Superparamagnetic Iron Oxide Nanoparticles Stabilized with

Article Cite This: ACS Appl. Nano Mater. 2018, 1, 894−907

www.acsanm.org

Superparamagnetic Iron Oxide Nanoparticles Stabilized with Multidentate Block Copolymers for Optimal Vascular Contrast in T1‑Weighted Magnetic Resonance Imaging Wangchuan Xiao,†,⊥ Philippe Legros,‡,§,∥ Pascale Chevallier,‡,§ Jean Lagueux,‡ Jung Kwon Oh,*,† and Marc-André Fortin*,‡,§,∥ †

Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H4B 1R6, Canada Centre de recherche du Centre hospitalier universitaire de Québec, Université Laval, axe Médecine Régénératrice, Québec City, Québec G1L 3L5, Canada § Centre de recherche sur les matériaux avancés and ∥Department of Mining, Metallurgy and Materials Engineering, Université Laval, Québec City, Québec G1V 0A6, Canada ⊥ College of Resource and Chemical Engineering, Sanming University, Sanming 365004, China ‡

S Supporting Information *

ABSTRACT: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) have been used as vascular contrast agents in magnetic resonance imaging (MRI), mainly for their capacity to generate negative contrast. To use USPIOs as positive contrast agents, it is necessary to achieve increased colloidal stability and signal-enhancement performance. Their molecular coatings must be carefully chosen, so that the vascular blood-pool contrast agents lead to long blood turnover times. However, to avoid long-term toxicological effects, they must also be cleared rapidly through the urinary or gastrointestinal pathways. In this context, highly stable USPIOs showing “positive” contrast in MRI and optimal clearance rates call for the development of robust biocompatible molecular coatings. In the present study, USPIOs were stabilized with a multidentate block copolymer (MDBC), using a one-pot polyol synthesis method in the presence of a MDBC. Two types of MDBCs having pendant COOH groups in the anchoring block were developed: a polymer with linear-poly(ethylene glycol) (PEG) blocks and a polymer containing brushed-PEG blocks. The synthesized superparamagnetic Fe3O4 crystals were uniform (5−8 nm in diameter), showed ultrasmall hydrodynamic diameters in dynamic light scattering, and were stable in physiological liquids. MDBC-coated USPIOs were analyzed in relaxometry, and the formulations showing the strongest potential for T1weighted vascular imaging (r2/r1: ∼4) were selected for in vivo MRI. Intravascular injections performed in the mouse model indicated long blood retention times and high signal enhancement in MRI for nanoparticles coated with linear-PEG block coatings. These results also indicate that MDBC/USPIOs could be used in vascular MRI applications, where the nanoparticles must transit the blood for several hours, followed by an efficient clearance in the next days following injection. The use of MDBCs as nanoparticle coatings could open new possibilities in the design of USPIOs for targeted molecular MRI. KEYWORDS: multidentate block copolymer, USPIOs, nanoparticle ligands, superparamagnetic iron oxide nanoparticles, MRI contrast agents, nanoparticle biodistribution, relaxivity

