Enhanced MRI T 2 Relaxivity in Contrast-Probed Anchor-Free PEGylated Iron Oxide Nanoparticles
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Superparamagnetic iron oxide nanoparticles (SPIONs, ~11-nm cores) were PEGylated without anchoring groups and studied as efficient MRI T 2 contrast agents (CAs). The ether group of PEG is efficiently and directly linked to the positively charged surface of SPIONs, and mediated through a dipole-cation covalent interaction. Anchor-free PEG-SPIONs exhibit a spin-spin relaxivity of 123 ± 6 mM−1s−1, which is higher than those of PEG-SPIONs anchored with intermediate biomolecules, iron oxide nanoworms, or Feridex. They do not induce a toxic response for Fe concentrations below 2.5 mM, as tested on four different cell lines with and without an external magnetic field. Magnetic resonance phantom imaging studies show that anchor-free PEG-SPIONs produce a significant contrast in the range of 0.1–0.4 [Fe] mM. Our findings reveal that the PEG molecules attached to the cores immobilize water molecules in large regions of ~85 nm, which would lead to blood half-life of a few tens of minutes. This piece of research represents a step forward in the development of next-generation CAs for nascent-stage cancer detection.
KeywordsPEGylation Magnetic iron oxide nanopaticles T2 relaxivity MRI contrast agents
Attenuated total reflectance
Dynamic light scattering
High-resolution transmission electron microscope
Magnetic resonance imaging
Selected area electron diffraction
Superparamagnetic iron oxide nanoparticles
Magnetic iron oxide nanoparticles (NPs) offer a wide scope of applications in nanomedicine, including magnetic resonance imaging (MRI), drug and gene delivery, tissue engineering, bioseparation, cell tracking and labeling, and innovative cancer therapeutics and diagnostics due to their excellent biocompatibility and unique magnetic properties [1, 2, 3, 4, 5, 6, 7]. In particular, magnetite (Fe3O4) has received the primary focus (when compared to other forms of iron oxide, such as maghemite, hematite, goethite and wustite) [8, 9] because its shape, size and surface-to-volume ratio can be synthetically modified, which facilitates larger payloads and superior stabilities useful for multiple applications in theranostics. However, the surface modification of this nanostructure using biocompatible polymers or inorganic components remains a challenge [10, 11, 12, 13, 14, 15, 16, 17, 18]. Greater efforts regarding the hydrophilicity concerns must be addressed in order to endow it with an enhanced colloidal stability in physiological media, low toxicity, low oxidation, and improved controlled agglomeration. Thus, superparamagnetic iron oxide NPs (SPIONs) surface-functionalized with specific biomolecules are indispensable for biomedical applications.
Polyethylene glycol (PEG) is one of the most common polymers used for surface functionalization of SPIONs due to its biocompatibility, hydrophilicity, nontoxicity, antifouling nature, and non-antigenicity and non-immunogenicity [24, 25, 26, 27]. PEGs are polyether-diols with two terminal hydroxyl groups and alternating ether linkages, and are soluble in water because of the hydrogen bonding of water molecules to electron-rich oxygen atoms in the polymer chain. It has been also demonstrated that PEGylation helps avoid recognition by the reticuloendothelial system (specifically the macrophage cells), and hence extends in vivo circulation time in blood pool for both imaging and drug delivery applications . In addition, PEG is inexpensive and has been approved by the US Food and Drug Administration (FDA) not only for pharmaceuticals, but also for other industries, such as food and cosmetics .
There have been several reports on PEGylation strategies for iron oxide based NPs [30, 31, 32, 33]. Hu  and Dai  and their respective co-workers reported the facile PEGylation of ultrasmall iron oxide nanoparticles and SPIONs using carboxyl homobifunctionalized (-COOH-PEG-COOH) to demonstrate an efficient MRI contrast enhancement. Wang and co-workers  also reported a facile PEGylation process of SPIONs with PEG/polyethyleneimine (PEI) for in vivo MRI of mouse brain. Further, Tong and co-workers  systematically studied SPION cores coated with DSPE-mPEG copolymer, and determined that a fine tuning of the core size and impermeable PEG coating of SPIONs can further increase the T 2 relaxivity per particle, which was ascribed to the fact that the PEG molecules can immobilize water molecules in a region much larger than the area of the actual iron oxide core. Similarly, Xie and co-workers  used dopamine as the anchoring group for an effective PEGylation of monodisperse Fe3O4 NPs through a covalent bond. They reported that the resulting NPs showed negligible aggregation for cell culture conditions, and much reduced non-specific uptake by macrophage cells . In order to provide an easier and more effective method for chemical coating, Larsen and co-workers  reported the preparation of biocompatible iron oxide magnetic NPs coated with PEG by replacing oleic acid with a commercially available silane-anchored PEG. They found an enhanced MRI contrast in the larger coated cores, which was associated to a combined effect of the size-dependent extravasation and the capture by macrophages in certain tumors . However, for such improvements, some intermediate molecules (anchoring groups) and complex PEGylation protocols are needed. There are few reports on anchor-free PEGylation of NPs [39, 40]. In addition, the feasibility of these PEGylated NPs as new-generation probing agents for MRI applications is still in its infancy. Here, we report the enhanced MRI relaxivity of 11-nm SPIONs PEGylated without anchoring groups, and its potential as an efficient T 2 CA for applications in vivo.
