Dextran-coated Ultrasmall Superparamagnetic Iron Oxide Research Articles

Feraheme (Ferumoxytol) Is Recognized by Proinflammatory and Anti-inflammatory Macrophages via Scavenger Receptor Type AI/II.

Feraheme (ferumoxytol), a negatively charged, carboxymethyl dextran-coated ultrasmall superparamagnetic iron oxide nanoparticle (USPIO, 30 nm, -16 mV), is clinically approved as an iron supplement and is used off-label for magnetic resonance imaging (MRI) of macrophage-rich lesions, but the mechanism of recognition is not known. We investigated mechanisms of uptake of Feraheme by various types of macrophages in vitro and in vivo. The uptake by mouse peritoneal macrophages was not inhibited in complement-deficient serum. In contrast, the uptake of larger and less charged SPIO nanoworms (60 nm, -5 mV; 120 nm, -5 mV, respectively) was completely inhibited in complement deficient serum, which could be attributed to more C3 molecules bound per nanoparticle than Feraheme. The uptake of Feraheme in vitro was blocked by scavenger receptor (SR) inhibitor polyinosinic acid (PIA) and by antibody against scavenger receptor type A I/II (SR-AI/II). Antibodies against other SRs including MARCO, CD14, SR-BI, and CD11b had no effect on Feraheme uptake. Intraperitoneally administered PIA inhibited the peritoneal macrophage uptake of Feraheme in vivo. Nonmacrophage cells transfected with SR-AI plasmid efficiently internalized Feraheme but not noncharged ultrasmall SPIO of the same size (26 nm, -6 mV), suggesting that the anionic carboxymethyl groups of Feraheme are responsible for the SR-AI recognition. The uptake by nondifferentiated bone marrow derived macrophages (BMDM) and by BMDM differentiated into M1 (proinflammatory) and M2 (anti-inflammatory) types was efficiently inhibited by PIA and anti-SR-AI/II antibody. Interestingly, all BMDM types expressed similar levels of SR-AI/II. In conclusion, Feraheme is efficiently recognized via SR-AI/II but not via complement by different macrophage types. The recognition by the common phagocytic receptor has implications for specificity of imaging of macrophage subtypes.

P6481Novel dextran-coated ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) - a safe contrast agent for magnetic resonance imaging of atherosclerosis

P6481Novel dextran-coated ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) - a safe contrast agent for magnetic resonance imaging of atherosclerosis

Ultrastructural characterization of mesenchymal stromal cells labeled with ultrasmall superparamagnetic iron-oxide nanoparticles for clinical tracking studies

Introduction. To evaluate survival and engraftment of mesenchymal stromal cells (MSCs) in vivo, it is necessary to track implanted cells non-invasively with a method, which does not influence cellular ultrastructure and functional characteristics. Iron-oxide particles have been applied for cell tracking for years, but knowledge regarding possible cytotoxic ultrastructural changes subsequent to iron-oxide particle labeling is limited. Hence, the purpose of this study was to label MSCs with dextran-coated ultrasmall super-paramagnetic iron-oxide (USPIO) particles conjugated with the transduction sequence of trans-activator of transcription (TAT) (IODEX-TAT) and evaluate the effect of labeling on ultrastructure, viability, phenotype and proliferative capacity of the cells. Materials and methods. MSCs were labeled with 5 and 10 μg IODEX-TAT/105 cells for 2, 6 and 21 hours. IODEX-TAT uptake and cellular ultrastructure were determined by electron microscopy. Cell viability was determined by propidium iodide staining and cell proliferation capacity by 5-bromo-2-deoxyuridine (BrdU) incorporation. Maintenance of stem cell surface markers was determined by flow cytometry. Results. IODEX-TAT labeling for 2, 6 and 21 h did not influence cellular ultrastructure or viability. Moreover, neither stem cell surface markers nor cell proliferation capacity was affected by labeling with IODEX-TAT. Conclusion. Our results demonstrate that labeling of MSCs for 21 h with a clinically relevant dose of 10 μg IODEX-TAT/105 cells is feasible and does not affect MSC ultrastructure, viability, phenotype or proliferation capacity.

Evaluating the effect of ultrasmall superparamagnetic iron oxide nanoparticles for a long-term magnetic cell labeling.

