Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles

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Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles: Engineering the Interface for Targeted Cellular Imaging Yavuz Oz, Mehmet Arslan, Tugce Nihal Gevrek, Rana Sanyal, and Amitav Sanyal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04664 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 15, 2016

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ACS Applied Materials & Interfaces

Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles: Engineering the Interface for Targeted Cellular Imaging Yavuz Oz,a Mehmet Arslan,a Tugce N. Gevrek,a Rana Sanyala,b and Amitav Sanyal*a,b a

b

Department of Chemistry, Bogazici University, Bebek, 34342, Istanbul, Turkey.

Center for Life Sciences and Technologies, Bogazici University, Istanbul, Turkey.

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 RAFT polymerization is modified to obtain nanoparticles with different ‘clickable’ groups. In this design, trithiocarbonate group terminated polymer brushes are ‘grafted from’ from MNPs using a catechol group bearing initiator. A post-polymerization radical exchange reaction allows installation of ‘clickable’

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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 multi-functional construct allows selective recognition of cancer cells that over express 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

INTRODUCTION 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 enables and enhances 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 towards biofouling under physiological conditions.6 For many applications, the surface coating on MNPs needs to be engineered in a manner that would allow


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attachment of specific ligands and biomolecules while exhibiting resistance towards 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 towards fabrication of functional MNPs.8,9 While there has been several advances in coating of MNPs with biocompatible 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 sufficient to effectively modify only the surface of the polymeric brush, since it is this interface that is presented to the surroundings. Traditionally, endfunctionalizable 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 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’ chemistry can be utilized to obtain a diverse library of functional nanoparticles.


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Scheme 1 Illustration of a modular approach to fabricate a library of functional MNPs through diversification of parent polymer-coated MNPs. A functional polymer brush based coating must satisfy some basic criterions: a) robust tethering onto the nanoparticle surface to provide stability in challenging biological milieu, b) an anti-biofouling nature to minimize non-specific attachment of proteins and c) must possess 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 phosphonates14 and silanes15 have been employed, recent interest has focused on catechol-based bio-inspired anchoring due to 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 ‘graft-to’ approach using


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side chain thiol-reactive copolymers containing catechol moiety at the chain end.20 Although ‘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 since it reduces non-specific 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 polymerization (SI-RAFT) 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 towards 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 radical cross-coupling reaction with appropriately functionalized azobased 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


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of 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 mixture of ‘clickable’ bisazo-compound along with non-reactive bisazo-compound such as 2,2’azobis(2-methylpropionitrile) (AIBN) during surface modification. Surface reactive MNPs thus generated provides flexibility with desirable modifications using different types of conjugation chemistries.

Scheme 2 Synthesis and modular post-modification of polymer-coated MNPs.


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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 gmol-1) was purchased from Sigma-Aldrich and purified over neutral aluminum oxide before use. 2,2’-azobis(2methylpropionitrile) (AIBN, Sigma) was recrystallized from methanol and dried at room temperature under vacuum prior to use. 1-octadecene, N-Hydroxysuccinimide (NHS) and 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were purchased from AlfaAesar. 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 was performed using silica gel plates (Kieselgel 60 F254, 0.2 mm, Merck). Furan protected maleimide containing alcohol,27 4,4-difluoro-1,3,5,7-tetramethyl-8-[(10-mercapto)]-4-bora-3a,4a-diaza-s-indacene thiol,28

BODIPY

alkyne,29

6-azido-1-hexanol,30

(BODIPY)

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 (DDMAT-NHS)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.


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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 experiments. MDA-MB-231 cells were maintained in RPMI-1640 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-(4-Aminophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was obtained from Sigma-Aldrich. Instrumentation. Zeta potential and dynamic light scattering (DLS) measurements were performed at 20 oC 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 with a heating rate of 10 oC/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 with using 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, USA). 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)


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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 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 340−850 nm with excitation filters installed at 405, 450, and 620 nm. Methods Synthesis of MNPs. Monodisperse MNPs were synthesized according to 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 ethanol, 20 ml distilled water and 47 ml hexane. The mixture was heated up to 70 oC and kept at that temperature for 4 hours. 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 1-octadecene (71 ml) by vigorous stirring. The mixture was heated up to 110 oC and kept at that temperature for 15 min in order to remove moisture. After 15 minutes, the mixture was heated to 320 oC with a constant heating rate 3 - 4 oC/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 1-octadecene residues and dispersed and stored in hexane for further use.


