Iron Oxide Nanoparticles with Grafted Polymeric Analogue of Dimethyl

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Biological and Medical Applications of Materials and Interfaces

Iron Oxide Nanoparticles with Grafted Polymeric Analogue of DMSO as Potential MRI Contrast Agents Jiajun Yan, Sipei Li, Francis Cartieri, Zongyu Wang, T. Kevin Hitchens, Jody Leonardo, Saadyah E. Averick, and Krzysztof Matyjaszewski ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06416 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 11, 2018

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ACS Applied Materials & Interfaces

Iron Oxide Nanoparticles with Grafted Polymeric Analogue of DMSO as Potential MRI Contrast Agents Jiajun Yan† , Sipei Li† , Francis Cartieri‡, Zongyu Wang†, T. Kevin Hitchens§, Jody Leonardo, Saadyah E. Averick‡, Krzysztof Matyjaszewski†* †

Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United

States ‡

Neuroscience Disruptive Research Lab, Allegheny Health Network Neuroscience Institute,

Pittsburgh, Pennsylvania 15212, United States §

Animal Imaging Center and Department of Neurobiology, University of Pittsburgh, Pittsburgh,

Pennsylvania 15203, United States 

Neuroscience Institute Allegheny Health Network, Pittsburgh, Pennsylvania 15212, United

States

KEYWORDS: Iron oxide nanoparticles, superparamagnetic nanoparticles, polymeric analogue of DMSO, magnetic resonance imaging, MRI contrast agent, T2-weighted contrast agent

ACS Paragon Plus Environment

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ABSTRACT Novel water-dispersible hybrid iron oxide nanoparticles grafted with a polymeric analogue of dimethylsulfoxide (DMSO) were prepared. Superparamagnetic iron oxide (SPIO) nanoparticles with immobilized atom transfer radical polymerization (ATRP) initiators were prepare via an in situ method using 12-(2-bromoisobutyramido)dodecanoic acid (BiBADA) as a surface ligand/initiator. The initiator-functionalized particles were employed in a surface-initiated initiator for continuous activator regeneration (ICAR) ATRP to graft poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA, a polyacrylate analogue of DMSO) from the surface. The resulting hybrid nanoparticles showed a high magnetic relaxivity ratio (r2/r1) of 600 at 7 T in fetal bovine serum, and a good biocompatibility up to 1000 mg L-1.

INTRODUCTION Magnetic Resonance Imaging (MRI) is a broadly employed noninvasive diagnostic imaging technique. MRI provides excellent soft tissue contrast and is not limited by penetration depth, contrary to optical or ultrasound imaging techniques, and does not use ionizing radiation typical for computed tomography (CT) or positron emission tomography (PET).1 The phenomenon of nuclear magnetic resonance forms the basis of MRI. It uses radio frequency (RF) pulses to excite proton nuclear spins polarized by a static magnetic field and exploits the spin density and relaxation properties of water in different tissues to generate image contrast. To enhance diagnostic capabilities, paramagnetic metal-ion-based MRI contrast agents can be used.2 The most prevalent MRI contrast agents are chelates or macrocyclic complexes of gadolinium (III). Paramagnetic gadolinium increases the longitudinal relaxation rate of surrounding water protons and leads to


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image hyperintensity on T1-weighted images. However, free gadolinium ions have been linked to nephrogenic systemic fibrosis.3 Alternatively, iron is generally considered benign, being an essential element and required for the body to make hemoglobin. Superparamagnetic iron oxide (SPIO) nanoparticles can be used as MRI contrast agents because they induce local magnetic field gradients, dephasing surrounding water protons and yielding image hypointensity on T2-weighted images. Therefore, SPIO-based MRI contrast agents have been extensively studied,1 but there is only one FDA-approved iron oxide nanoparticle, Feraheme (ferumoxytol), which is indicated for the treatment of iron deficiency anemia in adult patients with chronic kidney disease and off-label as an MRI contrast agent. Surface modification with hydrophilic and biocompatible ligands is necessary to disperse SPIO in a physiological environment.4 For example, Poly(ethylene glycol) (PEG)-grafted SPIO was applied to tracking T-cells in vivo.5 The synthesis of SPIO with in situ tethered ATRP initiators was recently described.6 Oligo(ethylene glycol methyl ether) acrylate (OEGA) monomer units were grafted from the surface. A high relaxivity ratio (r2/r1) was observed in a magnetic field of 0.47 T at 37 °C. Recently, biocompatible polymeric analogues of dimethylsulfoxide (DMSO) were introduced,7, 8 which feature both high hydrophilicity and low cytotoxicity. In comparison to poly(ethylene glycol) (PEG)-based polymeric ligand for magnetic nanoparticles, the polymeric analogue of DMSO potentially allows more water molecules in the vicinity of the nanoparticles. Hence, the influence of the nanoparticles to water molecules can be enhanced. In addition, the bulkier polyacrylate is expected to shield particle-particle interaction and its interaction with physiological macromolecules, and thus improve stability of the materials. The monomers are polymerizable in a controlled manner via atom transfer radical polymerization (ATRP).9,

