Ex vivo assessment of polyol coated-iron oxide nanoparticles for MRI diagnosis applications: toxicological and MRI contrast enhancement effects

Polyol synthesis is a promising method to obtain directly pharmaceutical grade colloidal dispersion of superparamagnetic iron oxide nanoparticles (SPIONs). Here, we study the biocompatibility and performance as T2-MRI contrast agents (CAs) of high quality magnetic colloidal dispersions (average hydrodynamic aggregate diameter of 16-27 nm) consisting of polyol-synthesized SPIONs (5 nm in mean particle size) coated with triethylene glycol (TEG) chains (TEG-SPIONs), which were subsequently functionalized to carboxyl-terminated meso-2-3-dimercaptosuccinic acid (DMSA) coated-iron oxide nanoparticles (DMSA-SPIONs). Standard MTT assays on HeLa, U87MG, and HepG2 cells revealed that colloidal dispersions of TEG-coated iron oxide nanoparticles did not induce any loss of cell viability after 3 days incubation with dose concentrations below 50 μg Fe/ml. However, after these nanoparticles were functionalized with DMSA molecules, an increase on their cytotoxicity was observed, so that particles bearing free terminal carboxyl groups on their surface were not cytotoxic only at low concentrations (<10 μg Fe/ml). Moreover, cell uptake assays on HeLa and U87MG and hemolysis tests have demonstrated that TEG-SPIONs and DMSA-SPIONs were well internalized by the cells and did not induce any adverse effect on the red blood cells at the tested concentrations. Finally, in vitro relaxivity measurements and post mortem MRI studies in mice indicated that both types of coated-iron oxide nanoparticles produced higher negative T2-MRI contrast enhancement than that measured for a similar commercial T2-MRI CAs consisting in dextran-coated ultra-small iron oxide nanoparticles (Ferumoxtran-10). In conclusion, the above attributes make both types of as synthesized coated-iron oxide nanoparticles, but especially DMSA-SPIONs, promising candidates as T2-MRI CAs for nanoparticle-enhanced MRI diagnosis applications.     

Biodistribution of GdCl3 and Gd-DTPA and their influence on proton magnetic relaxation in rat tissues

The biodistribution and relative molar effectiveness of the ionic (GdCl3) and chelated (Gd-DTPA) forms of gadolinium (Gd) to enhance proton relaxation rates in rat kidney, liver and spleen were evaluated. Rats were given intravenous injections of either GdCl3 (100 mumol/kg) or Gd-DTPA (178 mumol/kg). Gd-DTPA was primarily contained in the vascular compartment and was quickly accumulated in the kidney after injection with a relaxivity of 4.3 sec-1 (mumol/g kidney)-1. It was eliminated quickly from the body with only 2% of the injected dose remaining after 120 min. After GdCl3 injection, Gd was found primarily in liver and spleen. It accumulated continuously reaching 72% of the injected does in these two tissues after 120 min. Despite this continuous increase in tissue Gd concentration, the relaxation rates showed saturation in liver and spleen. The results suggest that after GdCl3 was injected it distributed either in a protein bound form that was effective at causing relaxation or in a colloid form that was not effective. The biodistribution of GdCl3 was such that it was determined by the phagocytic action of the recticuloendothelial system on a colloid. The biodistribution and tissue relaxivity of Gd-DTPA suggest it will be a useful vascular MRI contrast agent. However, the usefulness of GdCl3 as an MRI contrast agent is limited not only by its acute toxicity but also by its saturable effect on tissue relaxation rates. Consequently, GdCl3 has only a modest influence on tissue relaxivity.     

