Grafting of P(OEGA) Onto Magnetic Nanoparticles Using Cu(0

Article pubs.acs.org/Macromolecules

Grafting of P(OEGA) Onto Magnetic Nanoparticles Using Cu(0) Mediated Polymerization: Comparing Grafting “from” and “to” Approaches in the Search for the Optimal Material Design of Nanoparticle MRI Contrast Agents Johan S. Basuki,† Lars Esser,§ Per B. Zetterlund,‡ Michael R. Whittaker,§,* Cyrille Boyer,†,‡,* and Thomas P. Davis§,* †

Australian Centre for NanoMedicine, School of Chemical Engineering and ‡Centre for Advanced Macromolecular Design, University of New South Wales, 2052, Sydney, Australia § Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia S Supporting Information *

ABSTRACT: Superparamagnetic iron oxide nanoparticles (IONPs) have been studied extensively as negative contrast agents to enhance MRI efficacy. For optimal effective clinical use in T2/T2* weighted MRI imaging, the aim is to maximize relaxivity (r2) of IONPs, and minimize r1 relaxivity. A prerequisite for successful clinical use of magnetic nanoparticles is colloidal stability in biologically relevant media; biocompatible polymers with antifouling properties such as poly(ethylene glycol) (PEG) can be coated on the surface of IONPs, to improve stability and to impart longer blood circulation times. Our research aim was to optimize IONPs for use as contrast agents by achieving high grafting density and therefore colloidal stability, while retaining the magnetic properties of the IONP core. To attain the optimal material design the chemical functionalities and chain length of the polymeric layer must be precisely controlled. In this paper we describe the synthesis of poly(oligoethylene glycol acrylate) (P(OEGA)) functionalized magnetic iron oxide nanoparticles (IONP) made using a grafting “from” approach. Cu(0)-mediated living radical polymerization (LRP) was used to grow polymer chains of predetermined length from the surface of prefunctionalized IONPs. The polymers chain were further extended via an iterative addition of the same (or another) monomer with high efficiency demonstrating the retention of polymer chain end functionality. IONPs with different lengths of the P(OEGA) layer were also synthesized using a grafting “to” approach as a comparison study. Colloidal stabilities and MRI relaxivites of functionalized IONPs were investigated in both water and fetal calf serum (FCS). The grafting “from” approach proved to be superior to the grafting “to” approach as we were able to produce polymer coated IONPs with much higher r2 / r1 relaxivity ratios in water. At 9.4 T, the r2/r1 relaxivity values that we attained were about 6-fold higher than the commercial, clinically used, MRI contrast agent Resovist.

1. INTRODUCTION

relaxation times of the surrounding water protons are shortened as the local magnetic field gradient induced by magnetic nanoparticles accelerates the dephasing of proton spins in the water molecules.15 Higher relaxation rate (1/T2) of water in a specific tissue area (e.g., tumor), where the nanoparticles are localized, leads to more distinct contrast (dark/hypointense signals) in T2/T2*-weighted MRI.16 The ability to increase the relaxation rate of water protons imparted by the nanoparticulate contrast agent is expressed as r2 relaxivity, which normally remains relatively constant at increasing magnetic field. The longitudinal r1 relaxivity of superparamagnetic IONPs is usually much lower than r2 and decreases sharply at high magnetic field strength. In T2-weighted imaging it is essential

Iron oxide nanoparticles (IONPs) are of great interest for biomedical applications including magnetic resonance imaging (MRI),1,2 stem cell tracking,3,4 biomolecular separation (e.g., protein and DNA),5 hyperthermia,6 and drug delivery.7−9 IONPs have low toxicity,10 and they have been used in clinical applications as MRI negative contrast agents for the detection of liver lesions and adenocarcinoma.11,12 In comparison to other noninvasive imaging techniques, MRI has several advantages, including superb spatial resolution, good soft tissue contrast and zero irradiation (for example, when compared with CT scanning which uses X-rays).1,13 MRI uses a high strength magnetic field to align the nuclear magnetization of water protons in the body and a radio frequency pulse to alter the alignment in order to generate relaxation processes of these water nuclei.14 When superparamagnetic contrast agents like IONPs are introduced to a specific tissue, the T2 or transverse © XXXX American Chemical Society

Received: June 17, 2013 Revised: July 22, 2013

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dx.doi.org/10.1021/ma401250f | Macromolecules XXXX, XXX, XXX−XXX