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Surface-initiated ATRP (SI-ATRP) is one of the most robust methods to introduce polymers on an inorganic surface.11, 12 Use of initiator for continuous activator regeneration (ICAR) ATRP13 allows the minimal interaction with the reactive SPIO core and the coordinating sulfoxide group in the DMSO analogue. Herein, we present a novel iron-based potential MRI contrast agent synthesized via SIICAR ATRP of the DMSO mimicking monomer, 2-(methylsulfinyl)ethyl acrylate (MSEA), from the surface of the initiator-modified SPIO (Scheme 1). Scheme 1. Synthesis of the polymeric analogue of DMSO grafted from SPIO

(a) Synthesis of MSEA (the DMSO-like moiety is labeled in red). EDC: 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride; DMAP: 4-dimethylaminopyridine. (b) Synthesis of initiator-modified SPIO and subsequent SI-ICAR ATRP. AIBN: 2,2’azobisisobutyronitrile; Me6TREN: tris(2-dimethylaminoethyl)amine.

EXPERIMENTAL SECTION Materials Copper bromide (CuBr2, Aldrich, 99%), copper chloride (CuCl2, Aldrich, 99%), tris[2(dimethylamino)ethyl]amine (Me6TREN, Alfa, 99%), hydrochloric acid (HCl, Fisher, 36.5%38.0%), dimethylformamide (DMF, Fisher, 99.8%), sodium azide (NaN3, Aldrich, 99.5%)and fetal


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bovine serum (FBS, VWR, Seradigm Premium Grade) were used as received. 2,2’azobisisobutyronitrile (AIBN, Aldrich, 98%) was recrystallized from ethanol following literature procedures.14 The monomer, 2-(methylsulfinyl)ethyl acrylate (MSEA), and the hybrid macroinitiator, initiator-modified superparamagnetic iron oxide nanoparticles (SPIO-Br), were prepared according to our previous reports.6, 8 Deionized (DI) water was collected from a Millipore Milli-Q water purification system. Synthesis To synthesize SPIO-DMSO, 0.15/0.075/0.050 g of SPIO-Br, 1.05 g (6.47 mmol) of MSEA, and 28.9 µL of 20.0 mg/mL CuBr2 in DMF stock solution (2.58 µmol) were dispersed in 4.2 mL DMF. The dispersion was sonicated in a Branson M1800 ultrasonic cleaner overnight. 1.1 mg (6.5 µmol) of AIBN and 1.7 µL (6.5 µmol) of Me6TREN were added. The reaction mixture was degassed by 10 min of nitrogen bubbling. The reaction was transferred back into the ultrasonic cleaner set to 50 °C. The ultrasonication continued for 24 h before the reaction mixture was diluted with ~2 mL DMF. The diluted mixture was transferred into a regenerated cellulose dialysis tube and dialyzed against DI water for three cycles. Characterization The number-average molecular weight (Mn) and the dispersity (Mw/Mn) were determined by size exclusion chromatography (SEC). Polymer-grafted nanoparticles were dispersed in water and treated with HCl before SEC analysis. The SEC was conducted with a Waters 515 pump and Wyatt Optilab differential refractometer using PSS columns (Styrogel 105, 103, 102 Å) in 50 mM LiBr DMF solution as an eluent at 50 °C and at a flow rate of 1 mL min−1. Linear PMMA standards were used for GPC calibration. Transmission electron microscopy (TEM) was performed using a


