Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles

Jul 13, 2016 - Development of efficient and rapid protocols for diversification of ... brush coated magnetic nanoparticles find use in many such appli...
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Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles: Engineering the Interface for Targeted Cellular Imaging Yavuz Oz,† Mehmet Arslan,† Tugce N. Gevrek,† Rana Sanyal,†,‡ and Amitav Sanyal*,†,‡ †

Department of Chemistry, Bogazici University, Bebek, 34342 Istanbul, Turkey Center for Life Sciences and Technologies, Bogazici University, Istanbul, Turkey

‡

S Supporting Information *

ABSTRACT: Development of efficient and rapid protocols for diversification of functional magnetic nanoparticles (MNPs) would enable identification of promising candidates using high-throughput protocols for applications such as diagnostics and cure through early detection and localized delivery. Polymer brush coated magnetic nanoparticles find use in many such applications. A protocol that allows modular diversification of a pool of parent polymer coated nanoparticles will lead to a library of functional materials with improved uniformity. In the present study, polymer brush coated parent magnetic nanoparticles obtained using reversible addition−fragmentation chain transfer (RAFT) polymerization are modified to obtain nanoparticles with different “clickable” groups. In this design, trithiocarbonate group terminated polymer brushes are “grafted from” MNPs using a catechol group bearing initiator. A postpolymerization radical exchange reaction allows installation of “clickable” functional groups like azides and maleimides on the chain ends of the polymers. Thus, modified MNPs can be functionalized using alkyne-containing and thiol-containing moieties like peptides and dyes using the alkyne−azide cycloaddition and the thiol−ene conjugation, respectively. Using the approach outlined here, a cell surface receptor targeting cyclic peptide and a fluorescent dye are attached onto nanoparticle surface. This multifunctional construct allows selective recognition of cancer cells that overexpress integrin receptors. Furthermore, the approach outlined here is not limited to the installation of azide and maleimide functional groups but can be expanded to a variety of “clickable” groups to allow nanoparticle modification using a broad range of chemical conjugations. KEYWORDS: polymer brush, functional coating, magnetic nanoparticle, click chemistry, bioconjugation

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INTRODUCTION

tions, the surface coating on MNPs needs to be engineered in a manner that would allow attachment of specific ligands and biomolecules while exhibiting resistance toward undesired protein adsorption in the biological media.7 A variety of polymeric coatings ranging from ultrathin coatings of amphiphilic copolymers to block copolymers, as well as surface-tethered polymer brushes,have been utilized toward fabrication of functional MNPs.8,9 While there have been several advances in coating of MNPs with biocompatible

Magnetic nanoparticles have emerged as indispensable building blocks for a broad range of functional materials for biomedical applications.1−5 Oftentimes, these nanoparticles are coated with a layer of polymeric material to impart properties that enable and enhance their utility. Today, polymeric coatings on magnetic nanoparticles (MNPs) play a role beyond just simple prevention of their agglomeration. They impart these magnetic nanomaterials with functional attributes for various applications such as targeted drug delivery and magnetic resonance imaging (MRI) contrast agent. A judicious choice of biocompatible polymeric coating also provides the much needed resistance to biofouling under physiological conditions.6 For many applica© 2016 American Chemical Society

Received: April 20, 2016 Accepted: July 13, 2016 Published: July 13, 2016 19813

DOI: 10.1021/acsami.6b04664 ACS Appl. Mater. Interfaces 2016, 8, 19813−19826

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Illustration of a Modular Approach to Fabricate a Library of Functional MNPs through Diversification of Parent Polymer-Coated MNPs

Scheme 2. Synthesis and Modular Postmodification of Polymer-Coated MNPs

polymeric brushes, only a few reports have focused on functionalizable polymer brushes.10−12 Furthermore, depending on the application, it may be desirable as well as sufficient to effectively modify only the surface of the polymeric brush, as it is this interface that is presented to the surroundings.