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for a T2 contrast agent to exhibit high r2/r1 ratio for higher negative contrast.14,17 There have been previous studies on IONPs as T2 contrast agents (or negative contrast agents) investigating the relationship between the intrinsic properties of the nanoparticles (phase, size, shape, dopants, crystallinity and surface chemistry or coating18,19) and the resultant r2 relaxivity. Since the magnetic moment of a nanoparticle is proportional to its volume, the relaxation rate (1/T2) increases with nanoparticle size. Furthermore, the high surface-to-volume ratio can induce the surface spin canting effect causing the magnetization to decrease at smaller nanoparticle sizes.20 Smolensky et al.21 showed an increase of r2 relaxivity with increasing particle size (53 mM−1 s−1 for 4.9 nm and 125 mM−1 s−1 for 15 nm) compared to spherical nanocrystals (60 mM−1 s−1 for 16 nm). The increase of r2 until saturation was also observed in carboxydextran coated IONPs with higher hydrodynamic size (300 mM−1 s−1 for 60 nm) by Roohi et al.22 Since colloidal stability of the nanoparticles, especially in serum, is required for longer circulation time in the body, IONPs are usually coated to prevent agglomeration and subsequent uptake by the reticuloendothelial system.23,24 Coating parameters such as chemical structure, thickness, charge and hydrophilicity/ hydrophobicity may alter the r2 relaxivity of IONPs.25 Since the relaxation rate of water molecules depends on their interaction with the magnetic dipole of the IONPs (outer sphere spin−spin relaxation theory), the coating should facilitate the diffusion of water molecules.26 Polymer coatings such as PEG and dextran are normally used to stabilize IONPs, in particular PEG due to its “stealth” and antifouling properties.27 Tong et al. found a significant drop of r2 relaxivity when oleic acid functionalized IONPs (5 and 14 nm) were coated with PEG above a critical size/chain length (550 and 1 000 g/mol, respectively).28 A similar trend was observed by La Conte et al. using hydrophilic interactions with a phospholipid−PEG, resulting in an increase of r1 at longer chain lengths.29 On the other hand, when PEG coatings were grafted to IONPs via covalent bonds, the r2 increased with higher molecular weight of PEG (up to 5000 g/mol).30 Larsen et al.30 demonstrated that longer PEG chains improved the stability of IONPs increasing the r2 relaxivity and the blood circulation half-life time. Therefore, the effect of polymer coatings on IONPs does not solely depend on how the stabilizing polymer layer is anchored to the particle.31 Indeed, the anchoring group used to attach polymers onto IONPs can affect the spin organization of iron atoms, resulting in a decrease in magnetic properties (relaxivity). Polymer coatings on IONPs certainly influence the aggregation behavior of IONPs affecting the pharmacokinetics of IONPs as well as their in vivo efficiency as T2 contrast agents.22,30 There are many methods that can be used to synthesize functionalized superparamagnetic IONPs, such as coprecipitation and thermal decomposition, followed by polymer coating via a grafting “to” approach.32 Although the polymer can be attached using a range of modalities (e.g., hydrophobic, electrostatic or covalent bonding, i.e., via silane, dopamine or phosphonic acid),32 the grafting density is always limited for high molecular weight polymers.33,34 Surface-initiated controlled polymerization or grafting “from” approaches have been described using ATRP of PEG methacrylate from IONPs with immobilized initiators.35,36 Monomers such as dimethylaminoethyl methacrylate, glycidyl methacrylate and N-isopropylacrylamide have also been polymerized from the surface of

IONPs for various applications, e.g. antibacterial, cancer cell targeting with folate cellular receptors or to conjugate doxorubicin.37−39 To the best of our knowledge, a study on the effect of grafting “from” IONPs on the resultant r2 and r1 relaxivities has not been reported. In comparison to the conventional grafting “to” approach, higher grafting density might be expected that could improve both the stability of IONPs and their relaxivities.40,41 In this present work, we applied Cu(0)-mediated living radical polymerization (LRP) for the first time in a grafting “from” approach with IONPs. This polymerization technique, which was inspired by the previous work of Haddleton, Percec, and Matyjaszewski and coworkers, exhibits high control of the polymerization with high end-group fidelity at full monomer conversions.42−44 Haddleton et al. and our group demonstrated the “livingness” of this system by iterative multiblock polymerization of monomers without intermediate purification steps followed by end-group functionalization.45−47 Similarly, the iterative addition of sugar monomers to form quasi-block glycopolymers was also reported by Haddleton et al.48 Since poly(oligoethylene glycol acrylate) (P(OEGA)) has been utilized as an antifouling layer on IONPs, we investigated in this work the kinetics of Cu(0)-mediated LRP of this monomer using a phosphonate-bearing initiator. The resulting polymers were subsequently attached to IONPs by use of a grafting “to” approach. Furthermore, a grafting “from” approach was also employed to grow P(OEGA) from the surface of IONPs with successive addition of OEGA as well as different initial concentrations of OEGA. The colloidal stabilities of P(OEGA)-functionalized IONPs prepared using both approaches were then compared in serum. In addition, the effect of grafting method on the resulting magnetic properties (their r1 and r2 relaxivities) was investigated using MRI.

2. EXPERIMENTAL SECTION The synthesis, characterization and MRI measurement of P(OEGA)functionalized IONPs by grafting “to” and “from” approaches are reported in the Supporting Information.