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JEOL EX2000 electron microscope operated at 200 kV. Images were taken by amplitude and phase contrast using a Gatan Orius SC600 high-resolution camera. Aliquots of aqueous dispersions of SPIO containing ca. 5 mg/L Fe were cast on carbon-coated copper grids for the TEM. The hydrodynamic size distributions were measured at 25 °C by a Malvern Zetasize Nano ZS particle analyzer. The exact concentrations of Fe in the SPIO aqueous dispersions were determined by a Perkin Elmer AAnalyst 200 atomic absorption spectrometer (AAS) at 252.3 nm. The samples were diluted precisely to approximately 5 mg/L for accurate measurement. The SPIO-DMSO500, SPIODMSO1000, and SPIO-DMSO1500 samples were exactly diluted by a factor of 500, 250, and 500/3. Thermogravimetric analysis (TGA) was performed on a TA Instrument TGA 2950 and the data was processed with TA Universal Analysis software. The heating procedure involved four steps: (1) ramp up at 20 °C/min to 120 °C; (2) hold at 120 °C for 20 min; (3) high-resolution ramp up at 20 °C/min to 800 °C; (4) hold at 800 °C for 10 min. The inorganic fraction was determined by direct TGA measurement of magnetically precipitated samples. The organic contents of the samples were normalized to the weight loss between 120 °C and 800 °C. The grafting densities were calculated from the dry inorganic fraction using a previously reported equation.15 1

,

6

(1)

,

The value for fNP, in the equation, is the inorganic fraction measured by TGA after exclusion of any residual solvent; NA is the Avogadro number; Mn is the number average molecular weight of polymer brushes. To simplify the calculation, the SPIO particles were assumed to be Fe3O4 nanoparticles with diameter (d) of 10 nm and density (ρ) of 5.8×103 kg/m3.


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Magnetic Relaxivity Measurement The longitudinal relaxation time (T1) and transverse relaxation time (T2) were measured using a Bruker Minispec mq20 NMR Analyzer (20 MHz, 0.47 T) at iron concentrations of 1-40 mg/L in aqueous solutions. The T1 and T2 values of pure water were also measured and included into the linear fit of relaxivity in all samples. The MR probe temperature was set at 37.0 °C. T1 and T2 measurement were performed based on a reported procedure.16 Measurements were also made at 40 MHz using a 1.0 T Bruker Icon MRI running ParaVision 6.0.1 and at 300 MHz using a 7 T 30cm Bruker BioSpec AVANCE III scanner running ParaVision 6.0.1 and equipped with a 12 cm BGA12S2 gradient set and an 86mm birdcage RF coil. In the 1.0 T system, samples were samples were positioned as a row of 1.5 mL Eppendorf tubes for imaging with 50 × 50 mm FOV, 256 × 256 matrix, and 1.5 mm slice thickness for the T1 measurement and with 35 × 35 mm FOV, 60 × 60 matrix, and 1.25 mm slice thickness for the T2 measurement. T1 was measured with a RAREVTR sequence: TR ranging 25-6000 ms, TE = 12 ms, and 4 averages. T2 was measured with a MSME sequence: TR = 70 ms, TE ranging 7.79-60 ms, and 12 averages. In the 7 T system, samples were positioned as a row of 1.5 mL Eppendorf tubes for imaging with 60 × 30 mm FOV, 256 × 128 matrix, and 2 mm slice thickness. Temperature was maintained at 37 °C using warm air system with feedback from a fiber optic temperature probe positioned next to the samples (SA Instruments, Stony Brook, NY). T2 was measured with an MSME sequence: TR = 4000 ms, TE = 6.5 or 9 ms with an echo train of 64, and 16 averages. T1 was measured with a RAREVTR sequence: TR ranging from 300-5000 ms, TE = 8 ms, and 8 averages. ParaVision was used to determine the T1 and T2 values using a three parameter monoexponential fit of the signal intensity in each sample to the variable TR or TE, respectively. The recorded T1 and T2 are listed in Table S1 and S2.


