvent was removed under vacuum. The remaining residue was dissolved in 2 mL of CH2Cl2 and precipitated by addition of acetone (60 mL) at −20 °C. After centrifugation, the supernatant was removed, and the crude product was again purified by subsequent precipitations in acetone (60 mL) at −20 °C and diethyl ether (60 mL) at −20 °C. The product was dissolved in deionized water (25 mL) and then dried by lyophilization (yield: 1827 mg, 85%). 1H NMR (500 MHz, CDCl3, 25 °C): δ (ppm) = 5.97−5.55 (m, 1H per unit of 1,2-PI, −HCCH2), 5.30 (br s, 1H, −OCONH−), 5.11−4.49 (m, 1H per unit of 1,4-PI, −CCH−; 2H per unit of 3,4-PI, −CCH2; 2H per unit of 1,2-PI, −HCCH2), 4.20 (t, 2H, −CH2−OCONH−), 3.79−3.46 (m, 4H per PEO unit), 3.21 (m, 2H, −NH−CH2−), 2.80 (t, 2H, 3J = 5.7 Hz, −CH2−NH2), 2.27−1.70 (m, 1H per unit of 3.4PI, −CH−; 2H per unit of 1.4-PI, −HCCH−CH2−; 2H per unit of 1.4-PI, −CH2−CH(−CH3)=), 1.70−1.45 (m, 3H per unit of 3,4-PI, C−CH3; 3H per unit of 1.4-PI, −CH(−CH3)=), 1.45−1.04 (m, 3H, −CH(−CH3)−; 2H per unit of 1,2-PI, −C−(CH3)−CH2−; 2H, CH3−CH2−; 2H per unit of 3,4-PI, CH−CH2−), 1.04−0.86 (m, 1H, −CH(−CH3)−; 3H per unit of 1,2-PI, −C−(CH3)−), 0.86−0.74 (m, 3H, CH3−CH2−). Synthesis of PI-b-PEO-OMe. 254 mg (2.07 × 10−3 mol) of 3methoxypropionyl chloride (2.07 × 10−3 mol) was dissolved in 10 mL of THF and cooled to −30 °C. A solution of 700 mg (5.51 × 10−5 mol) of PI-b-PEO−OH in 10 mL of dry THF was injected dropwise using a syringe. The solution was stirred for 23 h at room temperature. After evaporation of the solvent, the syrup was dissolved in 2 mL of CH2Cl2 and precipitated by addition of acetone (40 mL) at −20 °C. The crude product was centrifuged off, and the precipitation process was repeated thrice. The product was dissolved in deionized water (10 mL) and dried by lyophilization (yield: 631 mg, 90%). 1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) = 5.97−5.55 (m, 1H per unit of 1,2-PI, −HCCH2), 5.11−4.49 (m, 1H per unit of 1,4-PI, −CCH−; 2H per unit of 3,4-PI, −CCH2; 2H per unit of 1,2-PI, −HCCH2), 4.26 (t, 2H, 3J = 4.8 Hz, −CH2−O−CO−), 3.79−3.46 (m, 4H per PEO unit), 3.76 (2H, t, 3J = 6.3 Hz, −CH2−OCH3), 3.35 (s, 3H, −OCH3), 2.61 (t, 2H, 3J = 6.3 Hz, −O−CO−CH2−), 2.27−1.70 (m, 1H per unit of 3.4-PI, −CH−; 2H per unit of 1.4-PI, −HCCH− CH2−; 2H per unit of 1.4-PI, −CH2−CH(−CH3)=), 1.70−1.45 (m, 3H per unit of 3,4-PI, C−CH3; 3H per unit of 1.4-PI, −CH(−CH3)=), 1.45−1.04 (m, 3H, −CH(−CH3)−; 2H per unit of 1,2-PI, −C− (CH3)−CH2−; 2H, CH3−CH2−; 2H per unit of 3,4-PI, CH−CH2−), 1.04−0.86 (m, 1H, −CH(−CH3)−; 3H per unit of 1,2-PI, −C− (CH3)−), 0.86−0.74 (m, 3H, CH3−CH2−). Seeded Emulsion Polymerization. In a standard preparation, 20 mg of iron oxide nanocrystals dispersed in CHCl3 were evaporated to dryness and redispersed in 3 mL of THF. Polysorbate-80 (30 mg) was added, and the THF solution injected into 30 mL of water. After removal of oxygen by alternating cycles of evacuating and flushing with nitrogen (20 cycles) 20 μL of 1-pentanol, 15 μL of styrene, 15 μL of divinylbenzene, and 1 mg of the radical initiator 2,2′-azobis[2-(2imidazolin-2-yl)propane] dihydrochloride were added, and the solution was stirred at 44 °C for 20 h (yield referring to iron oxide