Traditionally, end-functionalizable brushes have been obtained by chain-end modification of polymers produced using controlled radical polymerizations such as atom transfer radical polymerization (ATRP) and reversible addition−fragmentation chain transfer (RAFT) polymerization. In spite of advantages 19814

DOI: 10.1021/acsami.6b04664 ACS Appl. Mater. Interfaces 2016, 8, 19813−19826

Research Article

ACS Applied Materials & Interfaces

coupling reaction with appropriately functionalized azo-based compounds to install reactive “clickable” groups such as azide and maleimide. This approach leads to facile fabrication of water-dispersible MNPs that can be functionalized with (i) alkyne- or (ii) thiol-containing molecules. The azide functional end group enables the conjugation of an alkyne-containing compound via copper-catalyzed Huisgen-type click reaction. Likewise, functionalization of maleimide end groups on the polymer can be accomplished with thiol-bearing molecules via nucleophilic Michael addition reaction. Importantly, this approach also allows tailoring of the amount of “clickable” reactive functional group on the surface by using a mixture of “clickable” bisazo-compound along with a nonreactive bisazocompound such as 2,2′-azobis(2-methylpropionitrile) (AIBN) during surface modification. Surface-reactive MNPs thus generated provide flexibility with desirable modifications using different types of conjugation chemistries.

like control over coating thickness and their uniformity, this approach limits the types of conjugation chemistries that can be utilized to obtain a diversely functionalized collection of nanoparticles from a particular batch of polymer coated MNPs. Strategies to extend the methods of functionalization of the nanoparticle interface while minimizing batch-to-batch variability will be highly desirable. This can be accomplished through an approach that would entail fabrication of parent nanoparticles where the end group on the polymer can be converted into a variety of different “clickable” functional groups that undergo effective reaction under mild and benign conditions (Scheme 1). Thus, availability of a library of reactive polymer-coated nanoparticles that can be modified using a variety of different “click” chemistries can be utilized to obtain a diverse library of functional nanoparticles. A functional polymer brush based coating must satisfy some basic criteria: (a) robust tethering onto the nanoparticle surface to provide stability in challenging biological milieu, (b) an antibiofouling nature to minimize nonspecific attachment of proteins, and (c) stable reactive groups for facile and efficient attachment of functional moieties such as imaging agents, drugs, and targeting groups. Robustness of polymeric shell on the MNP’s surface is determined by the affinity of anchor group for iron oxide. While traditionally some common anchor groups such as carboxylic acids,13 phosphonates,14 and silanes15 have been employed, recent interest has focused on catechol-based bioinspired anchoring because of its high affinity for iron oxide and resistance to thermal and hydrolytic decomposition.16,17 In addition, the catechol unit can be readily introduced into a polymer or a polymerization initiator that bears an activated carboxylic acid.18,19 A “graft-from” or a “graft-to” approach can be utilized for obtaining polymer brush coating. In a recent work, we reported modification of MNPs through a “graft-to” approach using side-chain thiol-reactive copolymers containing a catechol moiety at the chain end.20 Although the “graft-to” approach provides a well-defined polymeric coating, obtaining dense polymeric coating is challenging due to steric hindrance. A “graft-from” technique, where all polymer chains simultaneously grow from the nanoparticle surface, provides a more robust and dense coating. The choice of polymeric material largely depends on the intended application. Polymer brushes composed of poly(ethylene glycol) (PEG)-based hydrophilic polymer have been extensively studied because it reduces nonspecific protein adsorption to the NP surface, which prolongs circulation of iron oxide NPs in the biological systems.21 PEG-coated NPs can be easily obtained using various controlled radical polymerization techniques.22,23 Surface-initiated RAFT (SI-RAFT) polymerization is one of the most widely used controlled polymerization techniques because it is compatible with a wide range of functional monomers and the polymerization proceeds under mild reaction conditions without the need of any metal catalyst. Importantly, the resulting polymer chains possess a thiocarbonylthio groups at the chain ω-end, which can be modified by a variety of methods to obtain desirable end groups or grow another block of polymer.24−26 In this study, we outline a modular approach toward generating a library of surface-functionalized MNPs coated with chain-end “clickable” polymer brushes that are produced using the “graft-from” approach (Scheme 2). Diversification of chain-end reactive groups on the parent nanoparticles is achieved through modification of the trithiocarbonate functional groups on the polymer chain ends using a radical cross-