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Stability Evaluation 20.0 µL of SPIO-DMSO500 aqueous dispersion containing 2.3×103 mg/L Fe was mixed with 10.0 µL 2.0% NaN3 aqueous dispersion and diluted to 1.0 mL with FBS. The size distribution was monitored by DLS over a period of 360 h while the sample was stored at 24 °C in a sealed disposable cuvette. Biocompatibility Test Human epithelial neuroblastoma cells (ATCC CRL#2266) were used to test the effect of the nanoparticle on cell viability, because of their sensitivity to cytotoxic polymers and because they represent both free-floating and adherent cell modalities. Cells were grown to a confluency of 80% and then sub-cultured with fresh growth medium into opaque 96-well culture plates at a concentration of 10,000 cells per well. Cells were grown in culture medium as per manufacturer recommendations (1:1 ratio of Eagle’s Minimum Essential Medium and F-12 growth medium with fetal calf serum added to 10% final volume). Two types of control wells were created without nanoparticle exposure: one treated with Triton X to kill 100% of cells, and the other with cells and media only. After 24-hours of incubation at 37 °C, 5% CO2, the cells were exposed to polymers of decreasing concentration by use of serial dilution, yielding dilution stages from 1 mg/mL to 0.5 µg/mL. Culture plates were incubated (conditions above) for an exposure period of 48 hours. We chose to estimate cell viability by means of adenosine triphosphate (ATP) assay based on the firefly luciferase enzyme (CellTiter-Glo® Luminescent Cell Viability Assay, Promega Corporation Cat.# G7570). Plates were removed from incubation and allowed to equilibrate to room temperature, after which an equal volume of ATP assay reagent (100 µL) was added to each well. Plates were shaken for 2 minutes to ensure cell lysis and exposure of membrane-bound ATP to stabilizer (ATPase inhibitor) and luciferase metabolism. Luminescent signal was allowed to


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stabilize for 10 minutes before luminance measurement and analysis via Biotek plate reader and Gen5 software. Nine wells/polymer dilution were read by averaging five luminance readings per well. Viability was estimated relative to luminance of control wells and reported as a percentage. This is appropriate given that the correspondence between average luminance and relative cell viability is very strong (R2 ~ 0.99). Average luminance variation data across triple replicates were used to calculate conservative estimates of error. Control and experimental data conforms to assumptions of normality with asymmetry and kurtosis between -2 and +2.17

RESULTS AND DISCUSSION Synthesis of Hybrid Nanoparticles The SPIO with in-situ immobilized SI-ATRP initiators (SPIO-Br) and the monomer, MSEA, were synthesized according to the previously reported procedures.6, 8 The SPIO-Br, MSEA, CuBr2, Me6TREN, and AIBN were dissolved in N,N-dimethylformamide (DMF) to prepare a dispersion with the initial monomer concentration [MSEA]0 = 1.5 mol/L. To simplify the calculation, the SPIO-Br particles were assumed to be spherical with an average diameter of 10 nm and an average initiator density of ~1 Br/nm-2. Three SI-ICAR ATRP reactions were performed with target degrees of polymerization (DPt) of 500, 1000, and 1500 over 24 h reaction period; annotated as SPIO-DMSO500, SPIO-DMSO1000, and SPIO-DMSO1500, respectively (Table 1). The DPt were tuned by only altering the SPIO-Br concentration to provide similar rates of polymerization. The reactions were performed in a 50 °C sonication bath to avoid the interaction between a stir bar and the SPIO particles. The resulting products were dialyzed against deionized (DI) water with a molecular weight cutoff of 25k to avoid aggregation.


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The synthesized hybrid nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Both SPIO-DMSO500 and SPIO-DMSO1000 displayed evenly distributed small clusters of SPIO nanoparticles with sizes of 100-200 nm. However, SPIODMSO1500 showed large clusters of aggregated nanoparticles (Figure 1). Table 1. Specifications of SPIO grafted with polymeric DMSO analoguesa Sample

DPt

Mnb

fSPIO,dryc

σ (nm-2)d

Dh (nm)e

SPIO-DMSO500

500

5.35×103 1.49

70.1%

0.46

199±3

SPIO-DMSO1000

1000

4.20×103 1.44

77.3%

0.41

158±2

SPIO-DMSO1500

1500

7.89×103 1.50

76.7%

0.22

704±51

Đb

a

Reaction condition: [SPIO-Br]0/[MSEA]0/[CuBr2]0/[Me6TREN]0/[AIBN]0 = b 1/500x/0.2x/0.5x/0.5x; [MSEA]0 = 1.5 mol/L in DMF; 50 °C; x = 1, 2, or 3. Determined by DMF SEC calibrated using linear PMMA standards. c Measured with TGA from magnetically separated samples. d Calculated from fSPIO,dry based on Eq. 1. e Z-averaged hydrodynamic size distribution determined by DLS in aqueous dispersions containing ca. 5 mg/L Fe.