nanocrystals: >99%). Purification of the product was performed by a magnetic column (see Supporting Information). Optionally, polysorbate-80 was replaced with PI-b-PEO or modifications of PI-b-PEO. Similarly, styrene and divinylbenzene were (partially) replaced with functional monomers or polymerizable linkers. Synthesis of Gold/Iron Oxide Hybrids. An aqueous solution of citrate stabilized gold nanocrystals (3 mL, 6.8 nM; d = 13.5) was mixed with 150 μL of 3-propyldisulfanylprop-1-ene-functionalized iron oxide nanocrystals (33 nM) and stirred for 10 min. 30 μL of an aqueous αmethoxypoly(ethylene oxide)-ω-(mercaptoundecanoate) solution (1 mM, M = 2000 g/mol)22 was added, and the hybrids were collected on a magnetic column. Dot-Blot Analysis. Dots of 1 μL avidin (1 mg/mL) dissolved in PBS (pH 4.2) were blotted on a nitrocellulose membrane. The spots were dried for 10 min at room temperature, and the procedure was repeated twice. Free binding sides were blocked using 0.1% TWEEN 20 in PBS (pH 7.4) for 30 min at room temperature. Binding was demonstrated by incubation with biotin-functionalized iron oxide nanocrystals in PBS (pH 7.4) for 1 h. BSA (4%) dissolved in PBS (pH 4.2) was used for the control experiments. Instrumentation. Total reflection X-ray fluorescence (TXRF) measurements were carried out with a Bruker S2 Picofox. The instrument is equipped with a Mo tube (750 mA, 50 kV), a curved multilayer monochromator, and a silicon drift detector with an active area of 30 mm2 and a resolution of 143 eV at 5.9 keV (Mn Kα). For all measurements, the Mo Kα line (17.4 keV) radiation was selected for excitation, and an integration time of 100 s was used. Transmission electron microscopy (TEM) and transmission electron microscopy− energy dispersive X-ray spectrometry (TEM-EDX) measurements were carried out with a Philips CM-300-UT microscope (200 keV) equipped with an EDAX DX-4 (Si(Li) detector with SUTW window). TEM measurements were also carried out with a Jeol JEM-1011 microscope (100 keV).The absorption spectra were recorded on a Cary 50 spectrometer (Varian). Zeta potential and dynamic light scattering (DLS) measurements were carried out with a Malvern Zetasizer Nano ZS system.
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RESULTS AND DISCUSSION We developed three different approaches for the preparation of ultrasmall functional nanocomposites, otherwise not achievable with classical ligand systems or other polymer coatings such as Pluronic that do not involve an emulsion polymerization:4−11 (1) functional monomers (Scheme 1, B), (2) functional and polymerizable linkers (C), and (3) functional surfmers (D). Especially the concomitant use of functional monomers and polymerizable linkers opens very attractive perspectives for a combinatorial approach, just by mixing the desired starting materials. Any functional molecule only requires a polymerizable group, typically an activated C−C double bond, which can be conveniently introduced by standard functional group transformations. Similarly, this concept is expanded if a linker chain is inserted between the polymerizable double bond and the desired functionality, hence providing a polymerizable functional linker. Both kinds of monomers can be simply added as comonomers to the shell-forming monomers, i.e., styrene and divinylbenzene, finally yielding a functionalized polystyrene copolymer shell around the inorganic core. Functionalities of small molecule monomers will be attached to the hydrophobic domain or to the hydrophobic−hydrophilic interface of the composite as illustrated in Scheme 1, B. By introducing an appropriate linker, the distance between the nanocrystal core and the functional group can be tuned, e.g., placing the functional group within the hydrophilic part or into the aqueous environment, respectively (Scheme 1, C). Also the amphiphilicity of the linker may be varied, hence expanding the concept to functional surfmers (Scheme 1, D). Evidently herein 4917
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Figure 3. Concentration-dependent dot-blot analysis of biotinfunctionalized iron oxide nanocrystals on avidin-coated nitrocellulose membrane (BSA coating as control).