3. RESULTS AND DISCUSSION Syntheses of P(OEGA)-functionalized IONPs via both Grafting “to” and “from” Approaches. Iron oxide nanoparticles (IONPs) were prepared by the coprecipitation method developed by Massart49,50 yielding magnetite Fe3O4 nanoparticles with an average size of 10 nm and a specific surface area of 105.5 ± 0.08 nm2 (characterization by XRD and BET isotherm as given in Figure S1, Supporting Information). In a second step, P(OEGA) polymers were grafted onto IONP surface to confer biocompatibility and colloidal stability in water and serum via two different techniques: grafting “to” and “from”. (a). Grafting “to”. In the grafting “to” approach polymers with phosphonic acid end-groups were prepared via Cu(0)mediated LRP. The phosphonic acid groups were subsequently employed for grafting “to” due to their strong affinity to the IONP surface. The Cu(0)-mediated polymerization was carried out in the presence of dimethyl-2-(2-bromoisobutyryloxy)ethyl)phosphonate (2) as initiator and OEGA as monomer with a mixture of copper(II) bromide and tris[2(dimethylamino)ethyl]amine (Me6TREN), using a molar ratio of [initiator 2]:[CuBr2]:[Me6TREN] = 1.00:0.10:0.36, and copper wire in a polar aprotic solvent, DMSO, based on a procedure reported in the literature (Scheme 1A).47,51 B


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Scheme 1. (A) Grafting “to” Approach with LRP of OEGA followed by Polymer Attachment to IONPs Surfaces and (B) Grafting “from” Approach with IONPs Surface-Initiated LRP of OEGA

Figure 1. (A) OEGA conversion and −ln (1-OEGA conversion) versus time; (B) evolution of the number-average molecular weight and PDI versus OEGA conversion; (C) molecular weight distributions of P(OEGA) homopolymers synthesized by Cu(0)-mediated LRP using [initiator 2]: [OEGA]:[CuBr2]:[Me6TREN] = 1.00:50:0.10:0.36; (D) molecular weight distributions of P(OEGA) homopolymers synthesized by Cu(0)mediated LRP using [initiator 2]:[OEGA] = 1.0:25, 1.0:50, and 1.0:100.0.

equation: Mn, theor. = ([OEGA]/[initiator]) × αOEGA × MWOEGA + MWinitiator, where αOEGA, MWOEGA, and MWinitiator donate OEGA conversion, molar mass of OEGA and initiator, respectively. However, at high monomer conversion, the SEC showed a typical high molecular weight shoulder, similar to what has been reported previously for Cu(0)-mediated LRP of OEGA.51 Nevertheless, a linear correlation between Mn and conversion as well as low dispersity (≤1.2) demonstrated the

Different ratios of [OEGA]:[initiator], i.e., 25:1, 50:1, and 100:1, were used to prepare phosphonate terminated P(OEGA) homopolymers with molecular weights ranging from 12 000 to 48 000 g/mol. The resulting first order plot was linear, and the number-average molecular weight (Mn) increased lineary with conversion (Figure 1, parts A and B). The experimental Mns obtained by SEC are in good agreement with the theoretical values, determined by the following C


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Table 1. Summary of Phosphonate Ester Terminated P(OEGA) Synthesized by Copper(0)-Mediated LRP polymers

αOEGA a

Mn,theor.b (g/mol)

Mn,NMRb (g/mol)

Mn,SECc (g/mol)

Đc

Mn,SECd (g/mol)

Đd

P(OEGA)-T25 P(OEGA)-T50 P(OEGA)-T100

0.92 0.88 0.86

11 300 21 400 41 500

11 500 20 300 37 600

11 700 20 800 31 200

1.11 1.14 1.18

10 500 21 800 34 000

1.14 1.19 1.19

OEGA conversion determined by NMR using the following equation: αOEGA = (I5.9−6.5 ppm/3)/(I4.0 ppm /2). bTheoretical molecular weights determined by the following equation: Mn, theor. = ([OEGA]/[initiator]) × αOEGA × MWOEGA + MWRAFT, with αOEGA, MWOEGA and MWRAFT correspond to OEGA conversion, molar mass of OEGA and RAFT, respectively. cNumber-average molecular weight of phosphonate ester terminated P(OEGA) determined by SEC using polystyrene calibration. dNumber-average molecular weight and dispersity (Đ) of phosphonic acid terminated P(OEGA) determined by SEC using polystyrene calibration. a