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Synthesis of dopamine functionalized chain transfer agent (Dopa-CTA). In 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 hours 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).

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C 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 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 –


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1.53 (m, 11H, C-CH2CH2-C, CH3), 1.48 – 1.32 (m, 8H, C-CH2CH2-C).

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C 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 place exchange 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 oC 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 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. Dopa-CTA 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 oC. After 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 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 ml of toluene, and purged with nitrogen for 15 min. The mixture was stirred in a preheated oil bath at 65 oC for 16 h. After the reaction, solvent was removed by rotary evaporation and surface modified NPs were precipitated from diethyl ether in order to remove unreacted species. The


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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 azobispMAL. 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 oC for 16 h. Then, the solvent mixture was removed by rotary evaporation.

End

group

modified

nanoparticles

(hereinafter

called

as

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 amount of protected maleimide groups were synthesized by using AIBN and azobis-pMAL. The first modification was carried out with 60 mg (0.09 mmol) azobispMAL, 44 mg (0.27 mmol) AIBN (called as Fe3O4@PEGMEA@pMAL1). Similarly, the second modification was done using 119 mg (0.18 mmol) azobis-pMAL, 30 mg (0.18 mmol) 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. Maleimide functionalized 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


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addition of CuSO4.5H2O (45 µg, 0.18 µmol) and sodium ascorbate (0.11 mg, 0.54 µmol). The mixture was stirred at 37 oC 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 BODIPY-SH (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 oC 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 thiolene 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 oC 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 from the dispersion by dialyzing against water for 48 h (MWCO 3500 Da). Bicinchoninic acid (BCA) protein quantification. The peptide (cRGD) conjugated onto MNPs’ surface was quantified using a BCA kit (ThermoScientific, USA) according to manufacturer’s instructions (details in the supporting information). Cell internalization experiment. Adenocarcinoma MDA-MB-231 human breast cells (50 000 cells/well) were seeded in a 12-well plate as triplicate in 1000 µl 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


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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 PBS three times and cells were mounted with PBS containing DAPI (5 mg/mL) at 37 °C for 30 min. Then 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 cRGD attached Fe3O4@PEGMEA@MAL3 MNPs was investigated by CCK-8 viability assay against MDA-MB-231 human breast adenocarcinoma. Cells were seeded in a 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 PBS solution twice. Cell viability was evaluated by CCK-8 assay by adding 10 µl CCK-8 reagent in 100 µl fresh medium onto wells.

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 240–300 oC.36 Obtained oleic acid stabilized iron oxide MNPs (Fe3O4@OA) were characterized by FT-IR spectroscopy (Fig. 1a), TGA (Fig. 1b), DLS and TEM (Fig. S7, S8). The size distribution of Fe3O4@OA MNPs was


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determined by DLS, revealing an average diameter 7.7 nm with a narrow size distribution as suggested by low polydispersity value of 0.028 for dilute hexane dispersion (Fig. S7). TEM image of Fe3O4@OA MNPs revealed spherical particles with an average diameter of 7.9 nm, with negligible aggregation (Fig S7). Nanoparticles were super-paramagnetic as deduced from their field depended magnetization curve obtained using VSM (Fig. S5). The coating of MNPs with poly(PEGMEA) was carried out by a two-step procedure. Firstly, 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 catechol group on various surfaces has been exploited in modification of MNPs and planar surfaces with polymeric materials.38-40 In the second step, surface initiated RAFT polymerization was used to obtain polymer brushes on the MNPs.

Scheme 3 Synthetic route of p(PEGMEA) coated iron oxide MNPs.


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The catechol modified CTA (Dopa-CTA) was synthesized from corresponding NHSactivated RAFT agent and dopamine (Scheme 3). Structure and purity of thus obtained DopaCTA was characterized by NMR spectroscopy (Fig. S1, S2). Thereafter, the CTA was immobilized onto the iron oxide MNP surface through place exchange reaction in chloroform at 40 oC under nitrogen atmosphere for 48 h. After through purification by precipitation in methanol to remove non-tethered Dopa-CTA species, FT-IR analysis was conducted to confirm successful introduction of Dopa-CTA onto MNP surface. While the FT-IR spectrum of oleic acid coated iron oxide NPs showed CH2 asymmetric and symmetric peaks at 2915 cm-1 and 2846 cm1

, respectively; the characteristic amide carbonyl group of Dopa-CTA was observed at 1642 cm-1

after the place exchange reaction (Fig. 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 low polydispersity value of 0.112, with a similar size in TEM analysis (Fig. S7 and S8).


