Biocompatible Low-Retention Superparamagnetic Iron Oxide

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dependent manner as effective contrast agents for the MRI of liver tumors. ... nanoclusters as a liver imaging contrast agent to resolve acute iron overload in the clinical usage of SPIOs ..... Quantitative analyses of the enhancement with these.

Article Journal of Biomedical Nanotechnology

Copyright © 2015 American Scientific Publishers All rights reserved Printed in the United States of America

Vol. 11, 854–864, 2015 www.aspbs.com/jbn

Biocompatible Low-Retention Superparamagnetic Iron Oxide Nanoclusters as Contrast Agents for Magnetic Resonance Imaging of Liver Tumor Yushuang Wei1 , Rufang Liao2 , Haijuan Liu1 , Huan Li2 , Haibo Xu2 , and Qibing Zhou1 ∗ 1

Department of Nanomedicine and Biopharmaceuticals, National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China 2 Department of Radiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China

Although superparamagnetic iron oxide (SPIO) nanoparticles have been developed as a contrast agent for magnetic resonance imaging (MRI), acute iron overload due to the persistently high retention of SPIOs in the liver and spleen that are slowly converted to ferroproteins is a serious safety concern. Here, we report that the addition of poly-L-lysine polymers to an SPIO hydroxyethyl starch solution produced tightly controlled, monodispersed nanoparticles in a sizedependent manner as effective contrast agents for the MRI of liver tumors. High MRI contrast was demonstrated with an orthotopic liver tumor model atbya Publishing low injection dose. Simultaneously, rapid Universiteit bioclearanceDelft of excess iron in the lung Delivered Technology to: Technische and spleen and in blood serum wasIP: observed within On: 24 h Fri, post-injection. The full excretion of excess iron was confirmed 145.94.2.95 10 Jul 2015 18:52:17 Copyright: American Scientific Publishers in urine post-intravenous injection, suggesting that the effective clearance of SPIOs could be achieved with our SPIO nanoclusters as a liver imaging contrast agent to resolve acute iron overload in the clinical usage of SPIOs as a contrast agent.

KEYWORDS: Superparamagnetic Iron Oxide Nanoparticles, Magnetic Resonance Imaging, Contrast Agent, Bioclearance, Urine Excretion, Liver Tumor Imaging.

INTRODUCTION Superparamagnetic iron oxide (SPIO) nanoparticles have been developed over recent decades for magnetic resonance imaging (MRI) as a contrast-enhancing agent to improve cancer diagnosis and prognosis, e.g., with oral ferumoxsil for the gastrointestinal tract and perfusion ferumoxtran and ferumoxides for the reticuloendothelial system.1–5 Some recent applications of SPIOs for nanomedicine include pancreatic tumor-targeted contrast SPIOs conjugated to antibodies,6 7 drug-loaded SPIO carriers of doxorubicin or temozolomide,8 9 dual-function SPIOs for imaging and hyperthermia treatment,10 11 and dendritic and cancer stem cell labeling for in vivo migration tracking.12–14 However, there is a serious safety concern with these SPIOs due to their in vivo biodistribution and metabolism.15 16 ∗

Author to whom correspondence should be addressed. Email: [email protected] Received: 23 March 2014 Accepted: 1 June 2014

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In animal studies, systemically delivered SPIOs persistently accumulated in the liver and spleen over more than 3 weeks.17–19 SPIO iron oxide cores in the spleen were shown to be slowly converted to ferritin over a period of 31 days rather than excreted directly via renal filtration.20 Most recently, Park and coworkers assessed the biodistribution and metabolism of a series of polyethylene glycol (PEG)-coated SPIOs with an iron oxide core size of 5, 15 and 30 nm.21 Consistently, iron levels remained high in the liver and spleen even after 30 days post-injection. The full clearance of the excess iron oxide in the liver and spleen to hemoglobin and ferritin would thus require months to complete. Even with tumor-targeted SPIOs, high levels of SPIO particles were persistently found in the liver and spleen.7 8 Therefore, acute iron overload would be a potential concern for SPIOs as a contrast agent when used in clinics. The biodistribution and bioclearance of SPIOs have been shown to be mainly determined by shell materials around the iron oxide core and the hydrodynamic size of the resulting particles.22 23 The most 1550-7033/2015/11/854/011

doi:10.1166/jbn.2015.2042

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Biocompatible Low-Retention SPIO Nanoclusters as Contrast Agents for MRI of Liver Tumor