Assessment of absolute blood volume in carcinoma by USPIO contrast-enhanced MRI

OBJECTIVES: The characterization of tumor vasculature is essential in studying tumor physiology. The aim of this study was to develop a new method - based on water proton MR density measurements, in combination with ultrasmall superparamagnetic iron oxide (USPIO) administration - to measure absolute blood volume (BV) in murine colon carcinoma. MATERIALS AND METHODS: MRI experiments were performed at 7 T. CPMG imaging was performed on subcutaneous murine colon carcinoma in six mice before and after administration of an USPIO blood-pool contrast agent. Density maps were obtained from the signal amplitude at TE=0 of the CPMG decay fit. Post-USPIO density maps were subtracted from pre-USPIO density maps to quantitatively yield absolute tumor BV maps. In a separate group of mice (n=6), the relative vascular area (RVA) of tumors was determined by immunohistochemistry. RESULTS: Ultrasmall superparamagnetic iron oxide administration resulted in a small decrease in the water proton MR density. The BV averaged over the six tumors was 4.6+/-1.6%. The value of the RVA measured by immunohistochemical staining was equal to 3.9+/-2.2%. CONCLUSIONS: After administration of an USPIO blood-pool agent (T(2) relaxivity > 100 mM(-1) s(-1)), the blood water protons become MRI invisible, and pixel-by-pixel BV map can be obtained by subtracting the calculated post-USPIO density map from the pre-USPIO density map. The value of absolute BV obtained with this novel MR approach is in good agreement with the value of the relative vascular measured by immunohistochemical staining.     

Surface properties of superparamagnetic iron oxide MR contrast agents: Ferumoxides,ferumoxtran, ferumoxsil

The surface properties of the active ingredients in AMI colloidal, superparamagnetic iron oxide magnetic resonance (MR) contrast agents are described. Scanning electron microscopy/energy dispersive X-ray elemental analyses and diffuse reflectance Fourier transform infrared spectroscopy (FTIR) spectra of ferumoxsil (AMI-121 drug substance) were consistent with the presence of a monolayer of H₂NCH₂CH₂NHCH₂CH₂CH₂Si(O⁻)₃ siloxane monomer or dimer. The X-ray photoelectron spectra (XPS) of ferumoxsil are also consistent with complete coverage of the iron oxide surface with a monolayer of siloxane. The static secondary ion mass spectra (SSIMS) of ferumoxsil showed that the siloxane film is covalently bonded (i.e., SiOFe bonds) to the iron oxide surface. The FTIR of ferumoxides (AMI-25) and Ferumoxtran (AMI-227) showed only adsorbed dextran. The XPS spectra of the dextrancoated colloids showed that Ferumoxtran has a thicker layer of dextran than ferumoxides iron oxide particles (∼5 and ∼3 nm, respectively). The SSIMS spectra of these dextran-coated colloids showed only low mass fragments due to the adsorbed dextran. The nature of the interactions of the dextran coating with the iron oxide surfaces of ferumoxides and Ferumoxtran is discussed.     

MR imaging of the liver and spleen: a comparison of the effects on signal intensity of two superparamagnetic iron oxide agents

We compared the effects of two superparamagnetic iron oxide (SPIO) contrast agents, ferumoxides and SHU-555A, in MR imaging of the liver and spleen. Thirty-six patients with known malignant lesions of the liver underwent T2W turbo spin-echo (TSE) and T1WGRE FLASH opposed-phase imaging before and after SPIO injection on a 1.0 T MR system. Post-ferumoxides images were obtained in 18 patients 90 min after infusion of 15 micrommol Fe/kg of the agent. In 18 other patients SHU-555A was administered as a rapid bolus at a dose of 7.0-12.9 micrommol Fe/kg. T1WGRE FLASH images were obtained immediately, 30 s and 480 s and T2WTSE images 10 min after injection. Signal intensity of the liver, spleen, and malignant liver lesions before and after SPIO was measured with operator-defined regions of interest. The effects of ferumoxides and SHU-555A were measured as the percentage signal intensity change (PSIC) and in the malignant liver lesions additionally as changes in lesion-to-liver contrast-to-noise ratio (deltaDCNR). On T2W TSE images, there was no significant difference between the two agents in signal loss of liver parenchyma (p > 0.05). The signal loss in the spleen produced by ferumoxides was greater than with SHU-555A (p < 0.05). Both SPIO agents produced a significant increase in the CNR of malignant liver lesions. Delta CNR was slightly greater with ferumoxides than with SHU-555A (p < 0.05). On T1WGRE FLASH images, a slight decrease of liver SI induced by both agents was found on late post-SPIO images. No significant difference of liver PSIC between the two SPIO agents was noted on T1W images. The SI of spleen was significantly increased with both agents on T1W images and no difference in PSIC of spleen was noted (p > 0.05). The T1 and T2 effects produced by ferumoxides and SHU-555A were comparable in the liver although ferumoxides produced a stronger T2 effect in the spleen.     