■

therefore they enable finer visualization of vascular signatures. The properties of USPIOs used as “positive” contrast agents have been reported in several reviews and in the recent literature.6−12 USPIOs can provide “positive” contrast enhancement in the blood preferably when their sizes are as small as 99%), hexane, anhydrous ethanol (EtOH), and sodium hydroxide (NaOH; >99%) from Sigma-Aldrich as well as a Pierce bicinchoninic acid (BCA) protein assay kit from Bio-Rad were used as received. NanoPure water (18.2 MΩ) was used in all experiments. The synthesis and procedure of P1/MDBC and P2/MDBC are detailed in the Supporting Information. Synthesis of Aqueous MDBC/USPIO Colloids. In this study, two variants of P1 and a single variant of P2 were prepared. The synthesis of P1-A as a typical example is as follows: A 2.5 M NaOH stock solution prepared by ultrasonification of a mixture consisting of NaOH and DEG was kept at 80 °C. A mixture consisting of the purified and dried MDBC (0.39 g, equivalent to 1.2 mmol of COOH), FeCl3 (195 mg, 1.2 mmol), and DEG (9 mL) was heated to 190 °C in a silicon oil bath, followed by the quick addition of 2.4 mL of the 2.5 M NaOH stock solution. The resulting black mixture was heated to 200 °C and kept there for 5 min. After cooling to room temperature, excess EtOH was added to precipitate the product, which was collected by centrifugation (5000 rpm × 5 min), washed with EtOH three times, and then dried at room temperature in a vacuum oven for 3 h. The resultant black solids were redispersed in water (10 mL). They were further ultrafiltered by a Millipore 8050 stirred cell to remove inorganic and organic impurities. P1-B was prepared using the same methodology but with two times the concentration of the MDBC polymers, i.e., 2.4 mmol of COOH. P2 was prepared with a 1.3 M NaOH solution in DEG. Characterization of MDBC/USPIO Colloids. The size of the NPs was measured from transmission electron microscopy (TEM) images obtained with a FEI Tecnai G2 F20 200 kV Cryo scanning/ transmission electron microscope, operated at 200 kV. To prepare specimens, colloidal dispersions were dropped onto copper TEM grids (400 mesh, carbon-coated) and then allowed to dry in air at room temperature. The hydrodynamic diameters of aqueous MDBC/USPIO colloids were determined by dynamic light scattering (DLS). The measurements were performed at a fixed scattering angle of 175° at 25 °C with a Malvern Instruments Nano S ZEN1600 equipped with a 633 nm He−Ne gas laser. The ζ potential was measured on a Malvern Nano Zetasizer. The polymer content in MDBC/USPIOs was determined by thermogravimetric analysis (TGA) using a TA Instruments Q50 analyzer. Typically, the freeze-dried samples (5−10 mg) were placed in a platinum pan and heated from 25 to 600 °C at a heating rate of 20 °C/min under a nitrogen flow. The crystal structure was determined by X-ray diffraction (XRD) using X’pert Pro with Cu Kα radiation (λ = 1.54056 Å) at 40 kV and 40 mA. Finally, the magnetic properties were evaluated using a Lakeshore 7400 vibration sample magnetometer at 298 K between −1.8 and +1.8 T. X-ray Photoelectron Spectroscopy (XPS). Drops of aqueous suspensions of MDBC/USPIO colloids were deposited on Si substrates cleaned with TL2 and TL1 solutions, according to previously reported methodologies.47 The samples were then analyzed by XPS using a PHI 5600-ci spectrometer (Physical Electronics, Eden Prairie, MN). An achromatic Al X-ray source (1486.6 eV and 300 W) was used to record the survey spectra (1400−0 eV), while highresolution XPS (HRXPS) spectra (C 1s and O 1s peaks) were obtained using an achromatic Mg X-ray source (1253.6 eV and 300 W). No charge neutralization was applied for both survey and HRXPS spectra. The detection angle was set at 45° with respect to the surface, and the analyzed area was 0.005 cm2. The curve-fitting procedures for C 1s and O 1s were performed by means of a least-squares Gaussian− Lorentzian peak-fitting procedure, after Shirley background subtraction. The C 1s peaks were referenced at 285 eV (C−C and C−H). Attenuated-Total-Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The measurements were performed using ATR mode in a FTIR spectrometer (Agilent Cary 660 FTIR, Agilent Technologies, Mulgrave, Victoria, Australia), equipped with a deuterated L-alanine-doped triglycine sulfate detector and a Ge-coated KBr beam splitter. Aqueous suspensions of MDBC/USPIO colloids were directly deposited on Si crystals. Spectra were recorded in