CO) signals were observed at 70.3 and 288.9 eV, respectively, in good agreement with the literature values.34b The amount of initiator (iByBr) on IONP@iByBr was quantified by thermal gravimetric analysis (TGA) (∼7% weight-loss, corresponding to 0.4 initiators/nm2). Cu(0)mediated LRP of OEGA was employed using the same [IONP@initiator]:[CuBr2]:[Me6TREN] ratio as for the grafting “to” approach in DMSO. For variation of chain lengths of P(OEGA), different molar ratios of [OEGA]:[IONP@ initiator] (25:1, 50:1, 100:1) were employed. Figure 3A shows conversion vs. time data as well as the corresponding first order plot for the grafting “from” approach. To determine the OEGA conversion, the addition of a standard (1,3,5-trioxane) to the reaction mixture was required (see Supporting Information). Interestingly, the polymerization appeared slower when the initiator was attached to the IONP surface. Indeed, about 91% conversion was reached after 7 h when the initiator was in solution, while 20 h was required to reach a similar conversion when the initiator was immobilized on IONPs. In addition, a minor retardation at low conversion (1 h) was observed for the Cu(0)-mediated LRP using IONP@ initiator. This might be attributed to the poor accessibility to monomer for the initiator attached to IONPs. The polymers were cleaved from the IONP surface using HCl (32 wt %), and subsequently analyzed by SEC. Figure 3B shows the molecular weight distribution of IONP@P(OEGA)-F25, -F50, and -F100, synthesized with [OEGA]:[initiator] = 25:1, 50:1 and 100:1, respectively. The molecular weight obtained by SEC (Table 2) is slightly higher than the theoretical values calculated using an initiator efficiency equal to 1 (i.e., all the initiators generate one polymer chain). This difference between the experimental molecular weight (determined by SEC) and the theoretical values is attributed to a low efficiency of the initiator attached to the IONP surface. As a control experiment, we treated P(OEGA) polymers in the presence of HCl to confirm that the polymers were not degraded (Figure S4). Iterative Addition of OEGA in IONPs Grafting “from” Approach. In order to investigate the end group fidelity of the grafted polymer via grafting “from”, iterative additions of OEGA monomers were performed.47 Once the Cu(0)mediated LRP reached a conversion of ∼95%, an additional 50 equiv of OEGA versus initiator and a CuBr2/Me6TREN mixture in DMSO were added. Fresh CuBr2/Me6TREN was injected to compensate for the amount of catalyst removed from the reaction mixture during sampling. This addition was repeated twice for IONP@P(OEGA)-F50 to yield IONP@ P(OEGA)-P(OEGA)-F100 and IONP@P(OEGA)-P(OEGA)P(OEGA)-F150 representing the first and second iterative additions of OEGA, respectively. The molecular weight distributions of these three polymers cleaved from their respective IONPs are given in Figure 4A. The increases of

control/“livingness” of this system. After polymer purification, the dimethyl ester phosphonate group was deprotected in the presence of trimethylsilyl bromide and purified by dialysis to yield phosphonic acid terminated P(OEGA) (Table 1). 31P NMR analysis confirmed the successful deprotection of the phosphonate ester group into a phosphonic acid group by a shift of phosphor signal from 29.3 to 20.8 ppm, while 1H NMR showed no degradation of the polymers. SEC analysis reveals a slight decrease of Mn with a similar dispersity after deprotection of phosphonate ester attributed to a change of hydrodynamic volume of the polymers. Subsequently, the phosphonic acid group was exploited to graft these polymers onto IONPs. The grafting “to” reaction is performed in water at 50 °C followed by nanoparticle purification by precipitation and redispersion. In the following, nanoparticles prepared by the grafting “to” approach are denoted IONP@P(OEGA)-Tn, whereas nanoparticles prepared by the grafting “from” approach are referred to as IONP@P(OEGA)-Fn (in both cases, n refers to the number of OEGA units). (b). Grafting “from”. In the grafting “from” approach, the phosphonate ester of initiator 2 was deprotected to yield phosphonic acid initiator 3, which was conjugated to the surface of IONPs (Scheme 1B).37 ATR-FTIR (Figure S2) confirmed the presence of initiator on IONPs with an ester signal (O−CO stretching) at 1730 cm−1 as well as the iron oxide bound phosphate signal at 1040 cm−1 (vibration of P− O−Fe).52 In addition, XPS confirmed the presence of bromine, phosphor and ester group (O−CO) on IONPs (Figure 2B). The P−O−Fe bond was characterized by the energy binding signal at 133.3 eV in [email protected] Bromine and ester (O−

Figure 2. XPS of (A) neat magnetite IONPs, (B) initiatorfunctionalized IONP@iByBr, (C) P(OEGA)-functionalized IONPs synthesized by grafting “from” approach, and (D) successive chain extensions with OEGA. D


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Figure 3. (A) OEGA conversion and −ln(1-OEGA conversion) versus time for Cu(0)-mediated LRP using grafting “from” with [OEGA]:[IONP@ initiator] = 50:1. (B) Molecular weight distribution of cleaved P(OEGA) homopolymers from IONPs using Cu(0)-mediated LRP, with [OEGA]: [IONP@initiator] = 25:1, 50:1, and 100:1, respectively.