Figure 1. Size distribution of SPIO-DMSO hybrid nanoparticles. (a-f) TEM images of SPIODMSO500 (a, b), SPIO-DMSO1000 (c, d), SPIO-DMSO1500 (e, f). Scale bars: (a, c, e) 200 nm; (b, d,


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f) 100 nm. (g) Intensity-weighted hydrodynamic size distribution of SPIO-DMSO500 (red), SPIODMSO1000 (cyan), SPIO-DMSO1500 (blue). Polymer grafts on the superparamagnetic nanoparticles allowed the thermal energy to resist the magnetic interaction. When a rare-earth magnet was placed next to a dispersion of SPIO-DMSO500, a bent surface of the dispersion was observed, while no obvious sedimentation on the wall was seen (Figure 2a). Indeed, it took as long as 72 h to precipitate a majority of SPIO-DMSO500 and SPIO-DMSO1000 from their aqueous dispersions. However, SPIO-DMSO1500 settled down spontaneously in a few minutes. An aliquot from each sample was etched with HCl to remove the SPIO core then the detached polymer brushes were analyzed with size exclusion chromatography (SEC) eluted with DMF, using linear poly(methyl methacrylate) (PMMA) standards. The inorganic fractions (fSPIO) were determined by direct TGA of the dry samples after magnetic precipitation. The weight loss pattern of all three SPIO-DMSO samples resembles that of the linear PMSEA. Two sharp decomposition stages starting at 200 °C and 500 °C, respectively, were observed in the linear polymer. However, both stages were to some extent delayed and broadened in the hybrid samples (Figure 3).18 The prolonged magnetic precipitation caused aggregation of the SPIO particles and probable detachment of polymer brushes from the surface. Therefore, the inorganic fractions could be overestimated (see Supplementary Information). The grafting densities were calculated from the number-average molecular weight (Mn) and fSPIO. As shown in Table 1, a much lower grafting density was likely the reason why SPIO-DMSO1500 tended to precipitate, while SPIO-DMSO500 showed no trace of sedimentation at a concentration of 90 mg/L Fe even after 12 months of storage under ambient condition (Figure 2b and c).


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Figure 2. Stability of SPIO-DMSO500 aqueous dispersion. (a) Photo of 20 min after a rare-earth magnet was placed next to a SPIO-DMSO500 aqueous dispersion containing 2.3×103 mg/L Fe. (b) Photo of a SPIO-DMSO500 aqueous dispersion containing 90 mg/L Fe was stored at ambient condition for 12 months. (c) Intensity-weighted size distribution of the corresponding SPIODMSO500 aqueous dispersion containing 90 mg/L Fe.

100

Normalized Weight (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80

SPIO-DMSO500

60

SPIO-DMSO1000 SPIO-DMSO1500

40

Linear PMSEA

20

0 0

200

400

600

800

Temperature (C)

Figure 3. TGA traces of SPIO-DMSO500 (red), SPIO-DMSO1000 (cyan), SPIO-DMSO1500 (blue), and linear PMSEA (gray). Sample weight after isotherm at 120 °C was normalized to 100%. The molecular weight distributions of grafted polymer brushes were rather broad. In a typical ICAR-ATRP reaction, the fraction of new chains from radicals fed into the reactions for activator


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regeneration is minor.13 However, in this case, the concentrations of all reagents, except for the macroinitiator, were kept parallel. Therefore, the contribution of AIBN to generation of new chains should be considered, especially for the reactions targeting high DPt. Therefore, a drop in the grafting density and loss of stability in an aqueous dispersion were seen in SPIO-DMSO1500, probably due to the superior reactivity of 2-bromoisobutyronitrile to that of the 2bromoisobutyramide.19 In addition to the free polymers, the mechanically induced background reaction may also contribute to the relative broad molecular weight distributions in all three products (see Supplementary Information).20, 21

