Superparamagnetic Iron Oxide Nanoparticles Stabilized with

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Article

Superparamagnetic Iron Oxide Nanoparticles Stabilized with Multidentate Block Copolymers for Optimal Vascular Contrast in T-weighted MRI 1

Wangchuan Xiao, Philippe Legros, Pascale Chevallier, Jean Lagueux, Jung Kwon Oh, and Marc-André Fortin ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00300 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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ACS Applied Nano Materials

Superparamagnetic Iron Oxide Nanoparticles Stabilized with Multidentate Block Copolymers for Optimal Vascular Contrast in T1-weighted MRI Wangchuan Xiao,

a,e

Philippe Legros,

b,c,d

Pascale Chevallier,

Fortin

b,c

b

Jean Lagueux, Jung Kwon Oh,

a,*

Marc-André

b,c,d,*

a. Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, H4B 1R6, Canada

b. Centre de recherche du Centre hospitalier universitaire de Québec – Université Laval (CR-CHUQ), axe Médecine Régénératrice, Québec, G1L 3L5, Canada;

c. Centre de recherche sur les matériaux avancés (CERMA), Université Laval, Québec, G1V 0A6, Canada

d. Department of Mining, Metallurgy and Materials Engineering, Université Laval, Québec, G1V 0A6, Canada

e. College of Resource and Chemical Engineering, Sanming University, Sanming 365004, China

Keywords:

Multidentate block copolymer • USPIOs • nanoparticle ligands • superparamagnetic iron oxide

nanoparticles • MRI contrast agents • nanoparticle biodistribution • relaxivity

ACS Paragon Plus Environment 1

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ABSTRACT

Ultra-small 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 the vascular blood-pool contrast agents must lead to long blood turnover times. However, to avoid long-term toxicological effects, they must also be cleared rapidly through the urinary or the 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 multidentate block copolymer (MDBC), using a one-pot polyol synthesis method in the presence of MDBC. Two types of MDBC having pendant COOH groups in the anchoring block were developed: a polymer with linear-PEG blocks and a polymer containing brushed-PEG blocks. The synthesized superparamagnetic Fe3O4 crystals were uniform (5-8 nm in diameters), showed ultra-small hydrodynamic diameters in DLS, and were stable in physiological liquids. MDBCcoated USPIOs were analyzed in relaxometry, and the formulations showing the strongest potential for T1-weighted 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 the nanoparticles coated with linear-PEG blocks coatings. These results also indicate that MDBC/USPIOs could be used in vascular MRI imaging 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 MDBC as nanoparticle coatings could open new possibilities in the design of USPIOs for targeted molecular MRI.


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Fe3O4

vs

Fe3O4



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INTRODUCTION

Ultra-small superparamagnetic iron oxide nanoparticles (USPIOs) have been successfully applied as vascular contrast agents in magnetic resonance imaging (MRI).1–5 These products change the relaxation time of hydrogen protons present in their immediate vicinity, thus enabling the detection of small accumulations of magnetic colloids by MRI. This strategy is the basic concept behind the use of USPIOs as “molecular” contrast agents in vascular injections. The contrast effects produced by T1-weighted imaging contrast agents (“positive” contrast agents) are less affected by susceptibility artifacts and 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 5.5 nm with a very good individualization of the nanoparticles to avoid too rapid renal clearance. The surface ligands must be appropriately selected to delay the sequestration of USPIOs by macrophages occurring in the blood.5,17 A large majority of contrast-enhanced MRI scans are performed nowadays with injections of gadolinium (Gd3+) chelates. However, these multivalent ions in the blood present significant toxicity risks, in particular a very close association with the occurrence of nephrogenic systemic fibrosis.18 The development of USPIOs that are highly stable in physiological environments is highly desired in order to eventually replace Gd-based products for targeted vascular imaging applications.