All reagents used in this investigation, ferric chloride hexahydrate (FeCl3⋅6H2O, ≥99%), ferrous chloride tetrahydrate (FeCl2⋅4H2O, 99.99%), poly (ethylene) glycol (average mol. wt. 3350 Da), and ammonium hydroxide (NH4OH, 28.0–30.0%), were analytical-grade reagents purchased from Sigma Aldrich, USA, and were directly used without any processing.
Synthesis of PEGylated Fe3O4
The PEG-coated magnetite NPs were synthesized via the co-precipitation method [38, 39] with minor modifications. Briefly, 1.3 mg of FeCl3.6H2O and 0.5 mg of FeCl2.4H2O were dissolved in 20 mL of deionized water, and the solution was then deoxygenated through N2 bubbling for 30 min, which produced a dark orange-colored solution. Afterwards, 2 mL of NH4OH was added dropwise under vigorous stirring in an inert environment. The solution was next heated up to 70 °C for 1 h and the flocculate was magnetically decanted several times, and then re-dispersed in deionized water obtaining a black solution of iron oxide. One third of the as-prepared solution was freeze-dried in vacuum. The resultant product was labeled as bare SPION in order to compare them to their PEG-coated counterparts. In a separate step, 40% (w/v) aqueous solution of PEG was added to two thirds of the remaining solution, being then rapidly probe-sonicated at room temperature at a frequency of 5 Hz. The resultant solution was washed many times with abundant water in order to remove uncoated PEG NPs via centrifugation at 8000 rpm for 30 min. Finally, it was freeze-dried in vacuum for 48 h obtaining powders of iron oxide labeled as PEG-SPION. The weight ratio of PEG/SPION was set at ~1:10.
The crystallographic phase and purity of the products were investigated with a Rigaku SmartLab X-Ray diffractometer (XRD) using CuKα (λ = 1.5406 Å) operating at 40 KV and 44 mA. The attenuated total reflectance (ATR) spectra of the products were obtained using a Bruker Tensor 27. The thermal behavior of SPION and PEG-SPION was studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) in the temperature range of 30 to 800 °C in presence of constant N2 flow of 20 ml/min using PerkinElmer STA 6000 Simultaneous Thermal Analyzer. The static size, morphology, crystallinity and size distribution of the products were recorded using a JEOL JEM-2200FS high-resolution transmission electron microscope (HRTEM) operating at 200 kV. To prepare the TEM samples, 30 μL of each product in solution (0.8 mM) was dropped on lacey carbon 300 M Cu grids, and dried overnight. The hydrodynamic diameter was determined by dynamic light scattering (DLS) technique and zeta potential of bare SPIONs and PEG-SPION were determined by using a Malvern Zetasizer Nanoseries Nano-ZS (Malvern Instruments, Malvern, UK). The dispersed samples in the solvents were measured without filtration. The magnetic properties of the products were studied using a vibrating sample magnetometer (VSM, Lakeshore 7400) and a Quantum Design (PPMS DynaCool). The relaxivity measurements were performed using an NMReady-60e benchtop relaxometer (Nanalysis Corp.) operating at 60 MHz and 1.40 T, whereas the T2-weighted MR phantom images were acquired using an Agilent 4.7 T/200 MHz MRI scanning system. The relaxivity measurements and MR phantom tests were conducted threefold and fourfold, respectively.