Besides polysorbate-80, we used an amphiphilic polyisoprene-block-poly(ethylene oxide) diblock copolymer (PI-bPEO) as a surfactant. Herein, the hydrophobic, polyunsaturated polyisoprene chains participate in the polymerization, whereas the functional groups are attached to the hydrophilic terminus of the PEO chains, thus offering a stable, covalent surface functionalization of the cross-linked polymer shell around the nanocrystal. In order to demonstrate the broad scope and versatility of the concept, we initially chose different kinds of functional monomers, namely halogenated styrenes, 3-propyldisulfanylprop-1-ene, 4-vinylbenzoic acid, and functionalized PEOlinkers, not only because of their interesting downstream reactivities but also in order to ensure analytical detectability in case of a successful functionalization. Halogen-containing styrenes such as 4-vinylbenzyl chloride or 2,3,4,5,6-pentafluorostyrene offer interesting reactivities for downstream coupling reactions or functional group transformation of the polymer shell.23−26 Furthermore, the halogen content can directly be determined by TXRF or TEM-EDX in the final nanoconstructs (see Supporting Information). In a further experiment dyestuff conjugated iron oxide nanocrystals were prepared by copolymerization of styrene/divinylbenzene and Rose Bengal, which contains four iodine and four chlorine atoms and was previously O-alkylated with 4-vinylbenzyl chloride in order to introduce a polymerizable double bond (see Supporting Information).27 Notably, the intensities of the X-ray fluorescence allowed the quantification of functional groups per particle. Thus, about 21 000 4-vinylbenzyl chloride moieties and 710 Rose Bengal molecules per iron oxide nanocrystal were calculated. In a second series of experiments we prepared gold−iron oxide hybrid nanoparticles, leveraging on the bonds, spontaneously formed between gold surfaces and thiols or disulfides.28,29 To this end, we copolymerized 3-propyldisulfanylprop-1-ene into the polystyrene shell of iron oxide nanocrystals, hence generating a disulfide functionalized surface. On addition of citrate stabilized gold nanocrystals the hybrid particles were formed, wherein the gold nanocrystals form satellites around each central iron oxide particle. The citrate gold surfaces facing outward can optionally be further functionalized with e.g. thiolated biomolecules.30−33 In an initial experiment, we successfully used α-methoxypoly(ethylene oxide)-ω-(mercaptoundecanoate) for the ligand exchange and passivation of reactive gold surfaces. Any unconjugated gold nanocrystals can be separated from conjugated ones by means of a magnetic column. This was proven by separation of hydroxy-functionalized iron oxide and gold nanocrystals, which showed no cross-affinity at all. Figure 1 shows a TEM image (a) and the absorption spectrum (b) of
Figure 1. (a) TEM image of the gold/iron oxide hybrids by functionalization of the polymer shell with 3-propyldisulfanylprop-1ene and subsequent grafting of gold nanocrystals. (b) Absorption spectrum of the gold/iron oxide hybrids (solid line), iron oxide nanocrystals (dotted line), and gold nanocrystals (dashed line).
Figure 2. Zeta potentials (at pH 7) of encapsulated nanocrystals functionalized with 4-vinylbenzoic acid, functional polymerizable linkers, or functional surfmers. Negative charged particles are tagged blue, neutral green, and positive red.
Scheme 2. Synthesis of Functional and Polymerizable Linkers
the functional group or affinity molecule is attached to the hydrophilic terminus of an amphiphilic linker, thus reaching out to the aqueous phase. 4918
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Figure 4. TXRF analysis (left) and zeta potential (right) of bifunctional iron oxide nanocrystals.
Alternatively, PEO chains were terminated with the biomolecule biotin. A dot-blot analysis on an avidin-coated nitrocellulose membrane proved the successful copolymerization and accessibility of the biotin on the nanoparticle. After 1 h incubation with biotin-functionalized iron oxide nanocrystals, a concentration-dependent staining was observed. In contrast, a blank replacing avidin with bovine serum albumin (BSA) showed no reactivity (Figure 3). With these methods in hand, we prepared hybrid nanoparticles bearing different functionalities on their surface, as demanded for combinatorial strategies in rapid nanoparticle optimization. Thus, in two additional experiments we combined 4-vinylbenzoic acid with 4-vinylbenzyl chloride as well as 4-vinylbenzyl chloride with amino-terminated PI-b-PEO for the encapsulation of iron oxide nanocrystals. As one can see, both series display a strong chloride K-line in the TXRF spectrum, indicating copolymerization of the 4-vinylbenzyl chloride. In contrast, the zeta potentials differ remarkably, as can be expected from the positive and negative charge of the comonomers and surfmers. Respectively, the acidic particles displayed strong negative zeta potential (−24 mV), whereas the amine-containing particles showed a strong positive zeta potential (+30 mV), thus proving the presence of the charged comonomer and surfmer (Figure 4).