studied shell materials are dextran, PEG and their respecfrom Sigma-Aldrich (USA), J&K Scientific Ltd. (China) or Sinopharm Chemical Reagent Co., Ltd. (China) unless tive derivatives of various lengths, which are recognized otherwise specified. by macrophages at a different level, resulting in a shortened or prolonged blood clearance time.24–28 In addition, shell materials also impact the reduced T2 relaxivity time Synthesis of SPIO-HES Nanoparticle Powder by SPIOs in MRI.23 26 In contrast, none of these studies Oil-based SPIOs (approximately 5 nm) were synthesized have sufficiently resolved the persistent long retention of based on a reported procedure with modifications.37 38 SPIOs in the liver and spleen. Furthermore, in contrast to Briefly, iron(III) acetyl acetonate (35.3 g, 0.1 mol), 1,2SPIOs used for brain tumor enhancement, which are typdodecanediol (101.2 g, 0.5 mol), oleic acid (84.5 g, ically less than 40 nm,6 10 SPIOs used for liver imaging 0.3 mol), oleylamine (80.2 g, 0.3 mol) and diphenyl ether such as Feridex are in the range of 50–120 nm, and there (1.0 L) were mixed under nitrogen at room temperature. is an even greater safety concern due to the significant The resulting suspension was heated at 200  C for 30 min 1–3 uptake of SPIOs by normal liver macrophages. under nitrogen and subsequently at 260  C for 30 min. Liver image enhancement by SPIO contrast agents is The reaction solution was slowly cooled down to room performed through the negative imaging method in the temperature under nitrogen. SPIOs were precipitated with T2-weighted MRI with a reduction of the T2 relaxivethanol (5 L) and collected by centrifugation (4500 g × 15 min). The obtained SPIOs were redissolved in hexanes ity in normal liver tissue upon the uptake of SPIOs by and precipitated again with ethanol. The precipitate was liver Kupffer cells.1 3 In contrast, SPIOs do not accumucollected by centrifugation and dried under vacuum. Liglate in liver tumor tissue due to the immunosuppression of and exchange on the resulting SPIOs was achieved by heatmacrophages. As a result, the MRI signals of tumor tissue ing a SPIO dimethylformamide (DMF) solution (30.0 g in ‘brighten up’ with decreased signals in the normal liver 2 L) with citric acid (10.0 g) at 75  C for 3 h under nitrotissue. Thus, SPIOs with high contrast capability and rapid gen. The obtained SPIO citrate-DMF solution was diluted bioclearance (in days) are highly desirable. For example, to a volume of 24 L with DMF followed by the addition SPIOs with a small iron oxide core could have a faster 21 of 1.0 L aqueous 10% w/w hydroxyethyl starch solution bioclearance of excess iron than the large ones. In addi(HES 200/0.5, Wuhan HUST Life Science & Technology tion, clusters of small SPIOs have been shown to produce 29 China) and incubationDelft at 80  C for 2 h. SPIO-HES a higher MRI T2 relaxivity rate than a single particle. Delivered by Publishing TechnologyCo. to: Ltd., Technische Universiteit particles were18:52:17 precipitated with t-butyl methyl ether, colWe report in this study a solutionIP:of145.94.2.95 SPIO nanoclusOn: Fri, 10 Jul 2015 lected by centrifugation (6000 g × 10 min) and lyophilized Copyright: American Scientific Publishers ters that exhibited a high MRI contrast property for liver to dryness as a fine powder (25.1 g) under vacuum. tumor imaging with rapid bioclearance of excess iron and The iron concentration of the SPIO-HES powder was full urine excretion in days. The formation of a series of determined by inductively coupled plasma (ICP) atomic SPIO nanoclusters was achieved via poly-L-lysine (PLL). absorbance analysis with a SpectrAA-40 spectrometer PLL showed low cytotoxicity at a concentration below (Varian, USA). Briefly, SPIO-HES powder (10.00 mg) was 20 g/mL toward both immune- and stem cells as the 12–14 digested with a 1:4 HClO –HNO acid mixture (5 mL), 4 3 shell material of SPIOs in the cell labeling application. and then the total volume was adjusted to 10.0 mL with Representative SPIO nanoclusters were investigated in the water in a volumetric flask. The iron concentration was MRI diagnosis of an orthotopic liver tumor model. Hepdetermined to be 0116 ± 0022 g Fe per gram SPIO-HES atocellular carcinoma (HCC) was the 3rd most common powder based on a standard curve of a series of iron solucause of death from cancer in the world in 2008.30 The curtions. The super-paramagnetic property of the obtained rent 5-year survival rate for advanced HCC patients with SPIO-HES powder was confirmed by a hysteresis loop a tumor size >2 cm is approximately 10%.31 32 The diagmeasurement with a Lake Shore 7404 vibrating sample nosis of early stage liver tumors less than 1 cm plays a magnetometer (Lake Shore Cryotronics, USA). critical role in surgical resection to significantly enhance the patient survival rate.33–35