polymer from the surface of IONPs was proven by the increase of carbon and oxygen signals after successive chain extensions. Indeed, the ratio of iron signal versus carbon or oxygen decreased with the chain length of P(OEGA) in IONP@ P(OEGA)-P(OEGA)-P(OEGA)-F150. Interestingly, we did not observe a signal for copper (Cu) in the XPS. Comparison of Grafting Densities for Grafting “to” and “from” Approaches. In this comparative study, we utilized IONPs with the same core sizes but with varying P(OEGA) layer thicknesses. The coating thickness and surface coupling approach have been previously found to influence stability and MRI relaxivities.18−20 The effect of the molecular weights of the P(OEGA) layers (synthesized by grafting “to” and “from” approaches) on the grafting density was investigated using TGA. In Figure 5, TGA measurements on the nanoparticles gave higher weight losses in IONP@ P(OEGA)-Fn (obtained by grafting “from”) than in IONP@ P(OEGA)-Tn (grafting “to”). The grafting density was calculated using the weight losses and the specific surface are of IONPs using the equation in Supporting Information. In the grafting “to” approach, a decrease of the grafting density was observed with an increase of the molecular weight attributed to an increase of the steric hindrance,34 while the grafting density for the grafting “from” approach did not change significantly with molecular weight (Table 3). It is noteworthy that the grafting density of IONP@ P(OEGA) obtained for IONPs@P(OEGA)-Fn (0.14−0.15

Table 2. SEC Results of Cleaved Polymers Synthesized by the Grafting “from” Approach polymers obtained after treatment with HCl

αOEGA a

Mn,theor.b (g/mol)

Mn,SECc (g/mol)

Đc

IONP@P(OEGA)-F25-HCl IONP@P(OEGA)-F50-HCl IONP@P(OEGA)-F100-HCl

0.90 0.87 0.69

11 100 21 300 33 400

19 000 23 400 30 000

1.19 1.2 1.25

a

OEGA conversion determined by NMR (see Supporting Information). bTheoretical molecular weights determined by the following equation: Mn,theor. = ([OEGA]/ [initiator]) × αOEGA × MWOEGA + MWinitiator, with αOEGA, MWOEGA, and MWinitiator correspond to OEGA conversion, molar mass of OEGA and initiator 3 respectively. c Number-average molecular weight and dispersity (Đ) of phosphonate ester terminated P(OEGA) determined by SEC using polystyrene calibration.

the chain lengths were consistent with the subsequent TGA results (Figure 4B), showing increasing weight losses at higher molecular weights. Nearly constant values in grafting density (∼0.10 chain.nm−2) were obtained for each chain extension (the calculation of grafting density based on the TGA results is explained in the Supporting Information). We compared TEM images of both neat magnetite IONPs and IONP@P(OEGA)-P(OEGA)-F100 revealing smaller aggregates, with a typical size around 50 nm, after polymer grafting (Figure S3). Characterization of the nanoparticles was also carried out using XPS (Figure 2C,D). The growth of

Figure 4. (A) Molecular weight distributions of cleaved P(OEGA) polymerized from the surface of IONPs following iterative additions of OEGA monomer. (B) TGA results of P(OEGA)-functionalized IONPs synthesized by the grafting “from” approach. E


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Figure 5. (A) TGA of IONP@P(OEGA)-Tn synthesized by grafting “to” approach. (B) TGA of IONP@P(OEGA)-Fn synthesized by grafting “from” approach.

significant decrease of accessibility to the neighboring initiator molecules grafted onto the IONP surface, limiting the overall efficiency of the initiation process. We therefore hypothesized that the grafting “from” approach could be made more efficient if the polymerization from the surface was started with less bulky monomers, followed by chain extension using OEGA, thus potentially generating higher grafting densities. Such a block copolymer approach from the surface of IONPs can be achieved easily by Cu(0)-mediated LRP without the burden of intermediate purification steps. We therefore started a grafting “from” approach using a hydrophilic monomer, 2-hydroxyethyl acrylate ([HEA]:[initiator] = 30:1) followed by the addition of OEGA ([OEGA]:[initiator] = 20:1) at full HEA conversion to yield IONP@P(HEA)-b-P(OEGA)-F20. Successful synthesis of block copolymers was confirmed by a shift of molecular weight distribution in the SEC. The resultant molecular weight of IONP@P(HEA)-b-P(OEGA)-F20 was comparable with IONP@P(OEGA)-F25 (Figure 6A). Despite similar degrees of polymerization of OEGA, higher weight loss was obtained in the TGA of IONP@P(HEA)-b-P(OEGA)-F20 when compared to IONP@P(OEGA)-F25 (Figure 6B). This is consistent with the grafting “from” approach using Cu(0)-mediated LRP of HEA enabling higher occupation of polymer on the surface of IONPs, giving higher access subsequently to the “bulky” OEGA monomer. As a result, the grafting density of IONP@P(HEA)b-P(OEGA)-F20 was increased by ∼2.5 fold relative to that of IONP@P(OEGA)-F25 and F50 (Table 3). The grafting “to”