SSI = ρ(1 − e−TR/ T1)e−TE/ T2 where ρ is the proton density (ρ = 1), TR is the repetition time (TR = 400 ms), and TE is the time to echo (TE = 10.8 ms). The relaxation rates (1/T1 and 1/T2) at which contrast agents accelerate the relaxation of protons are described with the following equation: 1 1 = + ri[Fe], Ti Tm

where i = 1, 2

where Tm is the relaxation time of the matrix, ri are the longitudinal and transversal relaxivities, and [Fe] is the molar concentration of iron. In Vitro MRI. Aliquots of MDBC/USPIO colloids were dispensed into a 96-well plate, inserted in a microplate radio-frequency (RF) coil, and imaged with a 1 T small-animal MRI system (M2M, Aspect Imaging, Shoham, Israel). A T1-weighted 2D spin−echo sequence was used as follows: TE = 10.8 ms; TR = 400 ms; fα = 90°; FOV = 70 mm; 1.4 mm slices with 0.1 mm gaps; dwell time = 16 μs; matrix = 200 × 200; 3 exc. In Vivo MRI. Six-week-old BALB/c female mice (Charles River, Montreal, Canada) were randomly divided into two groups of three animals each. All animal experiments were conducted under the guidelines of Université Laval and the Centre de recherche du Centre hospitalier universitaire de Québec’s animal ethical committee. One group was injected intravenously with the P2 compound and the other group with P1-A. Mice were first anaesthetized with 3% isoflurane in an induction box and transferred to the MRI mouse bed while kept 896

DOI: 10.1021/acsanm.7b00300 ACS Appl. Nano Mater. 2018, 1, 894−907

Article

ACS Applied Nano Materials under anesthesia by means of a nose cone integrated to the bed. The animals were continuously monitored for respiration with a smallanimal monitoring and gating system (model 1025T; SA Instruments, Stony Brook, NY). The mice were cannulated in the previously dilated caudal tail vein (30 G, winged needle), connected to a catheter prewashed with heparin 25 U mL−1 (0.25 cc of Heparin 10000 U diluted in 10 mL of 0.9% sodium chloride), and connected to the contrast media syringe (280 μm ID intramedic polyethylene tubing PE-10, 60 cm, total volume = 60 μL). The needle was secured with adhesive (3 M Vetbond). Protective gel (Lacri-Lube) was applied on the mice’s eyes. The mice were inserted in a 3.5-cm-diameter RF coil and scanned using an Aspect M2 compact high-performance MRI system (Aspect Imaging, Shoham, Israel). The mice were scanned using a T1-weighted 2D spin−echo sequence in coronal orientation. The scanning parameters were as follows: 26 slices of 0.8 mm each (0.1 mm slice gap); field-of-view = 100 mm, echo time/repetition time = 16.1/800 ms; dwell time = 25 μs; fα = 90°; total duration = 4 min 16 s. Two preinjection images were acquired as references (S0). The P1-A group (n = 3) was injected with 100 μL of NPs (8.3 mM Fe in 0.9% sodium chloride); the P2 group (n = 3) was also injected with 100 μL (2.3 mM Fe in 0.9% sodium chloride). The concentration of each suspension was adjusted up to a similar longitudinal relaxation time (T1, measured at 1.41 T; see the details in Relaxivity Measurements section). The mice were dynamically scanned for 2 h, followed by static acquisitions at 5, 24, 48 h, and then 7 days postinjection. For contrast enhancement analysis, regions of interest (ROIs) were drawn over sections of the abdominal aorta, left kidney, and liver. As controls, ROIs were drawn on leg muscles and on the background (air). The raw signal intensity (S) was extracted from the images using ImageJ software (version 1.50e; Wayne Rasband, National Institutes of Health, Washington, DC). Signal enhancement ratios were calculated as follows: S contrast enhancement = t = S0

In the present study, we used a one-pot synthesis to enable the rapid transfer of the colloids to aqueous conditions. A solution of NaOH as a hydrolytic reagent dissolved in DEG was injected into a mixture containing MDBCs, FeCl3, and DEG at 190−195 °C. A black solution was instantly formed, indicating nucleation of the iron oxide colloids. A series of MDBC/ USPIO variants were prepared by varying the experimental conditions such as the reaction time and precursor concentration ratios. Because the hydrodynamic size has possibly the strongest impact on blood retention in vivo, from our syntheses, we selected three products that show hydrodynamic diameters in a narrow range (4−7 nm; see Table 1). Two of Table 1. Characteristics and Properties of Aqueous MDBC/ USPIO Colloids Prepared by in Situ Fabrication method diameter (nm) MDBC/USPIOs P1-A P1-B P2 a

linear linear brushed

surface charge (mV)a

TEM

DLS

ζ potential

7.3 ± 1.4 6.5 ± 1.5 4.8 ± 1.3

12.8 ± 1.0 14.0 ± 0.2 9.9 ± 0.7

−28 −20 −1

ζ potential was measured in a buffer solution at a neutral pH.