the magneto-plasmonic hybrids. The central iron oxide nanocrystal appears lighter than the gold satellites on the polymer shell, in which both types of nanocrystals were embedded. The absorption spectrum of the hybrids clearly (solid line) displays the characteristic localized surface plasmon resonance band of the gold nanocrystals. In comparison to the localized surface plasmon resonance band of the pure gold nanocrystals a slight red-shift is observed, which can be ascribed to plasmon coupling between the gold nanocrystals within the hybrid.34 In a third series of experiments we tuned the zeta potential of polystyrene-coated iron oxide nanocrystals by copolymerization with varying amounts of 4-vinylbenzoic acid. The zeta potential is determinant for the interactions of nanoparticles with proteins and cells in vivo and in vitro.35 Hence, increasing the percentage of 4-vinylbenzoic acid from 0.7‰ to 3.6‰ and finally to 13.4‰ in the seeded emulsion polymerization effected a decrease of the zeta potential from −10 to −16 mV and eventually to −31 mV in a concentration-dependent manner (Figure 2). The hydrodynamic diameter in this series stays, as expected, almost constant and ranges from 29 to 34 nm (see Supporting Information). This clearly demonstrates that the number of functional groups on the nanoparticle can be controlled by the amount of functional monomer added during the emulsion polymerization. We further enhanced this concept by copolymerization of basic, acidic, and neutral functional surfmers and a polymerizable linker. Accordingly, copolymerized functional surfmers, like amino-terminated PI-b-PEO, provided a highly positive zeta potential (25 mV), whereas hydroxyl- and methoxyterminated PI-b-PEOs showed neutral and carboxy-terminated PI-b-PEO negative zeta potentials (−28 mV). Polymerizable PEO-linker provided a similar effect. Hence, particles containing amino-terminated allyl PEO (amin-PEO10-N-(allyl 2-hydroxypropyl ether)) showed a positive zeta potential of 12 mV (Figure 2). Finally, we developed a general strategy for the olefinic functionalization of aminated structures, e.g., amino-PEOs for subsequent copolymerization into the polystyrene shell (Scheme 2). Herein, a polymerizable allyl group was conveniently introduced to the respective amine via 1,4diazabicyclo[2.2.2]octane (DABCO)-catalyzed ring-opening of 1-allyloxy-2,3-epoxypropane. The broad scope of the reaction was demonstrated by introducing amino groups, thus modulating the zeta potential of nanoparticles (Figure 2) or providing reactive groups for amidation coupling chemistry. Furthermore, azide or alkyne moieties were attached for versatile “click” chemistry (see Supporting Information).
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CONCLUSION In conclusion, we demonstrated that seeded emulsion polymerization is a powerful tool to produce multiply functionalized PEO-coated nanocrystals. Advantageously, not only functional surfactants but also functional monomers and functional polymerizable linkers can be used, solely or in combinations thereof. Furthermore, the spatial position of the functional groups can be controlled by choosing the appropriate spacers. This methodology is highly amenable for combinatorial strategies and will spur rapid expedited synthesis and purposeful optimization of a broad scope of nanocrystals. The encapsulation of quantum dots via seeded emulsion polymerization is currently in work and will be reported elsewhere. Different spectroscopic and biochemical methods were applied to unambiguously characterize the samples and to prove successful functionalization. In particular, TXRF was used for the detection of halogen, whereas TEM was the method of choice for the characterization of gold/iron oxide hybrids. By copolymerization of a broad selection of acidic and basic monomers as well as surfmers, we were able to modulate the zeta potential in a range from −31 to +30 mV. Finally, biotin4919
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functionalized particles were validated by an avidin dot-blot assay.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental details about the preparation of alkylated Rose Bengal, azido-PEO10-N-(allyl 2-hydroxypropyl ether), alkynePEO70-N-(allyl 2-hydroxypropyl ether), and the magnetic purification as well as results about the copolymerization of halogen-containing styrenes and the diameter of 4-vinylbenzoic acid-containing samples. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address ⊥
N.G.B.: Institut Catalá de Nanotecnologia (ICN), Campus UAB, 08193 Bellaterra, Barcelona, Spain.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Mr. Oliver Dabrowski for the O-alkylation of Rose Bengal and Ms. Sylvia Bartholdi-Nawrath for TEM-EDX measurements. We also thank Mr. Christian Supej of CAN GmbH for supporting the dot-blot analysis. This work was supported by the European Union’s Seventh Framework Programme (VIBRANT, FP7-228933-2) and the state excellence initiative “Nanotechnology in Medicine”.
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REFERENCES
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