Table 3. Grafting Density of IONP@P(OEGA)-Tn (Grafting “to”) and IONP@P(OEGA)-Fn (Grafting “from”) grafting “to” and “from” Samples

Mn,SECa (g/mol)

Đa

% weight lossb

grafting densityc (chains/nm2)

IONP@P(OEGA)T25 IONP@P(OEGA)T50 IONP@P(OEGA)T100 IONP@P(OEGA)-F25 IONP@P(OEGA)-F50 IONP@P(OEGA)F100 IONP@P(HEA)-bP(OEGA)-T20 IONP@P(HEA)-bP(OEGA)-F20

11 700

1.11

20

0.10 ± 0.02

20 800

1.14

30

0.09 ± 0.03

31 200

1.18

34

0.07 ± 0.03

19 000 23 400 30 000

1.19 1.20 1.22

39 45 50

0.15 ± 0.03 0.15 ± 0.03 0.14 ± 0.03

18 300

1.18

36

0.11 ± 0.03

17 500

1.20

58

0.34 ± 0.03

a Number-average molecular weight and dispersity (Đ) of P(OEGA) determined by SEC using polystyrene calibration. bWeight-loss determined by TGA. cGrafting density calculated by grafting density = [(weight-loss/Mn,SEC) × Na]/[mIONPs × SIONPs], with Mn,SEC, Na, mIONPs, and SIONPs correspond to number-average molecular weight, Avogadro number, mass of IONPs, and the specific surface area of IONPs, respectively.

chain/nm2) was only slightly higher than for the grafting “to” approach (0.07−0.10 chain/nm2). The insertion of the first OEGA units at the early stage of the polymerization results in a

Figure 6. (A) Molecular weight distributions of IONP@P(HEA)-b-P(OEGA)-F20 synthesized by grafting “from” approach using an iterative Cu(0)mediated LRP in the presence of HEA ([HEA]:[initiator] = 30:1), followed by OEGA ([OEGA]:[initiator] = 20:1). (B) TGA of IONP@P(HEA)F30 and IONP@P(HEA)-b-P(OEGA)-F20 in comparison with IONP@P(OEGA)-F25. F


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Scheme 2. Synthesis of Block Copolymer Functionalized IONP@P(HEA)-b-P(OEGA)-F20 Using a Grafting “from” Approach

(OEGA)-T100, F50 and F100) showed a slight increase of particle size after 24 h, probably due to protein adsorption.54 Nevertheless, based on visual observation the nanoparticles were still stable even after 4 days of incubation in FCS at 37 °C. IONP@P(HEA)-b-P(OEGA)-F20 exhibited remarkable colloidal stability in serum. A comparison of SEM images between neat IONP, IONP@P(OEGA)-T50 and F50 confirmed an improvement in stability (less aggregation) in P(OEGA)functionalized IONPs (Figure S7). MRI Relaxivities of IONPs Made Using both Grafting “to” and “from” Approaches. In a T2-weighted MRI scan, superparamagnetic iron oxide nanoparticles decrease the signal intensity due to enhanced T2 relaxation of water molecules, resulting by a negative contrast in MRI. In our experimental approach the nanoparticles synthesized by grafting “to” and “from” approaches were dispersed in water and fetal calf serum at different concentrations of iron. ICP−OES (inductively coupled plasma−atomic emission spectroscopy) was used to determine the concentration of the stock solution used for the dilution series. T2 relaxation times were measured at 9.4 T using a Multi-Slice-Multi-Echo (MSME) sequence. The ability of a contrast agent to change the relaxation rate is represented quantitatively as relaxivity (r1 or r2) and is based on the relationship below, where 1/T2 of medium is the intrinsic relaxation rate of water or FCS.31

approach using block P(HEA)-b-P(OEGA)-T20 copolymer was performed as a control experiment (Figure S5). The grafting density was determined to be equal to 0.1 chain/nm2. This value is close to the grafting density obtained for P(OEGA)-T25 (Table 3) and inferior to the grafting “from” approach using P(HEA)-b-P(OEGA)-F20 (Figure S6). Comparing the Colloidal Stability of IONPs Made by Either the Grafting “to” or “from” Approaches. The particle size of IONPs@P(OEGA) prepared by both grafting “to” and “from” approaches were investigated using DLS at a concentration of 0.5 mg/mL (Figure 7) in both water and fetal

1 1 = + r2C Fe T2 T2 of medium

Figure 7. Colloidal stability study of IONP@P(OEGA)-Tn (grafting “to”) and IONP@P(OEGA)-Fn (grafting “from”) in water and fetal calf serum (FCS) based on number-average particle diameter (nm) by DLS. Note: error bars correspond to the distribution of the particle size, for triplicate experiments (n = 3).