Formation of multifunctional Fe_3O_4/Au composite nanoparticles for dual-mode MR/CT imaging applications

Recent advances with iron oxide/gold(Fe3O4/Au) composite nanoparticles(CNPs) in dual-modality magnetic resonance(MR) and computed tomography(CT) imaging applications are reviewed. The synthesis and assembly of "dumbbelllike" and "core/shell" Fe3O4/Au CNPs is introduced. Potential applications of some developed Fe3O4/Au CNPs as contrast agents for dual-mode MR/CT imaging applications are described in detail.     

MR enteroclysis using iron oxide particles (ferristene) as an endoluminal contrast agent: an open phase III trial

To evaluate efficacy and safety of a superparamagnetic iron oxide contrast agent (ferristene) as an endoluminal contrast medium for magnetic resonance (MR) enteroclysis in a phase III trial. Twenty-three patients with history of known or suspected small bowel Crohn's disease underwent MR imaging of the abdomen at 0.5 T unit. The imaging protocol included two phases: the first one without administration of any contrast agent and the second one, where the small bowel was filled by enteroclysis with 800 ml of the luminal iron oxide contrast medium and Gd-DTPA (0.1 mmol/Kg) was administered intravenously. Axial Spin-Echo (SE) T1-weighted (T1w), proton-density and T2w images, sagittal and coronal SE T1w and Short TI Inversion Recovery (STIR) sequences were subsequently obtained. Three investigators blindly evaluated images to determine small bowel distribution of ferristene, presence of artifacts, delineation of bowel lesion/wall and the diagnostic value of ferristene combined with gadolinium. Pre- and postcontrast signal intensity measurements of bowel lesion/wall, bowel lumen and background noise were also calculated. Three patients withdrew before the procedure, therefore 20 patients were effectively included in the study. No significant difference between the three investigators' evaluations of the improvement of the diagnostic information was found (percentage of improvement of 90% with 95% confidence limits of 68% and 99%). A statistically significant difference between the first and third investigators was found for grading of quality of delineation of bowel lesion/wall. Signal intensity measures showed a significant increase of the bowel lesion/wall and background noise/lesion for the SE T1w images. No serious adverse event was reported in our series. MR enteroclysis using ferristene as an endoluminal contrast agent appears to be a safe and efficient procedure for the study of the small bowel.     

Synthesis,characterization, and in vitro biological evaluation of highly stable diversely functionalized superparamagnetic iron oxide nanoparticles

In this article, we report the design and synthesis of a series of well-dispersed superparamagnetic iron oxide nanoparticles (SPIONs) using chitosan as a surface modifying agent to develop a potential T ₂ contrast probe for magnetic resonance imaging (MRI). The amine, carboxyl, hydroxyl, and thiol functionalities were introduced on chitosan-coated magnetic probe via simple reactions with small reactive organic molecules to afford a series of biofunctionalized nanoparticles. Physico-chemical characterizations of these functionalized nanoparticles were performed by TEM, XRD, DLS, FTIR, and VSM. The colloidal stability of these functionalized iron oxide nanoparticles was investigated in presence of phosphate buffer saline, high salt concentrations and different cell media for 1 week. MRI analysis of human cervical carcinoma (HeLa) cell lines treated with nanoparticles elucidated that the amine-functionalized nanoparticles exhibited higher amount of signal darkening and lower T ₂ relaxation in comparison to the others. The cellular internalization efficacy of these functionalized SPIONs was also investigated with HeLa cancer cell line by magnetically activated cell sorting (MACS) and fluorescence microscopy and results established selectively higher internalization efficacy of amine-functionalized nanoparticles to cancer cells. These positive attributes demonstrated that these nanoconjugates can be used as a promising platform for further in vitro and in vivo biological evaluations.     

MR imaging of pulmonary parenchyma and emboli by paramagnetic and superparamagnetic contrast agents

Using experimentally induced pulmonary emboli in an animal model, three intravenously administered contrast agents, Gd-DTPA-albumin microspheres (8-15 microns, 0.2 M particles/mg protein, 39-106 micrograms Gd/mg, 50 mg/ml), Gd-DTPA-liposomes (15-30 microns, 130 micrograms/mg lipid, 6 mg Gd/ml) and superparamagnetic ferrosome, (60 nm, 100 mM iron and 20 mg lipid/ml) were examined for MR imaging. Gd-DTPA entrapped in lung capillaries did not enhance the signal intensity of lung parenchyma, but liposomes (5 ml) served as better Gd-DTPA carriers and increased the parenchymal signal intensity by up to a factor of 2.3. However, neither agent improved delineation of pulmonary emboli. Ferrosome decreased the intensity of lung parenchyma, improving detectability of pulmonary emboli by several factors.     