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The colloidal stability of USPIOs has always been a matter of concern,19 as well as their capacity to remain in the blood for long times. In general, biocompatible hydrophilic coatings such as polyethylene glycol (PEG) in single layers, endow USPIOs with excellent colloidal stability in biological fluids and in physiological conditions.20–22 Upon injection in the blood, different types of proteins adsorb at the surface of nanoparticles.23 The nature and the thickness of these protein layers strongly depend on the type of polymer ligand, as well as on its surface charge.24,25 Welldesigned engineered surface coatings are essential to circumvent the undesired effects of certain protein corona forming at the surface of nanoparticles, which can cause a rapid elimination from the blood flow.23 Carboxylates, phosphonates, and catechol groups figure among the most common anchors integrated into the design of ligands for USPIO surfaces.7,26 Usually, small, oligomeric, and polymeric ligands contain either monodentate and bidentate groups. However, multidentate polymers having multiple carboxylates, phosphonates and catechol anchoring groups have also been designed. Thus-decorated polysaccharides, homopolymers, and random copolymers are commonly-used polymeric ligands.27–37 More recently, multidentate block copolymers (MDBCs) have been developed based on anchoring blocks that can bind to USPIO surfaces, and on a hydrophilic block that is made of polyethylene oxide (PEG). This component enhances the biocompatibility of the system while attenuating the adherence of proteins from the biological media.22,38,39 Overall, MDBC-coated USPIOs have shown remarkable colloidal stabilities in strongly saline aqueous media, including in biological conditions.37–40 Recent reports have demonstrated unique advantages for the use of MDBCs over monodentate and bidentate ligands and even homo or random polymeric ligands.41–44



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In recent years we have explored the robust MDBC strategy with well-defined block copolymers that are composed of poly(oligo(ethylene oxide) monomethyl ether methacrylate) (POEOMA) as a hydrophilic block. The POEOMA block was covalently linked with either poly(methacrylic acid) (PMAA) or a polymethacrylate with pendant catechol groups as an anchoring block.38–40,45 Through conventional and biphasic ligand exchange processes, the MDBC strategy allows for the fabrication of aqueous USPIO colloids (with core diameters 99%), hexane, anhydrous ethanol (EtOH), and sodium hydroxide (NaOH, >99%) from Sigma Aldrich as well as 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 and P2 MDBCs 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 ultra-sonification 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 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 up to 200 °C and kept for 5 min. After cooling down to room temperature, excess EtOH was added to precipitate the product, which was collected by centrifugation (5,000 rpm x 5 min), washed with EtOH three times, and then dried at room temperature under vacuum oven for 3 hrs. The resultant black solids were re-dispersed in water (10 mL). They were further ultrafilterated by Millipore 8050 Stirred Cell to remove inorganic and organic impurities. P1-B was prepared using the same methodology but with twice the concentration 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 nanoparticles was measured from Transmission Electron Microscopy (TEM) images obtained with a FEI Tecnai G2 F20 200 kV


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Cryo-S/TEM, 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. Hydrodynamic diameter by number of aqueous MDBC/USPIO colloids was 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. Zeta potential was measured on a Malvern Nano Zetasizer. 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 into a platinum pan and heated from 25 to 600°C at a heating rate of 20 °C/min under nitrogen flow. The crystal structure was determined by X-ray diffraction (XRD) using X′pert Pro with CuKα radiation (1.54056 Å) at 40 kV and 40 mA. Finally, magnetic properties were evaluated using a Lakeshore 7400 vibration sample magnetometer (VSM) at 298 K between -1.8 to 1.8 Tesla. X-ray Photoelectron Spectroscopy (XPS). Drops of aqueous suspensions of MDBC/USPIO colloids were deposited on silicon substrates cleaned with TL2 and TL1 solution, according to previously reported methodologies.47 The samples were then analyzed by XPS using a PHI 5600ci spectrometer (Physical Electronics, Eden Prairie, MN, USA). An achromatic aluminum X-ray source (1486.6 eV, 300 W) was used to record the survey spectra (1400-0 eV) while highresolution (HRXPS) spectra (C1s and O1s peaks) were obtained using an achromatic magnesium X-ray source (1253.6 eV, 300 W). No charge neutralization was applied for both survey and high-resolution 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 C1s and O1s were performed by means of a least-square Gaussian-Lorentzian peak fitting procedure, after Shirley background subtraction. The C1s peaks were referenced at 285 eV (C-C and C-H).