these MDBC/USPIOs were stabilized with linear-PEG chains (named P1-A and P1-B), and a single variant of USPIO was stabilized with brushed-PEG chains (named P2). The latter is a polymer synthesized for comparison purposes, already studied by our group, that had shown very high colloidal stability but relatively short blood retention times.38 After purification by precipitation and ultrafiltration, the MDBC/USPIO colloids were lyophilized to yield dark powders. For later steps, they were easily dissolved in water by simple shaking (no sonication). The average diameter and distribution of MDBC/USPIO colloids (Figure 2) were evaluated from TEM images. For the linear-PEG-coated NPs, the P1-A colloids had a USPIO core size of 7.3 ± 1.4 nm; P1-B had a slightly lower diameter of 6.5 ± 1.5 nm, which could be caused with the use of a higher amount of NaOH that could have induced more nucleation. Compared with P1 colloids, P2 is composed of brushlike POEOMA chains coating the NPs. The core of P2 was found to be very small (4.8 ± 1.3 nm). This small size could be due to either the use of less NaOH or the bulkiness of POEOMA chains as coronas on USPIO surfaces, which can limit the growth of the NP cores after nucleation in DEG. Given the TEM analysis of the USPIO core size in a dried state, the hydrodynamic size of MDBC/USPIO colloids (P1-A, P1-B, and P2) dispersed in aqueous solutions were evaluated by DLS. The three aqueous colloids exhibited an average diameter ranging from 9 to 14 nm, with monomodal and narrow hydrodynamic diameter profiles (Table 1 and Figure S1). One may notice the slightly larger size of P1-B compared to P1-A. This could be explained by a slightly denser surface coating, leading to an increase in the hydrodynamic size. Further, ζpotential analysis revealed surface charges of −20 and −28 mV for P1-A and P1-B, respectively. We hypothesized that the strong presence of ionic moieties from free carboxylic acid groups present at the surface of the linear-PEG-coated particles (P1) confers a strongly negative surface charge to USPIO compared with the brushed-PEG chains (surface charge of −1 mV). The colloidal stability in different chemical environments is discussed later in the text. In our previous studies, we found a

∑ pixel valuet ∑ ROI area ∑ pixel value0 ∑ ROI area

■

RESULTS AND DISCUSSION Well-defined P1 (linear-PEG chains; PEO-b-PMAA) and P2 (brushed-PEG chains; POEOMA-b-PMAA) MDBCs were synthesized according to methodologies described in our previous papers.49 Our previous reports suggest that the design of MDBCs with longer-anchoring (PMAA) and hydrophilic POEOMA blocks is important to retain the excellent colloidal stability of aqueous MDBC/USPIO colloids in physiologically relevant conditions.45 Thus, the degree of polymerization (DP) of PMAA (i.e., the number of MAA units in the PMAA block) was kept at 25, consequently, PEO113-b-PMAA25 for P1 and POEOMA35-b-PMAA25 for P2 (note that the subscripts denote the DP of each block). After extensive purification to remove unbound copolymers and other impurities, the MDBCs were characterized using 1H NMR for chemical structures and gel permeation chromatography for molecular weights (see the Supporting Information) prior to the in situ fabrication of superparamagnetic colloids. Synthesis of MDBC/USPIO Colloids Using an in Situ Approach. In our earlier work, we produced brushlike POEOMA “P2”/MDBC and then proceeded to the coating of USPIOs by a conventional ligand-exchange process.38,45 Oleic acids were thereby removed and replaced by MDBCs in organic solvents. Then, the resulting MDBC/USPIOs were transferred to aqueous solutions, with a final core diameter of 4.2 nm (TEM) and a hydrodynamic diameter (DLS) on the order of 25 nm. This ligand exchange and transfer to aqueous conditions was tedious and implied material losses. 897


Article

ACS Applied Nano Materials

Figure 2. TEM images and diameter histograms for MDBC/USPIO colloids (P1-A, P1-B, and P2). Inset: HRTEM image used for crystallographic characterization (scale bar: 5 nm).