Comparative analysis of the 1H NMR relaxation enhancement produced by iron oxide and core-shell iron-iron oxide nanoparticles

Physicochemical and magnetorelaxometric characterization of the colloidal suspensions consisting of Fe-based nanoparticles coated with dextran have been carried out. Iron oxide and iron core/iron oxide shell nanoparticles were obtained by laser-induced pyrolysis of Fe(CO)₅ vapours. Under different magnetic field strengths, the colloidal suspension formed by iron oxide nanoparticles showed longitudinal (R₁) and transverse (R₂) nuclear magnetic relaxation suspension (NMRD) profiles, similar to those previously reported for other commercial magnetic resonance imaging (MRI) contrast agents. However, colloidal suspension formed by ferromagnetic iron-core nanoparticles showed a strong increase of the R₁ values at low applied magnetic fields and a strong increase of the R₂ measured at high applied magnetic field. This behaviour was explained considering the larger magnetic aggregate size and saturation magnetization values measured for this sample, 92 nm and 31 emu/g Fe, respectively, with respect to those measured for the colloidal suspensions of iron oxide nanoparticles (61 nm and 23 emu/g Fe). This suspension can be used both as T₁ and T₂ contrast agent.     

Multifunctional Hybrids Based on 2D Fluorinated Graphene Oxide and Superparamagnetic Iron Oxide Nanoparticles

Carbon‐based nanomaterials have garnered a lot of attention in the research of yesteryear. Here this study reports a composite based on fluorinated graphene oxide—a multifunctional subsidiary of graphene; and iron oxide nanoparticles as a contrast agent for magnetic resonance imaging (MRI). Extensive structural and functional characterization is carried out to understand composite behavior toward biotoxicity and its performance as a contrast agent. The electron withdrawing fluorine group decreases the charge transfer to iron oxide increasing the magnetic saturation of the composite thus enhancing the contrast. The interaction of paramagnetic and superparamagnetic systems yields a superior contrast agent for MRI and fluorescent imaging.     

Effect of chain length and electrical charge on properties of ammonium-bearing bisphosphonate-coated superparamagnetic iron oxide nanoparticles: formulation and physicochemical studies

Bisphosphonates BP molecules have shown to be efficient for coating superparamagnetic iron oxide particles. In order to clarify the respective roles of electrical charge and the length of the molecules, bisphosphonates with one or two ammonium moieties with an intermediate aliphatic group of 3, 5 or 7 carbons were synthesized and iron oxide nanoparticles coated. The evaluation on their iron core properties was made by transmission electron microscopy (TEM), nuclear magnetic relaxation dispersion (NMRD) profiles and Mössbauer spectra. The core size is close to 5 nm, with a global superparamagnetic behaviour modified by a paramagnetic Fe-based layer, probably due to surface crystal alteration. The hydrodynamic sizes increase slightly with aliphatic chain length (from 9.8 to 18.6 nm). The presence of one or two ammonium group(s) lowers the negative electrophoretic mobility up to bear zero values but reduces their colloidal stability. These BP-coated iron oxide nanoparticles are promising Magnetic Resonance Imaging (MRI) contrast agents.     

Labelling of cultured macrophages with novel magnetic nanoparticles

Magnetic resonance (MR) imaging is capable of demonstrating human anatomy and pathological conditions. Iron oxide magnetic nanoparticles (MNPs) have been used in MR imaging as liver-specific contrast medium, cellular and molecular imaging probes. Because few studies focused on the MNPs other than iron oxides, we developed FeNi alloy MNPs coated with polyethylenimine (PEI). In this study, we demonstrated PEI-coated FeNi MNPs are able to label the cells, which could be detected in MR imaging. For labelling purpose, MNPs were incubated with mouse macrophage cell line (Raw 264.7) for 24 h and these PEI-labelled FeNi alloy MNPs can be uptaken by macrophages efficiently compared with Ferucarbotran, a commercialized superparamagnetic iron oxide (SPIO) under flow cytometry measurement. Besides, these cells labelled with MNPs could be imaged in MR with the identical potency as Ferucarbotran. Further investigation of the cells using Prussian blue staining revealed that FeNi alloy MNPs inside the cells is not oxidized. This phenomenon alleviated the consideration of potential risk of nickel toxicity. We conclude that PEI-coated FeNi MNPs could be candidate for MR contrast medium.     

MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents

Molecular imaging of tumor antigens using immunospecific magnetic resonance (MR) contrast agents is a rapidly evolving field, which can potentially aid in early disease detection, monitoring of treatment efficacy, and drug development. In this study, we designed, synthetized, and tested in vitro two novel monocrystalline iron oxide nanoparticles (MION) conjugated to antibodies against the her2/neu tyrosine kinase receptor and the 9.2.27 proteoglycane sulfate. MION was synthetized by coprecipitation of iron II and iron III salts in 12-kD dextran solution; antibody coupling was performed by reductive amination. The relaxivity of the conjugates was 24.1-29.1 mM(-1) s(-1), with 1.8 to 2.1 antibody molecules per nanoparticle. A panel of cultured melanoma and mammary cell lines was used for testing. The cells were incubated with the particles at 16-32 microg Fe/ml in culture medium for 3 h at 37 degrees C, and investigated with immune fluorescence, transmission electron microscopy (TEM), MRI of cell suspensions in gelatine, and spectrophotometric iron determination. All receptor-positive cell lines, but not the controls, showed receptor-specific immune fluorescence, and strong changes in T(2) signal intensity at 1.5 T. The changes in 1/T(2) were between 1.5 and 4.6 s(-1) and correlated with the amount of cell-bound iron (R = 0.92). The relaxivity of cell-bound MION increased to 55.9 +/- 10.4 mM(-1) s(-1). TEM showed anti-9.2.27 conjugates binding to the plasma membrane, while the anti-her2/neu conjugates underwent receptor-mediated endocytosis. In conclusion, we obtained receptor-specific T(2) MR contrast with novel covalently bound, multivalent MION conjugates with anti-9.2.27 and anti-her2/neu to image tumor surface antigens. This concept can potentially be expanded to a large number of targets and to in vivo applications.     

GoldMag nanoparticles with core/shell structure: characterization and application in MR molecular imaging

GoldMag is a kind of bi-functional nanoparticle, composed of a gold nanoshell and an iron oxide core. GoldMag combines the antibody immobilization property of gold nanoshell with the superparamagnetic feature of the iron oxide core. Rabbit anti-mouse IgG was immobilized on the surface of GoldMag to synthesize GoldMag-IgG in a single-step process. Transmission electron microscopy, UV/Vis spectrophotometry, zeta potential analysis, dynamic light scattering, enzyme-linked immunosorbent assay, and magnetic resonance imaging (MRI) were employed to characterize the nanostructures and the spectroscopic and magnetic properties of GoldMag and GoldMag-IgG. The antibody encapsulation efficiency of GoldMag was measured as 58.7%, and the antibody loading capacity was 88 μg IgG per milligram of GoldMag. The immunoactivity of GoldMag-IgG was estimated to be 43.3% of that of the original IgG. The cytotoxicity of GoldMag was assessed by MTT assay, which showed that it has only little influence on human dermal lymphatic endothelial cells. MR imaging of different concentrations of ultrasmall superparamagnetic iron oxide, GoldMag, and GoldMag-IgG showed that 3 μg/mL of nanoparticles could significantly affect the MRI signal intensity of GRE T2*WI. The results demonstrate that GoldMag nanoparticles can be effectively conjugated with biomacromolecules and possess great potential for MR molecular imaging.     

Inhalation MR lymphography: A new method for selective enhancement of the lung hilar and mediastinal lymph nodes

On MR lymphography, a new approach to the lung hilar and mediastinal nodes was developed in an animal model. Five rabbits were made to inhale iron colloid (cideferron) that was nebulized to aerosol. Two days after inhalation of the agent, the mediastinal lymph nodes decreased in signal on SE 2000/30 and SE 2000/60 images and proved to have iron on histological evaluation, whereas the popliteal nodes did not have any iron. Experimental results indicate that inhalational administration can deliver the agent to the pulmonary lymphatic system and has the potential of lung hilar and mediastinal MR lymphography.     