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Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR). The measurements were performed using Attenuated Total Reflectance (ATR) mode in FTIR (Agilent Cary 660 FTIR, Agilent technologies, Australia), equipped with a deuterated L-alaninedoped triglycine sulfate (DLa-TGS) detector and a Ge-coated KBr beam splitter. Aqueous suspensions of MDBC/USPIO colloids were directly deposited on Si crystals. Spectra were recorded in absorbance mode and 100 scans were recorded with a spectral resolution of 4 cm-1. Colloidal stability of aqueous MDBC/USPIO colloids. For colloidal stability assays, aliquots of aqueous colloids were mixed with 150 mM NaCl (pH = 5.6) and phosphate buffer saline (PBS, pH = 7.2) at 0.5 mg/mL. The size distribution was then assessed by DLS, and followed over 1 month (at day 0, 1, 2, 7, 14 and 30). For colloidal stability in the presence of proteins, aqueous colloids (1 mL, 2 mg/mL) were mixed with a bovine serum albumin (BSA) solution in PBS (1 mL, 50 mg/mL). The resulting mixtures were incubated at 37 °C for 24 and 48 hrs and then subjected to centrifugation (13,000 rpm x 30 min) to precipitate any undesirably-formed aggregates. Quantitative analysis was conducted using bicinchoninic acid (BCA) assay (Pierce® BCA Assay Kit) according to the manufacturer’s instructions as well as our previous report.48 The zeta-potential of the solution was also measured in various pH. All measured samples have a good quality factor, and the attenuation factor was set to 11. Relaxivity measurements. Aqueous MDBC/USPIO suspensions were prepared at 1.0 mg/mL using the similar protocol as described above. After aliquots of the dispersion were diluted with different volumes of water, the resulting dispersions were dispensed into 7.5 mm o.d. nuclear magnetic relaxation (NMR) tubes. Their longitudinal and transversal relaxation times (T1 and T2) were measured with a dedicated TD-NMR relaxometer (Bruker Minispec 60 mq, 60 MHz (1.41 T, 37 °C). For the measurement of T1, a standard inversion-recovery sequence was used (180°


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inversion pulse, followed by a certain delay (TI), then a 90° pulse to measure the intensity of the free induction decay signal). After a recovery period of at least 3 seconds, the sequence was repeated with a different delay (TI). The T1 curve was drawn using points acquired from at least 15 different delays. For T2 measurements, a standard Carr-Purcell-Meibom-Gill sequence was used (90° pulse, followed by 180° rephasing pulses to induce the echo; the T2 curve was drawn from measurements performed on at least 12 echos). Calculating the slope of 1/T1 and 1/T2 plotted against Fe concentration values provided the relaxivity values. The Fe content in the dispersions was measured by Inductively-Coupled Plasma Mass spectrometry (ICP-MS; Agilent 7500ce – Table S1). Prior to ICP measurements, aliquots of each MDBC/USPIO dispersion (≈5 mg/mL) were digested overnight at 80 °C in HNO3 (trace metal, Fisher Scientific A509–500) and 30% H2O2 (Sigma-Aldrich). The simulated signal intensity (SSI) was calculated using the conventional signal intensity equation for the spin-echo acquisition sequence. It is expressed as follows:38

= (1 −

)(

)

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; 1/T2) at which contrast agents accelerate the relaxation of protons are described with the following equation: 1 1 = + , ℎ = 1,2 where Tm is the relaxation time of the matrix, ri are the longitudinal and the transversal relaxivities and [Fe] is the molar concentration of iron. In vitro MR imaging. Aliquots of MDBC/USPIO colloids were dispensed into a 96 wells plate and inserted in a micro-plate RF coil and imaged with a 1 T small-animal MRI system (M2M,