Figure 3. Physicochemical characterization of the MDBC/USPIOs prepared: (A) XRD patterns, (B) FTIR spectra, (C) magnetization curves normalized to the total mass of the MDBC/USPIO product measured, and (D) per unit mass of Fe3O4 (data extracted from the organic-to-inorganic mass ratios from TGA measurements).

ζ potential of −10 mV for brushed-PEG-coated USPIOs, with good colloidal stability in physiological conditions.38,45 Quantitative Physicochemical Analysis of MDBC/ USPIOs. The inset in Figure 2 reveals a typical example of the high crystallinity of the magnetic iron oxide NPs achieved by this one-pot synthesis technique [high-resolution TEM (HRTEM) images of P1-B]. The high temperature (≈200 °C) used to synthesize the NPs in DEG resulted in the formation of highly crystalline structures. The distance between two adjacent lattice fringes was measured to be 0.256 nm, which corresponds to the lattice spacing of (311) planes of cubic USPIOs. To

complete the crystallographic investigation, XRD measurements were conducted to determine the crystalline structure and to determine the different phases present in the selected USPIO colloids (lyophilized samples). Figure 3A shows the XRD patterns of both P1/USPIO and P2/USPIO colloids with different core USPIO sizes. Each system shows the pattern of peaks for the (220), (311), (400), (411), and (440) lattice planes, which could be associated with the spinel structure known for Fe3O4 magnetite crystals.46,50,51 The Debye− Scherrer formula was used to calculate the nanocrystal sizes from XRD spectra obtained with low scanning speed (Figure 898


Article

ACS Applied Nano Materials Table 2. Surface Composition of MDBC/USPIOs Determined by XPS Analyses in Survey and TGA XPS element content (wt %)

TGA

MDBC/USPIOs

C 1s

O 1s

Fe

O/C ratio

C/Fe ratio

polymer weight (wt %)

P1-A P1-B P2

45.7 ± 0.2 61.3 ± 0.4 69.2 ± 0.4

43.2 ± 0.8 33.6 ± 0.4 30.0 ± 1.0

11.2 ± 0.8 5.1 ± 0.1 1.1 ± 0.6

1.0 0.6 0.4

4.1 12.0 61.1

31.3 33.8 80.6

the C/Fe ratio was higher on P1-B than P1-A, which is consistent with the FTIR results. For brushed PEG, P2 exhibited a very low concentration of Fe, 1.1%, and its C/Fe ratio was 61.1. This observation corroborated the previous observations from FTIR (Fe−O/CO ratio) and TGA, meaning that P2 has a thick MDBC coating. Further, the P2 coating exhibited an O/C ratio close to the one expected based on the PEG structure, which was also evidenced by the presence of the C−O band at 286.5 eV in high resolution C 1s (Table S3). Overall, the FTIR and XPS results clearly highlight the difference in the composition and also the density of the coatings: less Fe was detected on P1-B and a lower ratio of FeO/CO in FTIR, suggesting a higher density of the polymer on the surface of the NPs. In contrast, P1-A exhibited a probable thinner coating. In fact, the higher the FeO/CO ratio based on FTIR, the higher the percentage of Fe detected in XPS, even if a similar mass loss was found in TGA. At last, P2 had a very high C/Fe ratio in XPS, a very low contribution of Fe−O in FTIR, and a significant mass loss in TGA, which results in a high density of brushed polymer on the surface. Therefore, all of these results show that the organic covering is strongly dependent on the concentration of precursors and the polymer structure, linear and brushed. Colloidal Stability in Physiological Environments. USPIOs must be stable in the blood upon intravenous injection. They should have limited interactions with serum proteins present in the blood in order to avoid their aggregation and to prolong their circulation time. Otherwise, they are easily opsonized and thus eliminated from the blood circulation. Here, the colloidal stability of aqueous MDBC/USPIOs was investigated by incubating them at 37 °C in various physiological conditions. DLS was used to follow any changes in the hydrodynamic size distribution profile. The diameter of the colloids remained unchanged in water, saline, and PBS. Furthermore, they exhibit high shelf stability in saline and PBS conditions (Figure S4) but also as long as 4 weeks in saline conditions (Figure S5). Colloids incubated with BSA proteins at 8 mg mL−1 showed no significant precipitation over 96 h. Quantitative analysis using BCA assay48 indicated >95% BSA remaining in the supernatant of the mixtures. These results suggest that aqueous MDBC/USPIOs exhibit strong colloidal stability in saline and in the presence of BSA at physiological pH. Evidences abound in the recent literature, confirming the tolerance of several cells to MDBC/PEG-coated USPIOs, including MDBC containing −COOH groups. Our recent cytotoxicity studies performed with USPIOs coated with MDBC catechol groups confirmed that HEK 273 and HeLa cancer cells exposed 48 h to high concentrations (200 μg mL−1) of these superparamagnetic colloids were not significantly affected [>80% viability confirmed with 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay].39,40 USPIOs coated with a polymer made of poly-