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EXPERIMENTAL SECTION Materials. Iron(III) chloride hexahydrate (FeCl3·6H2O), oleic acid, 1-dodecanethiol, Aliquot 336, dopamine hydrochloride, triethylamine (TEA), thionyl chloride, and 4(dimethylamino)pyridine (DMAP) were purchased from Sigma-Aldrich and used without further purification. Poly(ethylene glycol) methyl ether acrylate (PEGMEA, Mn = 480 g mol−1) was purchased from Sigma-Aldrich and purified over neutral aluminum oxide before use. 2,2′-Azobis(2-methylpropionitrile) (AIBN, Sigma) was recrystallized from methanol and dried at room temperature under vacuum prior to use. 1Octadecene, N-hydroxysuccinimide (NHS), and 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI) were purchased from Alfa-Aesar. Sodium oleate was purchased from TCI. Carbon disulfide (CS2) and 4,4′-azobis(4cyanovaleric acid) (V-501) were purchased from Fluka. Column chromatography was performed using silica gel 60 (43−60 nm, Merck). Thin-layer chromatography (TLC) was performed using silica gel plates (Kieselgel 60 F254, 0.2 mm, Merck). Furan-protected maleimide-containing alcohol,27 4,4difluoro-1,3,5,7-tetramethyl-8-(10-mercapto)-4-bora-3a,4adiaza-s-indacene (BODIPY) thiol,28 BODIPY alkyne,29 6-azido1-hexanol, 30 4-(3-hydroxypropyl)-10-oxa-4-azatricyclo[5,2,1,02,6]dec-8-ene-3,5-dione (azobis-pMAL),31 cyclic arginine-glycine-aspartic acid-phenylalanine-cysteine (cRGDfC),32 2-(dodecylthiocarbonothioylthio)-2-methyl propionic acid (DDMAT),33 and DDMAT succinimide ester (DDMATNHS)34 were synthesized according to previous literature examples. Dichloromethane (DCM), ethanol, chloroform, dimethylformamide (DMF), n-hexane, toluene, and 1,4-dioxane were purchased from Merck. Anhydrous toluene, tetrahydrofuran (THF), and DCM were obtained from SciMatCo purification system, and other solvents were dried over molecular sieves. Cells. Human breast adenocarcinoma MDA-MB-231 cell line was obtained from ATCC (Wessel, Germany). Cells were kept in the logarithmic phase of cell growth for the duration of the experiments. MDA-MB-231 cells were maintained in RPMI1640 culture medium (Roswell Park Memorial Institute) (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Lonza), 100 U/mL penicillin, 100 g/mL streptomycin, and 0.25 g/mL amphotericin B at 37 °C, under 5% CO2 atmosphere and 95% relative humidity. 2-(4Aminophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was obtained from Sigma-Aldrich. 19815