MTS Cell Viability Assay
The cytotoxicity effects of PEG-coated magnetite NPs were performed on HeLa, A549, MDA-MB-231 and Jurkat cells using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), following our previously reported protocol . Briefly, the cells were cultured in Eagle’s minimum essential medium supplemented with 5% of human platelet lysate (EMD Millipore), 100 μg/mL, 100 U/mL penicillin, streptomycin, and 250 ng/mL amphotericin B (Cellgro) at 37 °C with 5% CO2. Cells were plated at a density of ~2 × 104 in 96-well plates and grown until reaching 80–90% confluence . Cell culture medium was then removed and 100 μL of complete medium supplemented with PEG-SPION at different concentrations (ranging from 0.1 to 10 [Fe] mM) was added, and three wells with only fresh complete medium were used as a positive control. The medium was removed after 24 h of incubation and a solution of fresh media containing 20% of CellTiter 96® AQueous One Solution reagent was added. Wells with fresh complete medium and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent without cells were used as a negative control . Cells were then incubated at 37 °C for 30 min. Afterwards, the 96-well plates were centrifuged at 2000 rpm for 10 min. The supernatant was transferred onto a clean microplate and the absorbance at 490 nm was recorded using a Synergy H1 Hybrid Multi-Mode Microplate Reader. The cell viability percentage was determined by means of the following equation: % = [(A490 of PEG-SPION-treated cells)/(A490 of untreated cells)] × 100. The same protocol was followed when the PEG-SPION/cells and controls were exposed to an external magnetic field (0.2 T). All of the experiments were done in triplicate.
Results and Discussion
We synthesized PEG-SPIONs free of anchoring groups via a modified co-precipitation approach. The XRD patterns of the as-synthesized products are depicted in Figure S1 (see Additional file 1). The bare XRD patterns of SPION and pure PEG were included for comparison. The diffraction peaks observed in PEG-SPION were indexed to the reflection planes of cubic inverse spinel Fe3O4 corresponding to the (111), (220), (311), (400), (422), (511), and (440) planes, compatible with the JCPDS file no. 79-0418 [41, 42, 43]. This indicates that the crystalline structure and phase of SPION are retained by PEG-SPION. Two additional diffraction peaks at 2θ = 19.2 and 23.3 that correspond to the crystalline planes of pure PEG were also observed , which confirm the successful surface functionalization of SPIONs. The diffraction peaks of PEG-SPIONs are less intense when compared to those of SPIONs, which further supports the adsorption of PEG by SPIONs (wt.% 1:10). The observed well-defined peaks, the absence of secondary phases (including maghemite and other forms of iron oxide) in the XRD patterns, and the co-existence of both phases in PEG-SPIONs are clear indicators of the successful formation of PEGylated Fe3O4 with high crystalline quality and purity. The broadening of the diffraction peaks of SPION and PEG-SPION is ascribed to their nanocrystalline nature. The calculated average crystal size of SPION was determined by means of Scherrer’s formula, yielding ~16 nm.
Figure 3b shows the HRTEM images and SAED patterns of PEG-SPION. Our observations indicate that when the capping ligand (PEG polymer) is added, the SPION cores retain their morphology, phase, narrow size distribution and crystallinity. Note that the irradiation of the 200-kV electron beam did not cause any damage, phase transformation or amorphous carbon deposition on the PEG-SPIONs. The agglomerated feature observed in PEG-SPION is associated to the drying process of the dispersions. Their relatively uniform dispersion in water is correlated to the effective PEG capping process (as displayed in Figure S2, see Additional file 1). This fact was confirmed by the HRTEM image displayed in the top right inset, in which a less-crystalline phase that surrounds the cores’ surface is easily differentiated by contrast. Thus, one would expect a bigger particle size at this PEG (3350)/SPION ratio (wt.% 1:10, PEG with mol. wt. > 3000 given the dense coating over the NPs surface) , which indicates the formation of adlayers on single particles. This was confirmed by the DLS measurements, which will be discussed in the next section.