S2). From the peaks indexed to (311) and (400), their diameters were determined to be 5.2 nm for P1-B, 6.4 nm for P1-A, and 4.1 nm for P2. These sizes appeared to be slightly smaller but close to those determined by TEM measurements. In addition to XRD analysis, their magnetic properties were measured in a vibrating sample magnetometer (Figure 3C,D). The magnetization was normalized to the mass of the magnetic NPs present in each system, as well as to the specific mass attributed to the Fe3O4 content, according to the inorganic content measured by TGA (see Table 2). The following saturation mass susceptibilities were found: ∼50, 39, and 26 emu g−1 Fe3O4 for P1-A (7.3 nm), P1-B (6.5 nm), and P2 (4.8 nm), respectively. These values closely correlate with previously reported values for USPIOs of 5.6 and 4.84 nm (∼45 and ∼20 Am2 kg−1 or emu g−1 ferrite, respectively).3 These results confirm that larger core sizes lead to higher magnetic moments, according to the theoretical magnetic behavior of USPIOs.10 Promisingly, all three samples show no remanence and coercivity, indicating that MDBC/USPIO colloids prepared by the in situ fabrication method here are superparamagnetic at room temperature. FTIR was used to study the physiochemical properties of MDBC/USPIO colloids (Figure 3B). The characteristic Fe−O vibration band at 625 cm−1 was observed for all samples, suggesting the formation of iron oxide. Both P1-A and P1-B coated with linear-PEG chains show similar FTIR spectra, with the characteristic band of PEG at 1175 cm−1 (C−O vibration) and the carbonyl (CO) vibrations from ester groups at 1709 and 1715 cm−1, respectively. The brushed-PEG-coated P2 NPs exhibit similar FTIR spectra. However, one may notice different band ratios, in particular the PEG peak, which is very intense compared to those of P1-A and P1-B. We hypothesize that the bands characteristic of the polymer coating (CO) and iron oxide (Fe−O), exhibiting different intensities, are associated with a difference in the polymer density. Indeed, the ratio of Fe−O/CO is around 0.2 for P2, whereas it is higher for P1-B and P1-A, reaching 1.2 and 2, respectively. This suggests that P2 has denser USPIO coverage with PEG coating, compared with P1-B and P1-A. This result is also confirmed by TGA (Figure S3), used to determine the content of the organic copolymers in the MDBC/USPIOs, showing a higher polymer content in P2 USPIO/MDBCs (Table 2). P2 USPIO/MDBCs had a polymer content as high as 80.6%, while it was lower for linear-PEG chain samples: 31.3 and 33.8 wt % for P1-A and P1B, respectively. Moreover, the surface coverage (ligands per square nanometer) was calculated and found to be similar for both P1/MDBCs (∼0.26 ligand nm−2 per USPIOs), while it was denser for P2 (0.81 ligand nm−2 per USPIOs) (Table S2). To strengthen the previous results, XPS analyses were performed to measure the surface atomic composition of MDBC/USPIOs (complete XPS data are available in Table S3). As summarized in Table 2, regarding P1 USPIOs with linear-PEG chains, the results from XPS analyses indicate that less Fe was detected for P1-B, compared with P1-A, although 899