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Figure 1 (a) FT-IR and (b) thermogravimetric analysis of Fe3O4@OA, Fe3O4@CTA and Fe3O4@PEGMEA. The polymer brush coating was obtained through surface initiated 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 ester carbonyl group of the PEGMEA repeat units, thus confirming successful growth of polymer chains from the surface (Fig. 1a). Additionally, the grafted poly(PEGMEA) displays a strong band at 1095 cm-1, ascribed to the C-O-C stretching.


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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 oC 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 iron oxide MNP surface (Fig. 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 dense polymer coating on the surface, MNPs showed good magnetic behavior by being attracted within less than 10 min in the presence of an external magnet (Fig. S6). The dispersion solution became almost clear after 12 h magnet attraction, which indicates that one can exploit magnetic behavior of Fe3O4@PEGMEA MNPs even after polymer coating (Fig. 2c). The particle size of nanoparticles before and after polymer grafting was also determined by DLS. After 16 h RAFT polymerization, the size of polymer-coated NPs increased to 13.7 nm with a narrow size distribution as suggested by low polydispersity value of 0.32 in dilute DMF dispersion (Fig. S7). Zeta potential was measured as -8.7 mV (data not shown), low enough to consider the particles as neutral. Furthermore, TEM analysis of polymer coated nanoparticles dispersed in water indicates that MNPs did not form any aggregates during the polymerization (Fig. 2).


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Figure 2 (a) TEM image of Fe3O4@PEGMEA MNPs, (b) Fe3O4@PEGMEA MNPs as aqueous dispersion and (c) after 12 h exposure to magnet.

The nanoparticle surface modification using the catechol bearing CTA and grafted polymer chains was also characterized using X-ray photoelectron spectroscopy (XPS). For the CTA derived nanoparticles, the peaks with binding energy (BE) of 56 (Fe 3p), 164 (S 2p), 285 (C 1s), 400 (N 1s), 532 (O 1s) and 711 (Fe 2p) were observed from wide scan spectrum (Fig. 3a). In the core level spectrum of C (1s), the peaks at 285.0, 286.6 and 288.9 eV were attributed to C-C/CH, C-O and C=O, respectively (Fig. 3b). The S (2p) peak belonging to sulfur in the trithiocarbonate groups was observed at 164 eV (Fig. 3c). As expected, the area ratio of N to S peaks was observed to be 1:3. The chemical composition of Fe3O4@PEGMEA MNPs was also characterized using XPS (Fig. 3d), with characteristic peaks of Fe (3p), S (2p), C (1s), N (1s), O (1s) and Fe (2p) observed at 56, 163, 285, 400, 532 and 710 eV, respectively. In the core level spectra of C (1s) and S (2p), binding energies of 285.0, 286.5 288.9 and 164.2 eV are attributed to C-C/C-H, C-O, C=O and S(2p), respectively (Fig. 3e and 3f).


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Figure 3 (a) XPS wide scan spectrum of Fe3O4@CTA, (b) C 1s core level spectra and (c) S 2p core level spectra of Fe3O4@CTA, (d) wide scan spectra of Fe3O4@PEGMEA, (e) C 1s core level spectra and (f) S 2p core level spectra of Fe3O4@PEGMEA. Installation and functionalization of ‘clickable’ functional groups. Post-polymerization chain-end modification was accomplished in a modular approach using radical exchange reactions in the presence of appropriately modified bisazo-based compounds bearing ‘clickable’ reactive groups. To introduce the azide groups, an azo-based azide-containing compound Azobis-N3 was synthesized and used for end group modification of polymer brush coated iron oxide MNPs. Using radical cross-coupling approach, polymer coated MNPs decorated with azide groups at the polymer chain termini (Fe3O4@PEGMEA@N3) were synthesized (Scheme 4). Similarly, polymer brush coated MNPs with protected maleimide end groups (Fe3O4@PEGMEA@pMAL) were synthesized using Azobis-pMAL (Scheme 4).