The linkage of PEG onto the surface of SPIONs was further corroborated by DLS and zeta potential measurements. The DLS profiles of bare SPION and PEG-SPION in water (at different pH values) and different solvents are depicted in Figure S3 (see Additional file 1). From this Figure, the hydrodynamic diameter (DH) of SPIONs was observed to be higher than that of PEG-SPION in most of the solvents. At neutral pH, the observed DH is 145.8 nm (PDI = 0.28) and 115 nm (PDI = 0.154) in water for SPION and PEG-SPION, respectively. These sizes are much higher than those observed in HRTEM. This is ascribed to the agglomeration of SPION as a result of their mutual magnetic attraction, whereas the formation of brush-type configuration of PEG (due to the high grafting density of 5 chains of PEG per nm2 surface area of SPION and large Flory radius of 4.7 nm) may partly contribute to the increase in hydrodynamic diameter for PEG-SPION. In acidic and basic conditions, the induction of surface hydration or the formation of electric double layer (EDL) on the surface of dispersed PEG-SPION may lead to the increased hydrodynamic diameter [48, 49]. Further, when dispersed in biological buffers, such as PBS 7.2 and NaCl solution, both SPION and PEG-SPION tend to flocculate due to Debye screening of electrostatic repulsion or adsorption of counter-ions .
The zeta potential measurements as a function of pH for SPION and PEG-SPION are shown in Figure S4 (see Additional file 1). It was observed that SPION acquire positive charges even at neutral pH with zeta potential of +2.15 mV, which gradually increases to +24.2 mV at acidic conditions (pH = 3). This clearly suggests the presence of positive charges on the surface of SPION and the preferential dissolution or deposition of H+ co-ions in acidic medium . The curve with solid circles in Figure S4 shows that the zeta potential of SPION almost drops to 0 mV at pH > 7 and pH < 8, and the SPION aggregate drastically. But an opposite trend was observed in basic conditions acquiring zeta potential of −37.7 mV at pH = 11. This particle stability is provided by electrostatic repulsion. Further, in case of PEG-SPION, the –OH group of the PEG is protonated in acidic medium (pH = 3) giving zeta potential of +10.8 mV, as shown in solid square in Figure S4. Below this pH, it was observed that the –OH group of PEG was gradually deprotonated and the isoelectric point (IEP) was determined to be at pH ~ 4.1 where the zeta potential becomes 0 mV. Similarly, the deprotonation of –OH group causes the formation of negative charges around the surface of PEG-SPION giving rise to large negative zeta potential at neutral and basic pH. The highest stability of PEG-SPION was noted at pH 11 with zeta potential of −30.6 mV, which is attributed to the electrostatic repulsion.
Comparison of r 2 relaxivity with the current study
Z ave. size (nm)
r 2 (mM−1s−1)
B 0 (T)
Fe3O4 /11 nm
We synthesized 11-nm SPION PEGylated without intermediate biomolecules that show enhanced T 2 relaxivity. The key strategy for this successful PEGylation is the existence of a dipole-cation binding (either covalent or ionic) between the ether group of PEG and the positively charged surface of the Fe3O4 cores. These hydrophilic NPs retain the morphology of the cores, and exhibit a good colloidal stability in both aqueous media and physiological environments due to the hydrogen bonding of water molecules to electron-rich oxygen atoms in the PEG chain. Our results indicate that the PEG-SPION generate MRI contrast enhancement (per-Fe atom relaxivity ~123 mM−1s−1) on T 2-weighted sequences, and hence can be considered as highly sensitive T 2 CA. This enhancement is correlated to the increased effective radii due to nanoparticle clustering and the local field inhomogeneity of the magnetic core. The fact that the anchor-free PEG-SPION do not require increased saturation magnetization values to produce high magnetic relaxivities can be a key in the development of next-generation CAs for nascent-stage cancer diagnosis. These findings have broader significance, since they can be extended for the effective conjugation of the NPs with a variety of macromolecules, making them suitable candidates for enhancing the signal-to-noise ratio in MRI and obtaining refined anatomical images.
The authors thank the valuable assistance of Mr. Oscar Resto for providing the HRTEM images, Dr. Huadong Zeng for magnetic resonance phantom images, and Prof. Ram S. Katiyar for his research facilities.
This project was partially supported by the Institute for Functional Nanomaterials (NSF EPSCoR Grant 1002410) and PR NASA EPSCoR (NASA Cooperative Agreement NNX15AK43A). A portion of this work was performed in the McKnight Brain Institute at the National High Magnetic Field Laboratory’s AMRIS Facility, which is supported by National Science Foundation Cooperative Agreement No. DMR-1157490 and the State of Florida.
BT, DDD and JB-H conceived and designed the study. BT, DDD, and JB-H performed the experiments. BT wrote the manuscript. BT, DDD, JB-H, BRW, and GM analyzed the data, reviewed and edited the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
All cell experiments were performed in accordance with protocols approved by the Biosafety Committee of the Molecular Sciences Research Center, University of Puerto Rico, USA.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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