Magnetic Relaxivity of Hybrid Particles A series of the three samples and Feraheme with Fe concentrations below 40 mg/L were prepared. The longitudinal and transverse magnetic relaxation times, T1 and T2, were first measured at 0.47 T and 37.0 °C (Figure 4a and Table 2).16 Transverse relaxivities (r2) of SPIO-DMSO500 and SPIODMSO1000 were found to be 339±16 and 564±29 mM-1 s-1, and relaxivity ratios (r2/r1) of 14.2±1.6 and 18.8±1.1, respectively, whereas Feraheme showed r2 = 80±4 mM-1 s-1 and r2/r1 = 2.07±0.03. In addition, the values were higher than other commercial iron-based contrast agents approved elsewhere or in development, including Feridex (r2 = 107 mM-1 s-1 and r2/r1 = 4.5),22 Resovist (r2 = 164 mM-1 s-1 and r2/r1 = 6.6),23 and Combidex (r2 = 78 mM-1 s-1 and r2/r1 = 2.1)24 under the same measurement conditions. The relaxivity ratios were also higher than the value achieved from the same SPIO grafted with poly(oligo(ethylene glycol) methyl ether acrylate-480).6 Such an improvement might be a result of the presence of smaller and more hydrophilic8 polymer ligands. The high hydrophilicity of polyMSEA and moderate aggregation of the SPIO in the hybrid material (Figure 1) might contribute to the enhancement of the r2.25,

26

The sample SPIO-


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DMSO1000 showed a similar extent of aggregation to that of SPIO-DMSO500 (Figure 1), but apparently more linear impurities generated from a higher concentration of AIBN was observed. Therefore, SPIO-DMSO500 was focused on in the further characterizations. Interestingly, the sample SPIO-DMSO1500 showed a positive trend of both T1 and T2 with the increase of Fe concentrations due to the extensive aggregation (Figure 1 and Table S1). The sample SPIO-DMSO500 and Feraheme were further characterized at 1.0 T and 7 T using preclinical MRI scanners. Under such conditions, closer to actual applications, r1 of both samples drop significantly as the magnetic flux density increased (Figure 4c and Table 2). The r2 of Feraheme decreased at 1.0 T and increased by two orders of magnitude at 7 T. A U-shaped r2 profile was observed in other commercial contrast agents.27 In contrast, r2 of SPIO-DMSO500 decreased steadily as the magnetic flux density increased, but it remained in the same order of magnitude.

Figure 4. (a) 1/T1 (open) and 1/T2 (solid) of water protons in Feraheme (gray), SPIO-DMSO500 (red), and SPIO-DMSO1000 (cyan) aqueous dispersions measured at 0.47 T plotted against the Fe concentrations, and their corresponding linear fitting (dashed and solid lines, respectively). (b) calculated magnetic relaxivity ratios based on the slope of the fitted linear relation. (c) Change of r1 (open squares and dashed lines) and r2 (solid circles and lines) of Feraheme (gray) and SPIODMSO500 (red) over a range of magnetic flux densities from 0.47 T to 7 T.


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Therefore, the relaxivities ratios of both samples were similar to those at 0.47 T, but they increased significantly at 7 T (Figure 5b and Table 2). At any of the three magnetic flux densities tested, the ratio of SPIO-DMSO500 was still approximately 10 times higher than that of Feraheme. The plots of signal decay versus echo time (Figure 5c) clearly showed how SPIO-DMSO500 at various Fe concentrations affected the relaxation of water protons. The T2-weighted image (Figure 5c, inset) with an echo time (TE) of 9 ms over samples at five different Fe concentrations demonstrates the contrast induced by the increase of Fe concentration. Both Feraheme and SPIO-DMSO500 were characterized in a physiological buffer, fetal bovine serum (FBS), to mimic in vivo conditions. The serum caused the r2 of both samples to drop, while the r1 of Feraheme seemed to be less influenced. Consequently, the relaxivity ratio of Feraheme dropped slightly in FBS, but that of SPIO-DMSO500 increased to 600 (Figure 5b).