Article


Figure 4. (A) Relaxation rates (1/T1 and 1/T2) of USPIO/MDBC in a saline medium at 1.41 T. (B) Theoretical (dotted line) and experimental (full line) contrast enhancement of in vitro T1-weighted MRI for P1 and P2 colloids at 1.0 T. (C) Corresponding 96-well-plate MRI for various Fe concentrations (numeric values given in millimolar of Fe; CTL = control).

obtained in the majority of studies reported on similar systems of USPIOs.38,39 On the other hand, the particles covered with brushed-PEG polymers (P2) showed lower longitudinal relaxivities, while keeping r2/r1 ratios on the same order of magnitude as those of P1 products: for P2, r1 = 5.3 mM−1 s−1, r2 = 20.7 mM−1 s−1, and r2/r1 = 3.8 (TEM diameter = 4.8 nm). Overall, these three products confirmed their strong potential as T1-weighted contrast agents; they are in line with our previous studies with brushed-PEG/MDBC-coated USPIOs (r1 = 12.1 mM−1 s−1 and r2/r1 = 3.5).38,45 By comparison, typical ultrasmall iron oxide contrast agents reported in the literature and showing good properties as “positive” contrast agents showed the following values: r2/r1 = 3.6 and r1 = 10.7 mM−1 s−1 (commercially available Supravist SHU-555C);52 r2/r1 = 3.4− 6.1 and r1 = 4.5−4.8 mM−1 s−1 for PEGylated ultrasmall NPs11,53 as well as USPIOs stabilized with COOH-bearing MDBCs.38,45 The positive contrast enhancement properties of these products were confirmed with a small-animal MRI scanner operating at clinical magnetic field strength (1.0 T; Figure 4B,C). The contrast enhancement was evaluated theoretically and compared to the actual experimental result.54 We found good correspondence with the experimental (done with a 96well plate) curve, suggesting an interesting potential as a

(isobutylene-alt-maleic anhydride) chains with PEG moieties and dopamine anchoring groups were well-tolerated by HeLa cells up to concentrations of 200 μg mL−1 (>90% viability confirmed with the MTT assay and 24 h incubation).37 Other studies have confirmed that USPIOs coated with PEG/MDBC containing phosphonate groups are tolerated at doses as high as 10 mM Fe in NIH/3T3 and RAW264.7 cells (WST-1 test; 24 h incubation; >80% viability).34 Relaxometric and in Vitro MRI Analyses. The relaxometric properties of the MDBC-stabilized colloids were measured at clinical magnetic field strengths (1.41 T, corresponding to 60 MHz; Figure 4). Longitudinal and transverse magnetic relaxation times (T1 and T2, respectively) were measured upon dilution of the colloids that showed both small particle size distributions (based on TEM) and narrow hydrodynamic diameter profiles (based on DLS), as listed in Table 1. From the slope of the relaxometric curves presented in Figure 4A, the colloids covered with linear-PEG blocks (P1) showed relaxivities as follows: P1-A, r1 = 18.8 mM−1 s−1, r2 = 76.7 mM−1 s−1, and r2/r1 = 4.1 (TEM diameter = 7.3 nm); for P1−B, r1 = 15.1 mM−1 s−1, r2 = 63.9 mM−1 s−1 and r2/r1 = 4.0 (TEM diameter = 6.5 nm), respectively. Both products revealed r2/r1 ratios close to 4.0, which is adequate for T1-weighted MRI, and their r1 values of 15−19 mM−1 s−1 were higher than those 900


Article


Figure 5. Dynamic contrast-enhanced MRI follow-up study after injection of (a) linear-PEG-coated iron oxide NPs (P1-A) and (b) brushed polymer-coated iron oxide NPs (P2). The arrows point to the liver, whereas the arrowheads point to the blood signal in the abdominal vessels.