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Scheme 4 Diversification of reactive surface modification of MNPs by azide or furan-protected maleimide derivatives. First, modification of polymer chain end group to a ‘clickable’ azide moiety was accomplished via radical cross coupling reaction at 65 oC in toluene using an excess of AzobisN3. Purification of the Fe3O4@PEGMA@N3 MNPs from reactants was achieved via precipitating from cold diethyl ether and collecting by centrifugation. The modification of trithiocarbonate end groups was verified by UV-vis spectroscopy. The absence of the absorption


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peak at 308 nm in the UV-vis spectrum confirmed the removal of the trithiocarbonate group (Fig. S9). DLS analyses revealed that the exchange reaction did not have any detrimental effect on the polydispersity of the polymer coated MNPs (Fig. S10). Likewise, the TEM analysis indicated that MNPs remained uniform and non-aggregated, which is important for the most of applications of MNPs (Fig. S11). The zeta potential of azide functionalized MNPs was measured from its water dispersion as -8.7 mV, revealing similar potential as Fe3O4@PEGMEA and can be considered neutral. Importantly, from the FT-IR spectrum, a band belonging to the stretching of the azide group was noticed at 2096 cm-1, which confirmed the chain-end modification through the cross-coupling reaction (Fig. 4a).

Figure 4 (a) FT-IR spectra of azide group terminated MNPs, (b) MNPs after BODIPY attachment by Huisgen click reaction and (c) dye conjugation onto azide groups terminated MNPs by Huisgen click chemistry; (c1) dye attached NPs, (c2) control, (c3) free dye. The DOPA-based anchor group has been omitted for clarity.


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Attachment of an alkyne-containing fluorescent dye onto iron oxide MNP surface was investigated to demonstrate the efficiency of post-polymerization end group modification of surface grafted polymers. Fe3O4@PEGMA@N3 MNPs were functionalized with alkyne groupcontaining BODIPY dye using the copper catalyzed Huisgen type azide-alkyne cycloaddition reaction (Fig. 4c). FT-IR spectrum of BODIPY attached MNPs showed the disappearance of the characteristic azide peak at 2096 cm-1 (Fig. 4b). As illustrated in Fig. 4c, the dye attached MNPs could be easily dispersed in water and were fluorescent under UV illumination, while the free dye is insoluble in water. As a control, the same procedure was carried out using azide functionalized MNPs and alkyne terminated BODIPY without a copper catalyst. The MNPs did not give any fluorescence under UV illumination indicating that there are no physical interactions between dye molecules and MNPs. Hence, the azide terminated polymer brush coated nanoparticles can be easily modified using the Huisgen type ‘click’ chemistry to obtain functional nanoparticles. Although, not demonstrated here, but one can expect the elaboration of azide group using other version such as the metal free strain-promoted azide alkyne cycloaddition reactions. Installation of the thiol reactive maleimide functional group was accomplished using a similar protocol by treatment of the polymer brush coated nanoparticles with azobis-pMAL. Attachment of furan-protected maleimide groups at the chain ends of grafted polymers was investigated using the radical cross-coupling modification with varying ratios of azobis-pMAL and AIBN to show efficiency of controlling end group density. Three sets of surface modified MNPs with increasing maleimide content were obtained using 25, 50 and 100% of the masked maleimide azobis-pMAL to yield Fe3O4@PEGMEA@pMAL1, Fe3O4@PEGMEA@pMAL2 and Fe3O4@PEGMEA@pMAL3, respectively. During the modification reaction, the temperature


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was kept at 65 oC to avoid any deprotection of maleimide groups. From UV-vis spectrum, end group modification of trithiocarbonate groups was confirmed as upon the cleavage of trithiocarbonate end groups by cross-coupling, the characteristic absorbance at 308 nm belonging to the trithiocarbonate group disappeared (Fig. S9).

Figure 5 (a) FT-IR spectra of furan protected maleimide terminated iron oxide MNPs and (b) deprotected maleimide terminated MNPs.

Fe3O4@PEGMEA@pMAL MNPs containing protected maleimide end groups were characterized by TEM and DLS. For TEM and DLS analysis, Fe3O4@PEGMA@pMAL3 MNPs


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dispersed in DMF were chosen. The hydrodynamic size of Fe3O4@PEGMA@pMAL3 was found to be 12.4 nm with a zeta potential of -9.5 mV, low enough to be considered neutral (Fig. S10). TEM analysis revealed that the Fe3O4@PEGMEA@pMAL3 MNPs were highly monodisperse after cross-coupling modification (Fig. S11). The end group ratios were probed by FT-IR measurements and found that transmittance of new carbonyl peak belonging to maleimide groups increased when the used azobis-pMAL ratio with respect to AIBN was increased (Fig. 5a). Characteristic peak of protected maleimide carbonyl group at 1701 cm-1 appeared after cross-coupling reaction of azobis-pMAL, and the peak intensity sharpened with the increase of protected maleimide end groups. This result indicated that it is possible to install a functional group onto iron oxide MNP surface with varying densities. Surface compositions and maleimide ratio of the NPs were also analyzed via XPS (Fig. 6). The photoelectron lines at binding energy (BE) of about 286 eV (C 1s), 399 eV (N 1s) and 531 eV (O 1s) were observed from wide scan spectrum. After end group modification of the MNPs, the S (2p) peak coming from the trithiocarbonate end groups of RAFT polymers completely disappeared and new N (1s) peak appeared. As expected, the XPS results clearly indicate an increase in the N atom content (from 0.31% to 0.44%) upon increasing the maleimide content.