Figure 5. (a) 1/T1 (open) and 1/T2 (solid) of water protons in Feraheme (gray and black) and SPIODMSO500 (red and cyan) aqueous dispersions (gray and red) and FBS dispersions (black and cyan) measured at 7 T plotted against the Fe concentrations, and their corresponding linear fitting (dashed and solid lines, respectively). (b) calculated magnetic relaxivity ratios based on the slope of the fitted linear relation. (c) Measured decay of normalized signal intensity (i.e. transverse magnetization, Mxy, red symbols) over the TE of water protons in SPIO-DMSO500 aqueous dispersions with various Fe concentrations and their corresponding signal exponential decay fitting


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(solid lines). Inset: T2-weight MRI image of the corresponding aqueous dispersions with an echo time of 9 ms. Table 2. Summary of magnetic relaxivities and relaxivity ratios at different field strengths and in different media. B (T)

Sample

r1 (mM-1 s-1)

r2 (mM-1 s-1)

r2/r1

0.47

Feraheme

38.5±0.4

80±4

2.07±0.03

SPIO-DMSO500

23.8±2.3

339±16

14.2±1.6

SPIO-DMSO1000

30.4±1.0

564±20

18.8±1.1

Feraheme

2.38±0.20

4.0±1.2

1.7±0.5

SPIO-DMSO500

20.3±1.7

332±26

16.4±1.9

Feraheme

2.56±0.07

114±3

45±2

Ferahemea

2.89±0.10

93.7±0.8

32±1

SPIO-DMSO500

0.62±0.10

296±9

480±78

SPIO-DMSO500a

0.33±0.05

198±1

600±91

1.0

7

a

Measured in FBS

Stability of Hybrid Nanoparticles in Physiological Buffer SPIO-DMSO500 displayed excellent stability in aqueous dispersions, making it difficult be separated (vide supra, Figure 2). To evaluate its tolerance to an environment closer to living organisms. A SPIO-DMSO500 dispersion containing 45 mg/L Fe in FBS was prepared. 0.02% sodium azide was added to suppress the growth of bacteria while maintaining the integrity of the proteins. The FBS dispersion was stored at 24 °C over a period of 360 h. No visible sedimentation was observed. DLS showed little shift in size distribution while almost identical single exponential decay of autocorrelation function was seen (Figure 6). At 360 h, a minor decrease in peak intensity


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of the hydrodynamic size distribution was observed. This implied a slight broadening of the size distribution, which was an indication of early-stage aggregation.

Figure 6. Stability evaluation by dynamic light scattering. (a) Intensity-weight hydrodynamic size distribution of 45 mg/L SPIO-DMSO500 in a FBS solution overtime. (b) The corresponding autocorrelation functions.

Biocompatibility of Hybrid Nanoparticles The sample SPIO-DMSO500 further underwent the 48-h adenosine triphosphate (ATP) biocompatibility assay using human epithelial neuroblastoma cells (Figure 7). The hybrid nanoparticles showed broad biocompatibility across the entire range of tested dilutions. Even at a concentration of 1 mg/mL, the viability was estimated to be 77.6% relative to control wells after


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48 hours of exposure. As the lower bound of the 95% confidence interval is above 70%, the hybrid nanoparticles are still non-cytotoxic at the highest tested concentration. For all other dilutions, 95% or more of the cells were viable after the exposure period. Indeed, cells treated with nanoparticles ≤333 mg/L result in viability estimates that do not differ significantly from untreated control cells. These viability results suggest that these new nanoparticles should be biocompatible in vivo and may be an effective preclinical or clinically T2 contrast agent, at a dose in the range used for other USPIO particles, for example, Feraheme is typically administered at a dose of 1-7.3 mg/kg.28 Fe concentration (mg/L) --

Survival Relative to Control (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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38

13

4.2

1.4

1000 333

111

37

0.46 0.15 0.05 0.02

--

100

50

0 12

4

1.4

0.5

--

Hybrid concentration (mg/L)

Figure 7. 48-h ATP biocompatibility assay of SPIO-DMSO500. CONCLUSION In summary, a novel type of potential MRI contrast agent was developed via combination of in situ synthesis of SPIO with immobilized SI-ATRP initiators and polymeric analogues of DMSO. The highly hydrophilic DMSO-based polymer enhanced the interaction between the magnetic particles and water protons. The moderately aggregated nature of the SPIO leads to higher values of r2 and r2/r1 ratio. The relaxivities and relaxivity ratios of the samples were evaluated at 0.47 T and 7 T and compared to those of Feraheme as well as reported values of other commercial contrast agents. Biocompatibility assay demonstrated the hybrid particles were non-cytotoxic in a wide


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concentration range. Therefore, the newly developed hybrid nanoparticles can be applied as a highly effective, low-dosage, and low-toxicity MRI contrast agent for preclinical or clinical imaging, such as cell tracking.