DOI: 10.1021/acsami.6b04664 ACS Appl. Mater. Interfaces 2016, 8, 19813−19826

Research Article

ACS Applied Materials & Interfaces Instrumentation. Zeta potential and dynamic light scattering (DLS) measurements were performed at 20 °C using a Malvern Zetasizer Nano ZS analyzer. Elemental analyses were performed using a Thermo Electron S.p.A. Flash EA 1112 elemental analyzer. Transmission electron microscopic (TEM) observations were carried out on a LVEM5 microscope operated at 5 kV. Thermogravimetric analyses (TGA) were conducted on a TA Instruments machine with a heating rate of 10 °C/min under a nitrogen atmosphere. X-ray photoelectron spectroscopy (XPS) was carried out using a K-Alpha instrument (Thermo Scientific). Fourier transform infrared (FT-IR) spectroscopy analyses were performed on a Nicolet 380 (Thermo Fisher Scientific, Inc.) instrument. Magnetization measurement was carried out using a vibrating sample magnetometer (VSM) on dry samples at room temperature. UV studies were performed with a Varian Cary 50 Scan UV/vis spectrophotometer. Fluorescent spectra were taken using a Varian Cary Eclipse Fluorescence spectrometer (Varian, Agilent, U.S.A.). 1H NMR and 13C NMR spectra were obtained using a Varian 400 MHz spectrometer. The dialysis bags (Spectra/Por Biotech Regenerated Cellulose Dialysis Membranes, MWCO 3500 Da) were purchased from Spectrum Laboratories. Cell internalization studies were performed with Zeiss Observer Z1 fluorescence microscope connected to Axiocam MRc5 using a Zeiss Filter set 38 (excitation band-pass (BP) 470/40, emission BP 525/50) for imaging the functionalized NPs and a Zeiss Filter set 49 (excitation G365, emission BP 445/50) was used for imaging DAPI stained nuclei. Images were obtained to visualize cell nuclei and morphology using Zeiss AxioVision software. Cell viability (CV) values for the cytotoxicity experiments were determined by measuring the absorbance of 96-well plates in use at 450 nm by Multiscan FC Microplate Photometer from Thermo Scientific equipped with a quartz halogen light source of a precision CV ≤ 0.2% (0.3− 3.0 Abs). It has an excitation wavelength range of 340−850 nm with excitation filters installed at 405, 450, and 620 nm. Methods. Synthesis of MNPs. Monodisperse MNPs were synthesized according to a previously reported procedure.35 Iron chloride (FeCl3·6H2O) (3.62 g, 13.4 mmol) and sodium oleate (12.18 g, 40 mmol) were dissolved in a mixture of 27 mL of ethanol, 20 mL of distilled water, and 47 mL of hexane. The mixture was heated up to 70 °C and kept at that temperature for 4 h. The organic layer containing iron oleate complex was washed with distilled water in a separatory funnel. The cleaning process was repeated at least three times, and excess hexane was removed via rotary evaporation. Iron oleate complex was obtained in a waxy solid form. Iron oleate complex (10 g, 11.1 mmol) and oleic acid (1.59 g, 5.6 mmol) were dissolved in 1octadecene (71 mL) by vigorous stirring. The mixture was heated up to 110 °C and kept at that temperature for 15 min to remove moisture. After 15 min, the mixture was heated to 320 °C with a constant heating rate 3−4 °C/min and kept at that temperature for 30 min. Then, the resulting MNPs were cooled to room temperature. MNPs were precipitated from ethanol and collected by centrifugation. Finally, the MNPs dispersed in hexane were again precipitated from acetone to remove 1octadecene residues and were dispersed and stored in hexane for further use. Synthesis of dopamine-functionalized chain transfer agent (Dopa-CTA). Into a round-bottom flask equipped with a magnetic stirrer was added Dopamine·HCl (0.41 g, 2.16 mmol) and TEA (0.22 g, 2.16 mmol) in DMF (14 mL), and the mixture was stirred for 15 min at room temperature. To this