Figure 6. Signal enhancement curves obtained from a dynamic contrast-enhanced MRI follow-up study after injection of linear-PEG-coated iron oxide NPs (P1-A; red curves) and brushed polymer-coated iron oxide NPs (P2; blue curves). The signal enhancement follow-up curves are displayed for the vascular system (A and B), liver (C and D), and spleen (E and F) for short-term time scale (minutes; left graphs) and long-term scale (days; right graphs).

covered with the brushed-PEG blocks (P2), the signal enhancement peaked at higher Fe concentrations (0.81−0.83 mM). The latter results are in agreement with a previous study in which we measured in similar conditions, the signal

contrast agent. The relaxometric properties of the P1 compounds (linear PEG) were optimal for T1-weighted imaging, with signal peaks obtained at concentrations as low as 0.27 mM Fe for P1-A and 0.37 mM Fe for P1-B. For NPs 901


Article


upon injection, the two injections were calibrated at the same T1 value. Because the relaxivities of both contrast agents were not equivalent, this implied different Fe concentrations per injection. More Fe was injected in P2 compared with the P1-A injection, for an equivalent blood signal enhancement effect in the first hour after injection (as confirmed in Figure 6A). The gradual blood signal enhancement increase observed for P2 in the first 2 h postinjection suggests that these particles are more susceptible to interaction with the surface of blood vessels, blood cells, and proteins, compared with their P1-A counterparts. In fact, the TGA data analysis confirmed a strong surface density of ligands for the P2 product (0.81 ligand nm−2), and this is precisely the ligand that contains a very large number of potentially reactive −COOH groups. The abundance of −COOH groups at the surface of these NPs, coupled with the blood signal increase trend observed in the first 2 h postinjection, suggests that these particles are subject to several interactions in the blood. These factors could explain the progressive increase of the blood signal for P2, as suggested in Figure 6A. In addition to this, the higher Fe concentration of P2 could possibly lead to a slight NP agglomeration effect in the blood pool upon injection. A slight agglomeration of USPIOs in the blood could affect the overall signal in the first minutes of the dynamic scan. These agglomerates would be filtered by the liver after a few minutes only, such that only ultrasmall NPs would remain in the blood to produce a high vascular signal. In the first 2 h of dynamic scans, the MRI acquisition window was kept at 5 min per scan. Because of the animal ethics requirements, the mice could not be kept under anesthesia for more than 2 h. Following the first dynamic scan acquisition, the animals were anaesthetized again at t = 5, 24, and 48 h and 7 days for static scans (with the same scanning conditions and the same time window of 5 min). The decrease in the signal intensity observed in Figure 6B between t = 2 and 5 h was revealed at the data analysis step. Even though the number of acquisition scans between t = 1 and 5 h is too low to allow a precise fitting, it is evident that the signal enhancement, which is a direct indicator of the concentration of NPs in the blood, is still very high at t = 2 h, followed by a significant decrease at t = 5 h. In fact, dynamic contrast-enhanced MRI is frequently used in clinics as an indicator of the concentration of contrast agents in the blood pool.60 Therefore, Figure 6B suggests blood halflives for P1 and P2, included between t = 2 and 5 h. A precise validation of the blood retention time could be performed by nuclear imaging (radiolabeled particles and positron emission tomography) or by frequent blood sampling followed by elemental analysis of the Fe content. Blood half-lives observed for other reported ultrasmall PEGylated iron oxide NPs figure in the range 30−160 min.61,62 For commercial dextran-coated iron oxide NPs such as Feridex, Supravist (SHU-555 C), Ferumoxtran-10, and Ferumoxytol-7228, blood half-lives of 2, 6, 24−36, and 10−14 h, respectively, were reported.63 The PEGylated starch-coated USPIOs (NC100150, feroglose) reported a half-life of 6 h.63 Finally, Hyeon’s group reported extremely small (

Superparamagnetic Iron Oxide Nanoparticles Stabilized with

Recommend Documents