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Aspect Imaging, 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 gap; dwell time = 16 µs; matrix: 200 x 200; 3 exc. In vivo MR imaging. Six-week-old BALB/c female mice (Charles River, Montreal, Canada) were randomly divided in two groups of three animals each. All animal experiments were conducted under the guidelines of Université Laval and CHUQ’s animal ethical committee. One group was injected intravenously with the P2 compound, and the other one with P1-A. Mice were first anaesthetized with 3% isoflurane in an induction box and transferred to the MRI mouse bed while kept under anesthesia by means of a nose cone integrated to the bed. The animals were continuously monitored for respiration with a small-animal monitoring and gating system (model 1025T, SA Instruments, Stony Brook, NY). Mice were cannulated in the previously dilated caudal tail vein (30 G, winged needle), connected to a catheter prewashed with heparin 25 U/mL (0.25 cc Heparin 10 000 U diluted in 10 mL sodium chloride 0.9%) 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 (3M 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 Aspect M2 compact high-performance MRI system (Aspect Imaging, Shoham, Israel). Mice were scanned using a T1-weighted 2D spin echo sequence in coronal orientation. The scanning parameters were: 26 slices of 0.8 mm each (0.1 mm slice gap); fieldof-view: 100 mm, echo time/repetition time: 16.1/800 ms; dwell time: 25 µs; fα = 90°; total duration: 4 min 16s. Two pre-injection images were acquired as references (S0). The P1-A group (n=3) was injected with 100 µL of nanoparticles (8.3 mM Fe in sodium chloride 0.9%); the P2 group (n=3) was also injected with 100 µL (2.3 mM Fe in sodium chloride 0.9%). The


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concentration of each suspension was adjusted up to a similar longitudinal relaxation time (T1, measured at 1.41 T: see details in section relaxivity measurements). Mice were dynamically scanned for 2 hours, followed by static acquisitions at 5, 24 and 48 hours, then 7 days post injection. For contrast enhancement analysis, regions of interest (ROI) 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, USA). Signal enhancement ratios were calculated as follows: ∑ Pixel Value, S, ∑ ROI Area Contrast Enhancement = = ∑ Pixel ValueS∑ ROI Area



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RESULTS AND DISCUSSION

Well-defined P1 (linear PEG chains; PEO-b-PMAA) and P2 (brushed PEG chains; POEOMA-bPMAA) MDBCs were synthesized according to methodologies described in our previous articles.49 Our previous reports suggest that the design of MDBC with longer anchoring (PMAA) and hydrophilic POEOMA blocks is important to retain 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 to be 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 supporting information) prior in situ fabrication of superparamagnetic colloids. Synthesis of MDBC/USPIO colloids using in-situ approach. In our earlier work, we produced brush-like POEOMA “P2”-MDBC, 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 final core diameters of 4.2 nm (TEM) and hydrodynamic diameters (DLS) in the order of 25 nm. This ligand exchange and transfer to aqueous conditions was tedious, and implied materials losses. 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


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instantly formed indicating the nucleation of iron oxide colloids. A series of MDBC/USPIO variants were prepared by varying experimental conditions such as reaction time and precursor concentration ratios. Because hydrodynamic size has possibly the strongest impact on blood retention in vivo, from our syntheses we selected three products that show hydrodynamic diameters in narrow ranges (4-7 nm, see Table 1). Two of 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 nanoparticles, the P1-A colloids had a USPIO core size of 7.3 ± 1.4 nm; the P1-B, had a slightly lower diameter of 6.5 ± 1.5 nm, which could be caused with the use of higher amount of NaOH that could have induced more nucleation. Compared with P1 colloids, P2 is composed of brush-like 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 NPs cores after nucleation in DEG.