solution, DDMAT-NHS ester (0.95 g, 2.06 mmol) was added, and the reaction mixture was stirred at room temperature for 24 h in the dark. After the reaction, the mixture was dripped into an aqueous phosphate solution (60 mL, pH 4.0) to obtain yellow precipitate. The precipitate was collected by filtration and dried by lyophilization. The product was then purified by column chromatography using chloroform/ethyl acetate (1:1) as eluent (0.9 g, 88% yield). 1H NMR (CDCl3) δ (ppm): 6.79 (d, 1H, CH−CHC−OH), 6.68 (d, 1H, C−CHC−OH), 6.56 (dd, 1H, CCH−CH), 5.64 (s, 1H, NH), 3.44 (q, 2H, NH−CH2−CH2), 3.25 (t, 2H, SC−S−CH2), 2.66 (t, 2H, NH−CH2−CH2−C), 1.71−1.57 (m, 8H, S−C−(CH3)2−C O, S−CH2−CH2−(CH2)9−CH3), 1.43−1.19 (m, 18H, CH2− (CH2)9−CH3), 0.88 (t, 3H, CH2−(CH2)9−CH3). 13C NMR (CDCl3) δ (ppm): 219.8, 173.3, 144.1, 142.9, 130.6, 120.6, 115.2, 115.1, 57.0, 41.7, 37.2, 34.5, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 29.0, 28.9, 27.7, 25.8, 22.7, 14.1. Anal. Calcd [C25H41NO3S3]: C, 60.08; H, 8.27; N, 2.80; S, 19.24. Found: C, 59.51; H, 8.55; N, 2.84; S, 20.24. Synthesis of 4,4′-azobis(azidohexanoyl 4-cyanopentanoate). 4,4′-Azobis(4-cyanovaleric acid) (V-501) (1.0 g, 3,6 mmol), 6-azido-1-hexanol (1.35 g, 9.4 mmol), DMAP (171 mg, 1.4 mmol), and EDC (1.5 g, 7.9 mmol) were dissolved in 10 mL of dry DCM at room temperature. After the reaction mixture was stirred for 24 h, the mixture was extracted with NaHCO3 and dried over sodium sulfate (Na2SO4). Then, the solvent was removed by rotary evaporation, and 4,4′-azobis(azidohexanoyl 4-cyanopentanoate) (azobis-N3) was purified by column chromatography with ethyl acetate/hexane (1:4) (0.92g, 48% yield). 1H NMR (CDCl3) δ (ppm): 4.11 (m, 4H, COOCH2), 3.37 (t, 4H, N3CH2), 2.58−2.30 (m, 8H, CO− CH2CH2−C), 1.73 (s, 3H, CH3), 1.70−1.53 (m, 11H, C− CH2CH2−C, CH3), 1.48−1.32 (m, 8H, C−CH2CH2−C). 13C NMR (CDCl3) δ (ppm): 171.3, 117.5, 71.8, 65.0, 51.3, 33.2, 29.1, 28.7, 26.3, 25.5, and 24.0. Anal. Calcd [C24H38N10O4]: C, 54.36; H, 7.17; N, 26.41. Found: C, 54.81; H, 7.92; N, 25.78. Immobilization of catechol-modified RAFT agent on MNPs. DDMAT-modified NPs were prepared by a placeexchange reaction. Oleic acid stabilized iron oxide NPs (50 mg) were dissolved in 5 mL of chloroform, and Dopa-CTA (250 mg) was added to the solution. The mixture was stirred at 40 °C for 48 h under nitrogen atmosphere. Then, the reaction mixture was concentrated by rotary evaporation, and methanol was added to the reaction mixture to collect precipitated magnetic NPs using a permanent magnet; and the mixture was then redispersed in THF. The precipitation process was repeated until all ungrafted Dopa-CTA was removed as indicated by TLC. Surface-initiated RAFT polymerization of PEGMEA. DopaCTA anchored NPs (25 mg), PEGMEA (1.6 g, 3.3 mmol), and AIBN (2.2 mg, 13.4 μmol) were dissolved in 3 mL of toluene, and the mixture was purged with nitrogen for 20 min. The reaction mixture was put in a preheated oil bath at 70 °C. After the desired polymerization time, the solvent was removed under reduced pressure, and polymer-coated NPs were precipitated from a mixture of THF/diethyl ether (1:5). Polymer-grafted NPs were collected by centrifugation at 8000 rpm. The cleaning procedure was repeated several times to remove all free polymers that originated from AIBN-initiated chains. Typical radical cross-coupling end-group modification of iron oxide NPs with azobis-N3. Azobis-N3 (191 mg, 0.36 mmol) and polymer-coated NPs (100 mg) were dispersed in 3 19816

DOI: 10.1021/acsami.6b04664 ACS Appl. Mater. Interfaces 2016, 8, 19813−19826

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from the dispersion by dialyzing against water for 48 h (MWCO 3500 Da). Bicinchoninic acid protein quantification. The peptide (cRGD) conjugated onto the MNPs’ surface was quantified using a bicinchoninic acid (BCA) kit (ThermoScientific, U.S.A.) according to manufacturer’s instructions (details in the Supporting Information). Cell internalization experiment. Adenocarcinoma MDAMB-231 human breast cells (50 000 cells/well) were seeded in a 12-well plate as triplicate in 1000 μL of appropriate culture medium. Cells were incubated at 37 °C for 24 h for complete adhesion. Then the culture medium was aspirated and replaced with fresh medium containing imaging agent functionalized NPs at a concentration of 1 × 10−5 M. The experiments were completed in triplicate. After incubation at 37 °C for several time points (3, 6, and 24 h), sample solutions were removed. Wells were washed with 100 μL of phosphate-buffered saline (PBS) three times, and cells were mounted with PBS containing DAPI (5 mg/mL) at 37 °C for 30 min. After being washed with PBS three times, cells were fixed with 4% formaline solution at 37 °C for 5 min. Images were collected using Zeiss Observer Z1 fluorescence microscope at room temperature. Cytotoxicity experiment. Cytotoxic activity of cRGDattached Fe3O4@PEGMEA@MAL3MNPs was investigated by CCK-8 viability assay against MDA-MB-231 human breast adenocarcinoma. Cells were seeded in 96-well plates and treated with the MNPs at 37 °C for 48 h. Seven different concentrations (from 1 μg/mL to 1 mg/mL) of MNPs were prepared by diluting the stock concentration. Cells were incubated after treatment with these samples. Wells were washed with 100 μL of PBS solution twice. Cell viability was evaluated by CCK-8 assay by adding 10 μL of CCK-8 reagent in 100 μL of fresh medium onto wells.