In order to evaluate the long-term viability, the iron content stability, and the labeling efficiency of mammalian cells using magnetic cell labeling; dextran-coated ultrasmall superparamagnetic iron oxide (USPIOs) nanoparticles with plain surfaces having a hydrodynamic size of 25 nm were used for this study. Tests were carried out in four groups each containing 5 flasks of 5.5 × 106 AD-293 embryonic kidney cells. The cell lines were incubated for 24 h using four different iron concentrations with and without protamine sulfate (Pro), washed with phosphate-buffered saline (PBS) and centrifuged three times to remove the unbounded USPIOs. Cell viability was also verified using USPIOs. There were no significant differences in the cell viability between the control group of cells and those groups with iron uptake at the specified iron concentrations. The average iron uptake ratio compared to that of the control group was (114 ± 1). The magnetic resonance images (MRI) at post-labeling day 1 and day 21 showed (75 ± 4)% and (22 ± 5)% signal decrements compared to that of the control, respectively. The Perl's Prussian blue test showed that 98% of the cells were labeled, and the iron concentration within the media did not affect the cell iron uptake. Magnetic cellular labeling with the USPIO-Pro complex had no short or medium term (3 weeks) toxic effects on AD-293 embryonic kidney cells.

Effect of ultrasmall superparamagnetic iron oxide nanoparticles (Ferumoxtran-10) on human monocyte-macrophages in vitro

Ferumoxtran-10, a dextran-coated ultrasmall superparamagnetic iron oxide particle, has the potential to reveal macrophages in vivo using magnetic resonance imaging potentially acting as a marker of inflammatory status. Pending clinical trials, we examined the interactions of Ferumoxtran-10 with human monocyte-macrophages (HMMs) in vitro to assess its safety and lack of pro-inflammatory activity. After 72 h, Ferumoxtran-10 was not toxic at 1 mg/ml and may be only mildly toxic at 10 mg/ml. Viability in cells with a high intracellular Ferumoxtran-10 load was not affected over 14 days. Ferumoxtran-10 did not interfere with baseline or stimulated cytokine (interleukin-12, interleukin-6, tumour necrosis factor- α or interleukin-1 β) or superoxide anion production or with Fc-receptor-mediated phagocytosis. Similarly, Ferumoxtran-10 did not induce cytokine production and was not chemotactic. High-resolution electron microscopy and selected-area electron diffraction confirmed the core of Ferumoxtran-10 is composed of crystalline magnetite. Bright field transmission electron microscopy of thin sections demonstrated that Ferumoxtran-10 was retained in lysosomes of HMM for several days. Ferumoxtran-10 is not toxic to HMMs in vitro, does not activate them to produce pro-inflammatory cytokines or superoxide anions, is not chemotactic and does not interfere with Fc-receptor-mediated phagocytosis. Furthermore, extremely high intracellular Ferumoxtran-10 concentrations had only slight or no effects on these key activities.

MAGNETIC RESONANCE IMAGING AFTER ADMINISTRATION OF FERUMOXTRAN-10 FOR EVALUATION OF PARAAORTIC LYMPH NODE METASTATASIS IN PATIENTS WITH PANCREATIC CANCER

Background: Presence or absence of paraaortic lymph node metastasis is one of the most critical factors to decide indication for surgical resection of pancreatic cancer. However, conventional imaging studies such as computed tomography (CT) and magnetic resonace imaging (MRI) often fail to make a correct diagnosis of paraaortic lymph node metastasis, because paraaortic lymph nodes can be enlarged even without metastasis and because size criteria alone is not good enough to discriminate metastatic lymph nodes from non-metastatic lymph nodes with nonspecific enlargement. Therefore, we have evaluated the paraaortic lymph nodes in six patients with pancreatic cancer by MRI after administration of ferumoxtran-10 (Combidex, Advanced Magnetics, Cambridge, MA), a dextran-coated ultrasmall superparamagnetic iron oxide particle that functions as a contrast agent for lymphatic tissue. Patients and Methods: Six patients with pancreatic cancer who were scheduled for surgical resection were enrolled. All patients underwent MRI scanning 24 hours after the intravenous administration of ferumoxtran-10 as well as multi-detector CT scanning. The presence or absence of metastasis in lymph nodes was assessed on the basis of their enhancement patterns. Briefly, non-malignant nodes contain macrophages that phagocytosed ferumoxtran-10 while, due to a decrease in phagocytic activity, metastaic nodes exhibit little or no uptake of ferumoxtran-10. As a consequence, negative nodes show low signal intensity and positive nodes show partial or entire high signal intensity. The results of MRIafter ferumoctran-10 administartion were compared with those of MD-CT and with the final histopathologic diagnosis. Results: In five of the six patients, surgical resctions either by pancreaticoduodenectomy or distal pancreatectomy were carried out. Histopathologic diagnosis of the paraaortic lymph nodes was all negative for metastasis in these patients. In contrast, intraoperative histopathological study of frozen sections resulted in a diagnsosis of metastasis to paraaortic lymph nodes and by-pass operation was performed in the remining one patient. Sensitivity, specificity, and accuracy of MD-CT were 100%, 25%, and 50%, respectively when enlarged paraaortic lymph nodes > 5 mm were considered positive. Sensitivity, specificity, and accuracy of MRI after ferumoxtran-10 infusion were all 100%. Conclusions: MRI after intravenous administration of ferumoxtran-10 is useful for evaluation of paraaortic lymph node metastasis in patients with pancreatic cancer.