P1-A

P2

P1-B

80

70

d = 7.3 nm

60

Particle number

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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70

d = 6.5 nm

70 60

50

d = 4.8 nm

60 50

50 40

40 40

30

30 30 20

20

20

10

10

10

0

0 0

1

2

3

4

5

6

7

8

9 10 11 12 13

Diameter (nm)

0 0

1

2

3

4

5

6

7

8

9 10 11 12 13

Diameter (nm)

0

1

2

3

4

5

6

7

8

9 10 11 12 13

Diameter (nm)

Figure 2. TEM images and diameter histograms for MDBC/USPIO colloids (P1-A, P1-B, and P2). Inset: HR-TEM used for crystallographic characterization (scale bar: 5 nm). Given the TEM analysis of USPIO core size on dried state, 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, zeta potential 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), confer a strongly negative surface charge to USPIO compared


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with the brushed PEG-chains (surface charge of -1 mV). The colloidal stability in different chemical environment is discussed later in the text. In our previous studies, we found zeta potentials of -10 mV for brushed PEG-coated USPIOs, with good colloidal stability in physiological conditions. 38,45 Table 1. Characteristics and properties of aqueous MDBC/USPIO colloids prepared by in situ fabrication method. Diameter (nm)

Surface charge (mV)a

MDBC/USPIOs

a

TEM

DLS

Zeta Potential

P1-A

Linear

7.3 ± 1.4

12.8 ± 1.0

-28

P1-B

Linear

6.5 ± 1.5

14.0 ± 0.2

-20

P2

Brushed

4.8 ± 1.3

9.9 ± 0.7

-1

Zeta-potential was measured in buffer solution at a neutral pH.

Quantitative physico-chemical analysis of MDBC/USPIOs. The inset in Figure 2 reveals a typical example of the high crystallinity of the magnetic iron oxide nanoparticles achieved by this one-pot synthesis technique (HRTEM images of P1-B). The high temperature (≈ 200 °C) used to synthesize the nanoparticles 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 pattern of both P1/USPIO and P2/USPIO colloids with different core USPIO sizes. Each system shows the pattern of peaks for the (220),



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(311), (400), (411), and (440) lattice planes, which could be associated to the spinel structure known for Fe3O4 magnetite crystals.46,50,51 The Debye-Scherrer formula was used to calculate nanocrystal sizes from XRD spectra obtained with low scanning speed (Figure S2). From the peaks indexed to (311) and (400), ther 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.

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


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Further 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 nanoparticles present in each system, as well as to the specific mass attributed to the Fe3O4 content, according to the inorganic content measured by TGA analysis (see Table 2). The following saturation mass susceptibilities were found: ~50, 39 and 26 emu/g Fe3O4 for P1-A (7.3 nm), P1-B (6.5 nm), and P2 (4.8 nm), respectively. These values closely correlate to previously reported values for USPIOs of 5.6 and 4.84 nm (∼45 and ∼20 Am2/kg or emu/g 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 in situ fabrication method here are superparamagnetic at room temperature. FTIR was used to study the physio-chemical 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) vibration from ester groups at 1709 and 1715 cm-1, respectively. The brushed PEG-coated P2 nanoparticles exhibit a similar FTIR spectrum. However, one may notice different band ratios, in particular the PEG peak which is very intense compared to that of P1-A and P1-B. We hypothesize that the bands characteristic of the polymer coating (C=O) and of the iron oxide (FeO), exhibiting different intensities, are associated to a difference in the polymer density. Indeed, the ratio of Fe-O/C=O are 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 analysis (Figure S3), used



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to determine the content of organic copolymers in the MDBC/USPIO, showing higher polymer content in P2 USPIO/MDBCs (Table 2). P2 USPIO/MDBCs had polymer content as high as 80.6% while it was lower for linear-PEG chains sample: 31.3 and 33.8 wt% for P1-A, and P1-B respectively. Moreover, surface coverage (ligands per nm2) was calculated and was found to be similar for both P1 MDBCs (~0.26 ligand/nm2 per USPIOs) while it was denser for the P2 (0.81 ligand/nm2 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 is available in Table S3). As summarized in Table 2, regarding P1 USPIOs with linear PEG chains, the results from XPS analyses indicate that less iron was detected for P1-B, compared with P1-A, although C/Fe ratio was higher on P1B than P1-A which is consistent with FTIR results. For brushed-PEG, P2 exhibited a very low concentration of iron, 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, P2 coating exhibited an O/C ratio closed from the one expected based on the PEG structure, which also evidenced by the presence of C-O band at 286.5 eV in HR C1s (Table S3).