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Figure 6 (a) XPS wide scan spectrum of Fe3O4@PEGMEA@pMAL1, (b) S 2p core level spectrum and (c) N 1s core level spectrum of Fe3O4@PEGMEA@pMAL1, (d) wide scan spectrum of Fe3O4@PEGMEA@pMAL2, (e) S 2p core level spectrum and (f) N 1s core level spectrum

of

Fe3O4@PEGMEA@pMAL2,

(g)

wide

scan

spectrum

of

Fe3O4@PEGMEA@pMAL3, (h) S 2p core level spectrum and (i) N 1s core level spectrum of Fe3O4@PEGMEA@pMAL3.


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Fe3O4@PEGMEA@pMAL3 MNPs were activated by retro Diels-Alder reaction by removing furan groups by heating MNPs to 110 oC (Fig. 7). DLS studies were examined for Fe3O4@PEGMA@MAL3 to show if any crosslinking occurred between maleimide groups during activation reaction. It was found that there was no aggregation, and the diameter of maleimide ended MNPs (Fe3O4@PEGMEA@MAL3) was 12.5 nm (Fig. S10). TEM image of Fe3O4@PEGMEA@MAL3 clearly indicated that MNPs were not aggregated during activation of maleimide end groups (Fig. S11). These MNPs were also characterized by FT-IR after deprotection of maleimide groups. As seen from FT-IR spectra in Fig. 5b, activated maleimide groups gave carbonyl peak at 1701 cm-1 after retro Diels Alder reaction

Figure 7 End group activation of MNPs by retro Diels-Alder reaction and dye conjugation onto maleimide groups terminated iron oxide MNPs by thiol-ene chemistry (a) dye attached MNPs, (b) control, (c) free dye.


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Maleimide groups modified MNPs were used for dye conjugation via Michael addition after deprotection of furan. Fe3O4@PEGMEA@MAL3 nanoparticles were conjugated with BODIPY-SH in chloroform at 37 oC for 16 h (Fig. 7). The functionalization could be achieved under mild and reagent free conditions. After the conjugation, MNPs were dispersed in water and were highly fluorescent under UV illumination, indicating successful functionalization (Fig. 7). As a control experiment, Fe3O4@PEGMEA@pMAL3 MNPs bearing protected maleimide groups were used. The experiment was conducted under the same conditions; however, thiol bearing dye did not react with protected maleimide groups as inferred from lack of any fluorescence under UV illumination. Dye functionalization onto MNPs’ surface was also examined by fluorescence spectroscopy using dilute THF dispersion. As expected, the MNPs gave a strong emission maximum at 505 nm, close to that of the free dye at 515 nm (Fig. S13). Dye functionalization of MNPs carrying different amount of maleimide groups were investigated

by

UV-vis

spectroscopy.

The

experiment

was

carried

out

for

Fe3O4@PEGMEA@MAL1 and Fe3O4@PEGMEA@MAL3 to observe a clear difference. Dye conjugated MNPs were dispersed in THF for UV-vis analysis. Characteristic absorbance of the BODIPY dye is observed at 499 nm. When the same amount of BODIPY dye was used for conjugation onto nanoparticles containing different amounts of maleimide group, a higher UV absorbance peak intensity belonging to the dye was observed for the MNP containing higher amount of maleimide groups on the MNP surface (Fig. S12).