ASSOCIATED CONTENT Supporting Information. Additional discussion, magnetic relaxation times, and raw data of the biocompatibility assay. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions J.Y. and S.L. contributed equally to the work. ORCID Jiajun Yan: 0000-0003-3286-3268 T. Kevin Hitchens: 0000-0001-6576-6742 Saadyah E. Averick: 0000-0003-4775-2317 Krzysztof Matyjaszewski: 0000-0003-1960-3402

ACKNOWLEDGMENT


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Page 20 of 22

We thank the National Science Foundation (DMR-1501324) for financial support. J.Y. acknowledges the support from the Richard King Mellon Foundation Presidential Fellowship. We acknowledge Robert Greco of Carnegie Mellon University for his help with the AAS, Prof. Chien Ho and Dr. Li Liu for access to the 0.47 T NMR spectrometer, and Brandon Kujawaski and Mark Biedka of Allegheny Health Network for the assistance on the 1.0 T MRI experiments. This project used the 7 T MRI scanner at the UPCI In Vivo Imaging Facility that is supported in part by award P30CA047904 REFERENCES 1. Lee, N.; Hyeon, T. Designed Synthesis of Uniformly Sized Iron Oxide Nanoparticles for Efficient Magnetic Resonance Imaging Contrast Agents. Chem. Soc. Rev. 2012, 41, 2575-2589. 2. Na, H. B.; Song, I. C.; Hyeon, T. Inorganic Nanoparticles for MRI Contrast Agents. Adv. Mater. 2009, 21, 2133-2148. 3. Sieber, M. A.; Steger-Hartmann, T.; Lengsfeld, P.; Pietsch, H. Gadolinium-Based Contrast Agents and NSF: Evidence from Animal Experience. J. Magn. Reson. Imaging 2009, 30, 12681276. 4. Basuki, J. S.; Esser, L.; Zetterlund, P. B.; Whittaker, M. R.; Boyer, C.; Davis, T. P. 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. Macromolecules 2013, 46, 6038-6047. 5. Liu, L.; Ye, Q.; Wu, Y.; Hsieh, W.-Y.; Chen, C.-L.; Shen, H.-H.; Wang, S.-J.; Zhang, H.; Hitchens, T. K.; Ho, C. Tracking T-Cells in Vivo with a New Nano-Sized Mri Contrast Agent. Nanomedicine: Nanotechnology, Biology and Medicine 2012, 8, 1345-1354. 6. Yan, J.; Pan, X.; Wang, Z.; Lu, Z.; Wang, Y.; Liu, L.; Zhang, J.; Ho, C.; Bockstaller, M. R.; Matyjaszewski, K. A Fatty Acid-Inspired Tetherable Initiator for Surface-Initiated Atom Transfer Radical Polymerization. Chem. Mater. 2017, 29, 4963-4969. 7. Hennaux, P.; Laschewsky, A. Novel Nonionic Surfactants Based on Sulfoxides. 2. Homoand Copolymers. Colloid. Polym. Sci. 2003, 281, 807-814. 8. Li, S.; Chung, H. S.; Simakova, A.; Wang, Z.; Park, S.; Fu, L.; Cohen-Karni, D.; Averick, S.; Matyjaszewski, K. Biocompatible Polymeric Analogues of Dmso Prepared by Atom Transfer Radical Polymerization. Biomacromolecules 2017, 18, 475-482. 9. Wang, J. S.; Matyjaszewski, K. Controlled Radical Polymerization - Atom-Transfer Radical Polymerization in the Presence of Transition-Metal Complexes. J. Am. Chem. Soc. 1995, 117, 5614-5615. 10. Matyjaszewski, K.; Xia, J. Atom Transfer Radical Polymerization. Chem. Rev. 2001, 101, 2921-2990.


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Iron Oxide Nanoparticles with Grafted Polymeric Analogue of Dimethyl

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