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Table 2. Surface composition of MDBC/USPIOs determined by XPS analyses in survey and TGA

XPS

TGA Polymer weight (wt %)

Element content (wt %) MDBC/USPIOs C1s

O1s

Fe

O/C ratio

C/Fe ratio

P1-A

45.7 ± 0.2

43.2 ± 0.8

11.2 ± 0.8

1.0

4.1

31.3

P1-B

61.3 ± 0.4

33.6 ± 0.4

5.1 ± 0.1

0.6

12.0

33.8

P2

69.2 ± 0.4

30.0 ± 1.0

1.1 ± 0.6

0.4

61.1

80.6

Overall, FTIR and XPS results clearly highlight the difference in composition, and also on density of coatings: less iron was detected on P1-B and a lower ratio FeO/C=O in FTIR suggesting a higher density of polymer on the surface of nanoparticles. By opposite, P1-A exhibited a probable thinner coating. In fact, a higher FeO/C=O ratio based on FTIR, a higher %Fe detected in XPS, even if a similar mass loss was found in TGA. At last, P2 had a very high amount of C/Fe ratio in XPS, a very low contribution of Fe-O in FTIR and a significant mass loss in TGA which concludes in a high density of brushed polymer on the surface. Therefore, all 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



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of aqueous MDBC/USPIO 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, in saline, in in PBS. Furthermore, they exhibit high shelf-stability in saline and PBS conditions (Figure S4), but also as long as 4 weeks in saline condition (Figure S5). Colloids incubated with BSA proteins at 8 mg/mL showed no significant precipitation over 96 hrs. Quantitative analysis using the BCA assay48 indicated >95% BSA remaining in the supernatant of the mixtures. These results suggest that aqueous MDBC/USPIO 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 MDBCPEG-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 hours to high concentrations (200 µg/mL) of these superparamagnetic colloids, were not significantly affected (>80% viability confirmed with the MTT assay).39,40 USPIOs coated with a polymer made of poly(isobutylene-alt-maleic anhydride) (PIMA) chains with PEG moieties and dopamine anchoring groups, were well-tolerated by HeLa cells up to concentrations of 200 µg/mL (>90% viability confirmed with the MTT assay, 24h 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,


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Figure 4). Longitudinal and transverse magnetic relaxation times (T1 and T2 respectively) were measured upon dilutions 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-1s-1, r2 = 76.7 mM-1s1

, and r2/r1 = 4.1 (TEM diameter: 7.3 nm); for P1-B: r1 = 15.1 mM-1s-1, r2 = 63.9 mM-1s-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-1s-1 were higher than those 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 in the same order of magnitudes as those of P1 products: P2: r1 = 5.3 mM-1s-1, r2 = 20.7 mM-1s-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-1s-1 and r2/r1 = 3.5).38,45 By comparison, typical ultra-small 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-1s-1 (commercially available Supravist - SHU-555C)52; r2/r1 = 3.4 – 6.1 and r1 = 4.5-4.8 mM-1s-1 for PEGylated ultra-small nanoparticles11,53 as well as USPIOs stabilized with COOH-bearing MDBCs.38,45 The positive contrast enhancement properties of these products were confirmed with a smallanimal MRI scanner operating at clinical magnetic field strength (1.0 T, Figure 4B-C). The contrast enhancement was evaluated theoretically and compared to actual experimental.54 We found good correspondence with the experimental (done with 96-well plate) curve suggesting an



interesting potential as 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 nanoparticles covered with the brushed PEG blocks (P2), the signal enhancement peaked at higher iron concentrations (0.810 - 0.828 mM). The latter results are in agreement with a previous study in which we measured in similar conditions, the signal enhancement peak generated by COOH-MDBC-coated iron oxide nanoparticles prepared by a conventional ligand exchange process (attained at 0.9-1.9 mM Fe).38 These results highlight the fact that P1 provides a more efficient positive contrast enhancement effect at very low iron concentrations, that for USPIOs coated with brushed polymers (P2).