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Scheme 5 Attachment of targeting peptide (cRGDfC) and imaging dye (BODIPY-SH) onto the Fe3O4@PEGMEA@MAL3 MNPs. Lastly, cRGDfC, a cyclic peptide targeting the overexpressed integrin receptors in several types of cancer, and the fluorescent dye, BODIPY were conjugated onto the MNP surface (Fe3O4@PEGMEA@MAL3) via Michael addition reaction to evaluate these multifunctional constructs for targeted cellular imaging (Scheme 5). Conjugation onto the nanoparticle surface was carried out using cRGDfC and BODIPY-SH simultaneously. The attachment of the cRGDfC peptide onto nanoparticle surface was confirmed by bicinchoninic acid assay (BCA) analysis (Fig. S14). Amount attached onto MNP surface was calculated as 9.78 µg/mg nanoparticle by using a calibration curve using UV absorbance values for standard peptide solutions at 562 nm. Cytotoxicity of cRGD attached MNPs was evaluated on MDA-MB-231 human breast cancer cell lines. The results showed that cRGD conjugated MNPs did not have cytotoxic effect on the cell lines as indicated in Fig. 8. This safe aspect of the peptide functionalized polymer coated suggests them to be appropriate for biological applications.


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Figure 8 Cell cytotoxicity evaluation of cRGD attached Fe3O4@PEGMEA@MAL3 MNPs with different concentrations over MDA-MB-231 cells.

The uptake of cRGDfC conjugated MNPs was examined over integrin overpressing adenocarcinoma MDA-MB-231 human breast cell line. Cellular uptake of fluorescent magnetic nanoparticles was investigated using fluorescence microscopy. Cultured cells were treated with dye conjugated Fe3O4@PEGMEA@MAL3 MNPs and incubated at 37 °C. Cell internalization experiments were investigated at 3 h, 6 h and 24 h. Cell nuclei were detected by staining with 4′,6-diamidino-2-phenylindole (DAPI), which can be visualized by its blue fluorescence. As expected, it was observed that the cellular internalization increased upon increasing the time of incubation for both MNPs. However, it can be seen that after 6 h and 24 h, the green fluorescence was remarkably higher for cells treated with cRGD- and BODIPY-conjugated MNPs when compared to the cells treated with MNPs conjugated with only BODIPY (Fig. 9). Thus, MDA-MB-231 cells with over-expressed integrin receptors could be targeted and visualized using the targeting ligand and imaging agent containing MNPs that were readily obtained using the polymer coated MNPs.


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Figure 9 Merged fluorescence images of MDA-MB-231 cells following treatment with a) BODIPY attached, b) BODIPY and cRGD attached Fe3O4@PEGMEA@MAL3 NPs.

CONCLUSIONS The approach to obtain a library of ‘clickable’ polymer-coated magnetic nanoparticles reported here expands the tool box of building blocks for rapid fabrication of multifunctional nanoparticles. Polymer brush coated parent magnetic nanoparticles were modified to obtain different types of surface reactive ‘clickable’ nanoparticles. This expands the availability of various conjugation strategies, thus allowing facile generation of a diverse library of materials originating from the same batch of nanoparticles. In particular, polymeric brushes synthesized using RAFT can be modified with clickable functional groups like azides and maleimides. Thus modified MNPs can be decorated with alkyne-containing and thiol-containing functional


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moieties like peptides and dyes. The approach outlined here is not limited to the installation of azides and maleimides but can be expanded to a variety of ‘clickable’ groups. Multifunctional materials fabricated here benefit from the magnetic nature of the metal-oxide core that can enable localization as well as imaging; the polymeric shell that enhances dispersibility in biological media as well as surface functionalities that enables targeting and optical imaging. Development of such efficient and rapid protocols for diversification of functional nanomaterials promises to enable identification of lead candidates for various applications such as imaging and delivery using high throughput protocols. ASSOCIATED CONTENT The supporting information is available free of charge on the ACS Publications Website at DOI:xxxxxxx. 1

H and

13

C NMR spectra of DOPA-CTA; synthetic scheme and 1H and

13

C NMR spectra of

azobis-N3; magnetization curve of MNPs; images of water dispersions of MNPs; DLS and TEM analysis of Fe3O4@OA, Fe3O4@CTA and Fe3O4@PEGMEA; UV-vis spectra of MNPs before and after radical exchange reaction; DLS and TEM analysis of end group modified MNPs; absorption and fluorescence spectra of dye conjugated MNPs and BCA protein quantification procedure. AUTHOR INFORMATION Corresponding Author: E-mail: [email protected], Tel: 902123597613. Notes The authors declare no competing financial interest.


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ACKNOWLEDGEMENTS R.S. acknowledges funding from Ministry of Development of Turkey for grant No. 2009K120520. The authors thank Ahmet Genc for help with the cell uptake studies.

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TABLE OF CONTENT GRAPHIC


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Modular Fabrication of Polymer Brush Coated Magnetic Nanoparticles

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