Plots of 1/T2 against iron concentration (CFe) are shown in the Supporting Information (IONP@P(OEGA)-T25 vs F25 in Figure S8 in the Supporting Information, IONP@P(OEGA)T50 vs F50 in Figure S9, IONP@P(OEGA)-T100 vs F100 in Figure S10 and IONP@P(HEA)-b-P(OEGA)-F20 in Figure S11). On the basis of the slope of the graphs, the r2 relaxivity values of each nanoparticle in water as well as in FCS were calculated for comparison (Figure 8). In water, r2 relaxivity of IONP@P(OEGA)-T25 was calculated to be ∼150 mM−1 s−1, which is typical for IONPs with particle size of 10−15 nm.29 The r2 relaxivity of these grafted “to” nanoparticles increased slightly with longer polymer chains probably resulting from the colloidal stability of the nanoparticles. IONP@P(OEGA)-Fn that was synthesized by the grafting “from” approach exhibited even higher r2 relaxivity in water. The r2 relaxivity IONP@ P(OEGA)-F50 (∼340 mM−1 s−1) was found comparable to the commercial IONP product Resovist at 9.4 T.55 The higher r2 relaxivity value obtained for IONP@P(OEGA)-Fn could be caused by a higher grafting density of the hydrophilic polymer increasing the retention of water molecules in the polymer layers, close to the magnetic field periphery of iron oxide.26 In serum, IONP@P(OEGA)-Tn formed larger aggregates presumably caused by the relatively low grafting density leading to protein adsorption. Although our MRI relaxivity measure-

calf serum (FCS). In water all the nanoparticles showed good colloidal stability except for IONP@P(OEGA)-T25. When the IONPs made by grafting “from” IONP@P(OEGA)-Fn were studied, an increase of particle size was observed with increasing Mn as expected. IONP@P(OEGA)-F100 was comparable in particle size to IONP@P(OEGA)-T100 and T50 in water. However, in FCS we observed a significant increase of particle size in IONP@ P(OEGA)-T50 and T25 as well as F25, which we attributed to protein adsorption indicating incomplete PEG shielding (short grafted chains53). Nanoparticles, such as IONP@P(OEGA)F50 and F100, were found to be stable even for a few months. Block copolymer functionalized IONP@P(HEA)-b-P(OEGA)F20 had a larger initial particle size than IONP@P(OEGA)-F25 and F50, consistent with a high density of hydrated P(HEA). In order to mimic the retention of nanoparticles in blood serum, all samples in FCS were incubated at 37 °C. After 24 h, slight precipitation of aggregated nanoparticles was visible (IONP@ P(OEGA)-T50, T25 and F25), caused by the adsorption of serum proteins. The remaining nanoparticles (IONP@PG


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through outer sphere spin−spin relaxation, the diffusion of the water molecules could be affected by the polymer layers.26 The r1 relaxivities of IONP@P(OEGA)-Tn in water were calculated to give 0.4−0.5 mM−1 s−1, which was lower than that reported for dextran coated Resovist (1.67 mM−1 s−1).1,21 This might be attributed to a more limited diffusion of water molecules in the PEG layers of IONPs, probably due to hydrogen bonds and steric hindrance imparted by PEG chains.2,60 In serum, IONP@P(OEGA)-Tn showed a slight reduction in r1 relaxivity, probably following protein adsorption leading to nanoparticle aggregation. Lower r1 relaxivity in the grafting “from” nanoparticles IONP@P(OEGA)-Fn was observed (0.2−0.3 mM−1 s−1). In addition to the effect of P(OEGA) chains on water diffusion, the iron oxide surface of IONP@ P(OEGA)-Fn was occupied with unreacted initiators. This hydrophobic layer could diminish interactions between the water molecules and iron oxide.61,62 In serum we did not observe any changes in the r1 relaxivity due to less protein adsorption for IONP@P(OEGA)-Fn. It was of interest that the r1 relaxivity of IONP@P(HEA)-b-P(OEGA) is slightly higher than IONP@P(OEGA)-Fn, which could be attributed to the hydrophilic layer of P(HEA) favoring the diffusion of water molecules to the surface of IONPs. The r2 and r1 relaxivity values in water and FCS are all summarized in Table S1 in the Supporting Information. The calculation of r2/r1 ratio was required to evaluate the efficiency of negative contrast in MRI. In comparison with the commercial product Resovist (r2 / r1 = 224),55 IONP@ P(OEGA)-Tn showed a slightly higher ratio, especially T100 with r2 / r1 = 494 in water (Figure 10A). In contrast, due to a

Figure 8. T2 relaxivity study of IONP@P(OEGA)-Tn (grafting “to”) and IONP@P(OEGA)-Fn (grafting “from”) in water and fetal calf serum (FCS) using 9.4 T MRI. Note: error bars correspond to the standard deviation for triplicate experiments (n = 3).