mL of toluene and purged with nitrogen for 15 min. The mixture was stirred in a preheated oil bath at 65 °C for 16 h. After the reaction, solvent was removed by rotary evaporation and surface-modified NPs were precipitated from diethyl ether to remove unreacted species. The purified azide-functionalized nanoparticles (Fe3O4@PEGMEA@N3) were kept in THF for further reaction. Typical radical cross-coupling end-group modification of iron oxide NPs with azobis-pMAL. Azobis-pMAL (239 mg, 0.36 mmol) and polymer-coated NPs (100 mg) were dispersed in 3 mL of toluene/DMF (1:1) and purged with nitrogen for 15 min. The reaction mixture was stirred in a preheated oil bath at 65 °C for 16 h. Then, the solvent mixture was removed by rotary evaporation. End-group-modified nanoparticles (hereinafter called Fe3O4@PEGMEA@pMAL3) were dissolved in methanol, and the precipitate was removed by centrifugation. Finally, surface-modified NPs were dialyzed against methanol using dialysis membrane (MWCO 3500 Da) for 72 h. NPs bearing different amounts of protected maleimide groups were synthesized by using AIBN and azobis-pMAL. The first modification was carried out with 60 mg (0.09 mmol) of azobis-pMAL and 44 mg (0.27 mmol) of AIBN (called Fe3O4@PEGMEA@pMAL1). Similarly, the second modification was done using 119 mg (0.18 mmol) of azobis-pMAL and 30 mg (0.18 mmol) of AIBN to obtain Fe3O4@PEGMEA@ pMAL2. Typical procedure for activation of maleimide functional groups via retro-Diels−Alder reaction. Protected-maleimide end-group-containing iron oxide NPs (75 mg) were dispersed in 40 mL of anhydrous toluene and refluxed under nitrogen atmosphere for 16 h. After the solution cooled to room temperature, the solvent was evaporated to dryness. Maleimidefunctionalized magnetic (Fe3O4@PEGMEA@MAL) nanoparticles were redispersed in THF for further use. Surface functionalization of azide-containing MNPs by azide−alkyne Huisgen cycloaddition. Fe3O4@PEGMA@N3 NPs (10 mg) were dispersed in 200 μL of THF/H2O (3:1) mixture. Then, alkyne-functionalized BODIPY (0.59 mg, 1.8 μmol) was added to the mixture, followed by addition of CuSO4·5H2O (45 μg, 0.18 μmol) and sodium ascorbate (0.11 mg, 0.54 μmol). The mixture was stirred at 37 °C for 16 h under nitrogen atmosphere. The dye-conjugated nanoparticles were purified by precipitating into cold diethyl ether. Surface functionalization of maleimide-containing MNPs by thiol−ene reaction. End-group maleimide-modified magnetic NPs (Fe3O4@PEGMA@MAL3) (10 mg) and BODIPYSH (0.76 mg, 1.8 μmol) were dissolved in 200 μL of chloroform under nitrogen atmosphere. Then, triethylamine (0.25 μL, 1.8 μmol) was added into the reaction mixture. The mixture was stirred at 37 °C for 14 h. Unreacted dye molecules were purified by adding diethyl ether to the solution and collecting NPs by means of a magnet. Simultaneous conjugation of maleimide-containing MNPs with dye and cRGDfC via thiol−ene reaction. Fe3O4@PEGMA-MAL3 NPs (5 mg), cRGDfC (0.22 mg, 0.38 μmol), and BODIPY-SH (0.16 mg, 0.38 μmol) were dissolved in 300 μL of DMF under nitrogen atmosphere. Then, triethylamine (0.56 μL, 4 μmol) was added into the reaction mixture. The reaction mixture was stirred at 37 °C for 16 h. NPs were precipitated from diethyl ether and centrifuged at 4000 rpm to remove unreacted dye. After dye purification, NPs were dispersed in water and unreacted peptide was removed