160

P2

120

r 2=76.7

r2/r 1=4.1

100

80

80

60

60

40 20

150 100

r 1=15.9

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

0 0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

2

P1-B 4000

4000

3000

3000

3000

2000

2000

2000

1000

1000

1000

0

0 2.0

2.5

[Fe] (mM)

3.0

3.5

4.0

10

12

14

16

P2

4000

1.5

8

5000

P1-A

1.0

6

[Fe] (mM)

5000

5000

0.5

4

[Fe] (mM)

[Fe] (mM)

0.0

r 1=5.41

50

20

0

r 2/r 1=3.8

200

40

r 1=18.8

r2=20.7

r2/r 1=4.0

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0.0

0.5

1.0

1.5

[Fe] (mM)

2.0

2.5

3.0

3.5

4.0

[Fe] (mM)

C)

CTL

100

250

r 2=63.9

P1-A

CTL

1/T (s-1)

P1-B

140

120

B)

300

160

P1-A

140

P1-B

CTL

A)

Signal intensity

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

Page 24 of 53

P2


Page 25 of 53 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


Figure 4. A) Relaxation rates (1/T1 and 1/T2) of USPIO/MDBC in saline medium at 1.41 T. B) Theoretical (dotted line) and experimental (full line) contrast enhancement in vitro T1-weighted MRI for P1 and P2 colloids at 1.0 T and C) corresponding 96-well plate MRI for various iron concentration (numeric values given in mM of Fe; CTL = Control). In vivo dynamic contrast-enhanced MRI study. The signal enhancement provided by the ultrasmall iron oxide nanoparticles in T1-weighted MRI, was assessed in a mouse model. The two products having shown similar r2/r1 relaxometric ratios, as well as the closest combination of TEM and DLS results (P1-A and P2, see Table 1), were selected for MRI studies in vivo. Two groups of animals were injected (n = 3 each). Each animal was injected with 100 µL of iron oxide nanoparticles, in an estimated total blood volume of 1.6 mL in 20 g animals. The doses were prepared to provide equivalent longitudinal relaxation times, (T1 = 22.1 – 23.8 ms; 0.23 µmol Fe (11.4 µmol/g of body weight) for P1-A and 0.83 µmol Fe for P2 (34.3 µmol/g body weight), respectively. In fact, the expected values of T1 in the blood are one of the main factors guiding the “positive” contrast enhancement in MRI. These values correspond to a total dose of 12.7 µg, and 46.2 µg of iron per animal (0.64 mg/kg, and 2.31 mg/kg body weight). Overall, the total quantity of iron injected in each mouse was well below the typical dose administrated for blood-pool iron oxide contrast agents reported elsewhere.55–57 They were in the same range as strong doses injected for liver retention studies in the pre-clinical studies of commercial SPIO and USPIOs (e.g. feruglose, ferumoxutol, and and ferumoxytes).58 As a comparison, Gd-labeled dendrimers are typically injected at a dosage of 30 µmol Gd/kg body weight to perform vascular studies.59 The T1-weighted MRI before and after the injection with these two compounds, are shown in Figure 5, whereas Figure 6 reveals the quantitative signal analysis performed on these images, by



Page 26 of 53

extracting the MRI signal from well-selected regions of interest (ROIs: described in the materials and method section). A rapid inspection of the images obtained in MRI revealed that, for animals injected with P1-A (linear polymer-coated nanoparticles), the blood signal remained very high for at least 5 hours after the injection. A very progressive darkening was observed in the liver up to t = 48 h. However, after 7 days, the signal of the liver appeared to be at the same intensity as prior to the injections. For the P2 nanoparticles after 7 days, the signal in the liver and in the spleen is still much lower compared with the controls (before injection). To further quantify these findings, the MR signal values observed in the blood (abdominal vessels), in the liver and in the spleen, were also quantified and plotted as signal-enhancement curves (Figure 6). The evolution of contrast observed in these organs is detailed in the following sections.