A Non-Invasive Approach to Detecting Organ Rejection by MRI: Monitoring the Accumulation of Immune Cells At the Transplanted Organ

Organ transplantation is the generally preferred medical procedure of treatment for patients with end-stage organ failure. The immunological reaction of rejection is a major cause of functional failure in transplant patients. The current "gold standard" for detecting or confirming graft rejection following solid organ transplantation requires biopsy samples in order to detect immune cell (e.g., T-cells, macrophages, etc.) infiltration into the graft and other pathological changes. This procedure is not only invasive, having associated risks, but is also prone to sampling errors that can yield false negative results. To circumvent the need for biopsies, we are developing magnetic resonance imaging (MRI) techniques to monitor the accumulation of immune cells at the transplanted organ as a means to detect graft rejection. By labeling immune cells with an MRI contrast agent, dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles, we can monitor the accumulation of these labeled immune cells at the rejecting graft as a non-invasive method to detect graft rejection. Cells can be labeled ex vivo and then infused into the animal, or MRI contrast agents can be introduced directly into the animal in vivo. Our results show excellent correlation among the MRI signal intensity due to the USPIO-labeled macrophages at the rejecting graft, immuno-staining for macrophages, histo-pathology for graft rejection, and the iron staining of tissue samples. In this article, we shall give a summary of our progress from detecting single immune cells in vitro to monitoring the accumulation of immune cells in vivo at the transplanted kidneys, hearts, and lungs in our rat models for organ transplantation by MRI.

In vivo detection of acute rat renal allograft rejection by MRI with USPIO particles

Magnetic resonance imaging (MRI) for non-invasively detecting renal rejection was developed by monitoring the accumulation of macrophages labeled with dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles at the rat renal allografts during acute rejection. Five groups of male rats with DA-->BN renal allografts and one group with BN-->BN renal isografts were investigated by MRI before, immediately after, and 24 hr after intravenous infusion with different doses of USPIO particles. All infusions were done on post-operative day 4. MRI experiments were carried out in a 4.7-Tesla instrument using a gradient echo sequence. MR signal intensity (MRSI) of the cortex was found to decrease with higher dosages of USPIO particles. In the absence of USPIO infusion, a decrease in MRSI was seen in the medulla region, presumably due to hemorrhage associated with renal graft rejection, while no significant change was observed in the cortex. The optimal dose of USPIO particles for visualizing rejection-associated changes in our rat kidney model appears to be 6 mg Fe/kg body weight. Iron staining results correlated with the MRSI data, indicating that the signal reduction in the MR images was due to the presence of iron. Immunohistochemical results indicated that USPIO particles were mostly taken up by infiltrating macrophages in the rejecting grafts. Our results suggest that MRI with intravenous administration of dextran-coated USPIO particles appears to be a valuable and promising tool that can be used as a non-invasive and sensitive method to detect graft rejection in renal transplantation.

Macrophage accumulation associated with rat cardiac allograft rejection detected by magnetic resonance imaging with ultrasmall superparamagnetic iron oxide particles.

Acute cardiac allograft rejection continues to be the cause of graft loss and contributes to the morbidity and mortality after cardiac transplantation. In this study, we report a new method for detecting organ rejection in transplantation with an MR-based technique using dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles. These particles ( approximately 27 nm in diameter) are known to shorten relaxation times in MRI experiments. A new rat model of heterotopic heart and lung transplantation has been developed for MRI experiments. Allotransplantations (DA-->BN) were performed (n=8), with syngeneic transplantations (BN-->BN) serving as controls (n=8). MR images were obtained with a gradient echo method. At postoperative day 7, allotransplants developed moderate rejection as determined histopathologically. A significant reduction in MR signal intensity was observed after USPIO injection into rats with allotransplanted hearts. Syngeneic transplants showed no differences in MR signal intensity before and after USPIO injections. After injection of USPIO particles at postoperative day 6, a group of allotransplanted rats was treated with cyclosporin A (3 mg/kg). Animals treated with cyclosporin A for 7 days showed no reduction in MR signal intensity after USPIO reinjection at day 14, whereas animals treated for 4 days showed a significant decrease in MR signal intensity in the transplanted hearts indicative of acute graft rejection. Pathological analysis of these animals revealed that dextran-coated USPIO particles were taken up by the infiltrating macrophages that accumulated within the rejecting cardiac graft. This MRI method offers promise as a noninvasive method for detecting transplant allograft rejection.