ments were conducted at low concentrations (≤0.05 mg/mL IONPs), the magnetic field induced the precipitation of IONP@P(OEGA)-T25 in FCS, thus lowering r2. Higher r2 relaxivity values in T50 and T100 were most likely caused by the clustering of the nanoparticles.56,57 In contrast, IONP@ P(OEGA)-Fn (grafting “from”) did not show major changes in r2 relaxivity from water to FCS as the medium, confirming the superior stability of these nanoparticles in serum. Our observations on the correlation between r2 relaxivity and the colloidal stability or aggregation behavior of IONPs was in accord with previous reports in the literature.58,59 A slight increase of r2 relaxivity was observed for IONP@P(HEA)-bP(OEGA) on going from water to serum. Nevertheless, its r2 relaxivity was slightly higher than IONP@P(OEGA)-F25 and similar to IONP@P(OEGA)-F100, which exhibited a similar number-average particle diameter. Since negative contrast agents require a high ratio of r2 / r1 relaxivities in T2-weighted MRI scans, we were interested in the measurement of T1 relaxation times as well. Measurements were conducted at different iron concentrations using a RAREVTR (rapid acquisition rapid echo with variable repetition time) sequence, to determine r1 relaxivities both in water and in FCS (Figure 9). Since r1 relaxivity is more influenced by the interaction between water molecules and iron oxide surfaces

Figure 9. T1 relaxivity study of IONP@P(OEGA)-Tn (grafting “to”) and IONP@P(OEGA)-Fn (grafting “from”) in water and fetal calf serum (FCS) using 9.4 T MRI. Note: error bars correspond to the standard deviation for triplicate experiments (n = 3).

Figure 10. r2/r1 ratio of IONP@P(OEGA)-Tn (grafting “to”) and IONP@P(OEGA)-Fn (grafting “from”): (A) in water and (B) fetal calf serum using 9.4 T MRI. H


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lower r1 relaxivity IONP@P(OEGA)-Fn exhibited 5−6 times higher r2/r1 ratio than Resovist, especially F50 with r2/r1 = 1412. In serum the r2/r1 ratio of IONP@P(OEGA)-Tn improved due to increasing r2 relaxivity (Figure 10B). Ultimately, the r2 and r1 relaxivity values of both nanoparticles synthesized by grafting “to” and “from” approaches were actually comparable in serum. A higher r2/r1 ratio of IONP@ P(OEGA)-F25 and T50 was most likely caused by aggregation in serum. Although the MRI relaxivity measurements were conducted in serum for clinical simulation, in reality, the body temperature (37 °C) would affect the r2/r1 ratio. It has been reported that higher temperatures decrease the r2 and increase the r1 relaxivity of IONPs.21 Therefore, it is important to develop stable IONPs that exhibit high enough r2/r1 ratio in serum for in vivo applications.63 Stable nanoparticles like IONP@P(OEGA)-F50, F100 and T100 have the potential to be utilized as negative MRI contrast agents in clinical applications because of their colloidal stability and high r2/r1 ratio in serum.

helpful discussions and advice on the design of the experimental setup. CB is thankful for his fellowships from Australian Research Council (APD-ARC and Future Fellowship).

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4. CONCLUSIONS In this article, two different grafting strategies, “from” and “to”, were employed to functionalize IONPs with P(OEGA). In the grafting from, we have successfully demonstrated that Cu(0)mediated living radical polymerization (LRP) of OEGA could be used to grow polymers from superparamagnetic IONPs. The grafting “from” approach allowed functionalization of IONPs with higher grafting density than grafting “to” methods. IONPs synthesized by grafting “from” approach exhibited better colloidal stability in serum than those made by a grafting “to” approach, especially at longer polymer chain lengths. In water the r2 relaxivity of IONPs synthesized by a grafting “from” approach was higher and the r1 relaxivity was lower than the IONPS made by a grafting “to” approach. In serum the r2 and r1 relaxivities of IONPs synthesized by grafting “from” approach did not deviate much from the values in water, unlike IONPs synthesized by the grafting “to” approach. This work demonstrates the promise of stabilizing and functionalizing IONPs with well controlled polymer layers, enabled using living radical polymerization methods.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, XRD, N2 BET isotherm, additional NMR spectra of initiator, P(OH)2-POEGA and block copolymers, ATR-FTIR spectra, dynamic light scattering (DLS), SEM pictures, TEM pictures, 1/T2 relaxivity measurement and thermal gravimetric analysis (TGA) (Figures S1−11 and Table S1). This material is available free of charge via the Internet at http://pubs.acs.org/.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: (M.R.W.) [email protected]; (C.B.) [email protected]; (T.P.D.) [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Nuclear Magnetic Resonance facility, the Biomedical Resource Imaging Laboratory and the Electron Microscope Unit at the Mark Wainwright Analytical Centre for I


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Grafting of P(OEGA) Onto Magnetic Nanoparticles Using Cu(0

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