A)

P1-A

B)

P2

Figure 5. Dynamic contrast-enhanced MRI follow-up study after injection of a) linear PEGcoated iron oxide nanoparticles (P1-A), and b) brushed polymer-coated iron oxide nanoparticles


Page 27 of 53 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


(P2). The arrows point to the liver, whereas the arrowhead point to the blood signal in the abdominal vessels.

Vascular signal enhancement and blood clearance. As revealed in Figure 6.A, strong vascular signal enhancement is found for both types of particles (P1-A and P2) in the first 125 minutes following injections. Then, Figure 6B, indicates a decrease in UPSIOs concentration in the blood between the end of dynamic acquisition (2 h), and the first static measurement performed (5 hours). After 5 hours, the decrease of signal in the blood correlates well with the strong signal decrease in the liver due to T2/T2* effects (described in the next section). For the brushed-PEG-coated nanoparticles (P2), the vascular signal gradually increases within the first 2 hours to reach a similar contrast enhancement as that observed for P1-A (Figure 6A). In order to produce an equivalent signal increase in the blood 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 iron 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 hours post-injection, suggest that these particles are more susceptible to interact with the surface of blood vessels, with blood cells and with 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/nm2) 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 nanoparticles, coupled with the blood signal increase trend



Page 28 of 53

observed in the first 2 h post-injection, suggest that these particles are subject to several interactions in the blood. These factors could explain the progressive increase of blood signal for P2, as suggested in Figure 6A. In addition to this, the higher iron concentration of P2 could possibly lead to a slight nanoparticles 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 filtrated by the liver after a few minutes only, such that only ultra-small nanoparticles would remain in the blood to produce high vascular signal.


Page 29 of 53

Vascular System 2.2

2.2

A)

2.0

B)

2.0

1.8

1.8

1.6

1.6

1.4

1.4

1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4 0.2

0.2 0

Signal enhancement

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


25

50

75

100

0

125

1

2

3

4

5

6

7

2

3

4

5

6

7

2

3

4

5

6

7

Liver 2.2

2.2

C)

2.0

D)

2.0

1.8

1.8

1.6

1.6

1.4

1.4

1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2 0

25

50

75

100

125

0

1

Spleen 2.2

2.2

E)

2.0

F)

2.0

1.8

1.8

1.6

1.6

1.4

1.4

1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4 0.2

0.2 0

25

50

75

Time (min)

100

125

0

1

Time (day)

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



Page 30 of 53

In the first 2 hours of dynamic scans, the MRI acquisition window was kept at 5 minutes per scan. Because of the animal ethics requirements, mice could not be kept in anesthesia for more than 2h. Following the first dynamic scan acquisition, the animals were anaesthetized again at t = 5, 24, 48 h and 7 days, for static scans (same scanning conditions, same time window of 5 minutes). The decrease in signal intensity observed in Figure 6B between t = 2 h and t = 5 h, was revealed at the data analysis step. Even though the number of acquisition scans between t = 1 h and t = 5 h is too low to allow a precise fitting, it is evident that signal enhancement which is a direct indicator of the concentration of nanoparticles in the blood, is still very high at t = 2 h, followed by a significant decrease at t = 5 h. In fact, DCE-MR imaging is frequently used in the clinics, as an indicator of the concentration of contrast agents in the blood pool.60 Therefore, Figure 6B suggests blood half-lives for P1 and P2, included between t = 2 h and t = 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 iron content. Blood half-lives observed for other reported ultra-small PEGylated iron oxide nanoparticles figure in the range 30-160 min.61,62 For commercial dextran-coated iron oxide nanoparticles such as Feridex, Supravist (SHU-555 C), Ferumoxtran-10, Ferumoxytol-7228, blood half-lives of 2 h, 6 h, 24-36 h and 10-14 h were reported, respectively.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

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