Relaxation efficacy of paramagnetic and superparamagnetic microspheres in liver and spleen

A distinct knowledge of the relationship between physiochemical properties, cellular distribution and relaxation efficacy of particulate MR contrast media is needed for the development of tissue specific contrast compounds. To study these relations paramagnetic gadolinium labelled microspheres and superparamagnetic iron oxide microspheres (MSM) were injected intravenously to rats. The T1 and T2 relaxation times of the liver and spleen were recorded and the gadolinium tissue content quantified. A clear relationship between the gadolinium dose and the gadolinium concentration of the liver and spleen was observed while the T1 of the tissues remained unchanged. After injection of MSM, T2 of liver and both T1 and T2 of spleen decreased dose-dependently. The splenic relaxation efficacy of MSM was higher compared with that of liver, probably due to the morphology of the spleen allowing a scattered cellular sequestration of MSM. To mimic a uniform tissue distribution of the contrast agents, the liver and spleen samples were homogenized and a marked increase in the intrinsic relaxation efficacy of both the paramagnetic and superparamagnetic microspheres was observed.     

Investigation of atherosclerotic plaques with MRI at 3 T using ultrasmall superparamagnetic particles of iron oxide

This study aims to investigate the uptake of the experimental ultrasmall superparamagnetic particles of iron oxide (USPIO) contrast agent DDM43/34 (Schering AG, Berlin, Germany) by aortic atherosclerotic plaques using magnetic resonance imaging (MRI) at 3 T. Six Watanabe heritable hyperlipidemic rabbits were injected with USPIO at doses of 0.1–1.0 mmol/kg Fe. Parasagittal magnetic resonance angiography (MRA) scans were acquired using 3D gradient-echo sequences before and after USPIO administration, then again after 6 h, 1 day, 2 days and 5 days. At later time points, when the USPIO concentration was too low to enhance blood signal, additional MRA scans were acquired during the infusion of gadopentate dimeglumine (Magnevist; Schering AG). In the images, widespread susceptibility artifacts demonstrated readily detectable USPIO uptake in the liver, bone marrow and lymphatic vessels. Surprisingly, however, no such effects could be associated specifically with the aortic vessel wall, in contrast to previous studies that showed strong uptake with similar pulse sequences. Histological analysis was performed on aortic slices from two animals, demonstrating that aortic plaques were active but showed very little USPIO uptake, consistent with MRI findings. We conclude that, despite the exciting potential of plaque detection using USPIO, some caution is advised since the absence of susceptibility effects does not necessarily imply the absence of plaque, even at 3 T, which offers increased sensitivity to susceptibility. Future work will investigate the dependence of such results on stage of plaque development, magnetic field strength and choice of contrast agent.     

Uptake of magnetic nanoparticles into cells for cell tracking

A challenge for future applications in nanotechnology is the functional integration of nano-sized materials into cellular structures. Here we investigated superparamagnetic Fe₃O₄ iron oxide nanoparticles coated with a lipid bilayer for uptake into cells and for targeting subcellular compartments. It was found that magnetic nanoparticles (MNPs) are effectively taken up into cells and make cells acquire magnetic activity. Biotin-conjugated MNPs were further functionalized by binding of the fluorescent tag streptavidin–fluorescein isothiocyanate (FITC) and, following uptake into cells, shown to confer magnetic activity and fluorescence labeling. Such FITC-MNPs were localized in the lysosomal compartment of cells which suggests a receptor-mediated uptake mechanism.     

Immunospecific NMR contrast agents

Most NMR contrast agents suggested to date have been paramagnetic. These agents, which include the transition and lanthanide metal ions as well as stable organic free radicals, do not provide effective contrast at concentrations much below 1 mM. However, the use of macromolecular ferromagnetic and superparamagnetic particles provides, for the first time, an NMR relaxation agent that is effective at subnanomolar concentrations. Two different sized superparamagnetic particles have been coupled to monoclonal antibodies with high affinity for a neuroblastoma-specific cell surface antigen. The specific binding of these particles, both in vivo and in vitro is demonstrated and the consequences for immunospecific NMR contrast are discussed.