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RESULTS AND DISCUSSION Synthesis of polymer brush coated parent MNPs. Magnetic iron oxide NPs with narrow size distribution were obtained by thermal decomposition of iron oleate complex in the presence of oleic acid in 1-octadecene, a high-boiling hydrocarbon solvent, using literature protocols. Briefly, iron oleate complex was synthesized using iron(III) chloride and sodium oleate. Nucleation and major growth of the MNPs takes place by dissociation of oleate ligands between 240 and 300 °C.36 Obtained oleic acid stabilized iron oxide MNPs (Fe3O4@OA) were characterized by FT-IR spectroscopy (Figure 1a), TGA (Figure 1b), DLS, and TEM (Figures S7 and S8). The size distribution of Fe3O4@OA MNPs was determined by DLS, revealing an average diameter of 7.7 nm with a narrow size distribution as suggested by a low polydispersity value of 0.028 for dilute hexane dispersion (Figure S7). The TEM image of Fe3O4@OA MNPs revealed spherical particles with an average diameter of 7.9 nm, with negligible aggregation (Figure S7). Nanoparticles were superparamagnetic as deduced from their field-depended magnetization curve obtained using VSM (Figure S5). The coating of MNPs with poly(PEGMEA) was carried out by a two-step procedure. First, a trithiocarbonate-based chain transfer agent (CTA) bearing catechol functionality was utilized to anchor MNP surface (Scheme 3). The catechol group is a well-known anchor group for various metal and metal oxides surfaces,37 and the strong binding ability of the catechol group on various surfaces has been exploited in modification of MNPs and planar 19817

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surface. While the FT-IR spectrum of oleic acid coated iron oxide NPs showed CH2 asymmetric and symmetric peaks at 2915 and 2846 cm−1, respectively, the characteristic amide carbonyl group of Dopa-CTA was observed at 1642 cm−1 after the place-exchange reaction (Figure 1a). From DLS measurements of CTA-immobilized MNPs (Fe3O4@CTA) in dilute DMF dispersion, the hydrodynamic size was found to be 7.9 nm with a narrow size distribution, as suggested by the low polydispersity value of 0.112, with a similar size in TEM analysis (Figures S7 and S8). The polymer brush coating was obtained through surfaceinitiated RAFT polymerization from the Fe3O4@CTA MNPs in the presence of a hydrophilic monomer poly(ethylene glycol) methyl ether acrylate (PEGMEA) via the “graft-from” approach (Scheme 3). Analysis of these nanoparticles using FT-IR spectroscopy revealed a new peak at 1731 cm−1 belonging to the ester carbonyl group of the PEGMEA repeat units, thus confirming successful growth of polymer chains from the surface (Figure 1a). Additionally, the grafted poly(PEGMEA) displays a strong band at 1095 cm−1, ascribed to the C−O−C stretching. Additionally, MNP surface modification with CTA and polymers was also investigated using thermogravimetric analysis (TGA). The mass of organic shell on the Fe3O4@OA MNP surface was estimated by TGA, showing a weight loss of 24% from 180 to 400 °C attributed to the oxidation of oleic acid groups, while Fe3O4@CTA gave a two-stage decomposition profile belonging to oleic acid residuals and Dopa-CTA on the iron oxide MNP surface (Figure 1b). Following the polymerization, the weight loss of Fe3O4@PEGMEA NPs was found to be 90% after 16 h polymerization time, which is, as expected, significantly higher than those of Fe3O4@OA and Fe3O4@CTA MNPs. Albeit having a dense polymer coating on the surface, MNPs showed good magnetic behavior by being attracted within