METHODS Materials Murine liver cancer H22 cells were obtained from Shanghai Institute of Life Science Cell Culture Center (China). Cells were maintained in RPMI-1640 (Invitrogen, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 25 mM HEPES, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 50 U/mL penicillin and 50 g/mL streptomycin at 37  C and 5% CO2 . All chemicals were purchased J. Biomed. Nanotechnol. 11, 854–864, 2015

Preparation of SPIO Nanocluster Solutions An SPIO aqueous stock solution (1.16 mg Fe/mL) was first prepared by dissolving 1.00 g of SPIO HES powder in 100 mL of sterile phosphate buffered saline solution (PBS, pH 7.4). A serial sterile PLL (30–70 kDa, Sigma-Aldrich, USA) in the PBS were prepared at concentrations of 0, 2, 3, 4, 5, 6, 7 and 8 g/mL. SPIO nanocluster solutions were prepared by slowly adding 5 mL of PLL solution into 5 mL of SPIO-HES solution in a sonicating water bath. Thus, the iron concentration of each PLL SPIO solution was 0.58 mg Fe/mL based on the dilution factor with PLL solutions. 855

Biocompatible Low-Retention SPIO Nanoclusters as Contrast Agents for MRI of Liver Tumor

Physical Characterization of SPIO Nanocluster Solutions The hydrodynamic sizes of the SPIO nanocluster solutions were determined by directly measuring the obtained solutions with a Nano-ZS90 particle analyzer (Malvern, United Kingdom) after pre-equilibration at 25  C for 5 min. The zeta-potentials of the SPIO nanocluster solutions were measured in a phosphate buffered solution (pH 7.4) with a Nano-ZS90 particle analyzer. A transmission electron microscopic analysis of the SPIO cluster solutions was carried out with phosphotungstic acid staining on a Tecnai G2 20 spectrometer (FEI Incorporated, USA).

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subcutaneously (3 ×106 cells per mouse) in the lower back of BALB/c mice.36 After the tumor reached an average size of 15 mm × 15 mm, mice were euthanized by cervical dislocation. Tumor tissues were harvested, cut into small pieces ( 005) among these Liver three SPIO solutions at the same time point. For the kidLiver ney, a similar increase of the iron level by approximately 10 g Fe/g was consistently observed at all the time points with all three SPIO nanoclusters (Fig. 4(c)). In the spleen samples, a general trend was observed showing that the Figure 2. Images of the dose study for T2-weighted MRI. Delivered by1Publishing Technology to: Technische DelftSPIOs with 3.5 g/mL level of iron wasUniversiteit the lowest for Images of normal mice prior to and at h post-iv injection of IP: different 145.94.2.95 Jul 2015 18:52:17 SPIO solutions with 0 g/mL PLL at four doses:On: 5.0,Fri, 10 PLL and the highest for SPIOs with no PLL at 0.5 and 3 h Publishers 2.5, 0.5 and 0.25 mg Fe/kg bodyweight. Copyright: American Scientific post-injection (Fig. 4(d)). Unfortunately, variations in samples resulted in P > 005 in the Bonferroni posttest analyFig. 3(a)). Pathological analysis also confirmed the pressis. In contrast, a significantly lower iron level was found ence of tumor tissues. in the lung tissue at 0.5 h post-injection for SPIOs with Quantitative analyses of the enhancement with these 3.5 g/mL PLL compared to the other two SPIO solutions three SPIO solutions were carried out by measuring the (Fig. 4(e)). Rapid clearance of excess iron was observed signal intensity of multiple locations in the normal liver in the lungs with all these SPIO solutions after 3 h posttissue versus that of the muscle tissue of the same mouse injection. Similarly, the iron level in blood serum was also as a reference control (Fig. 3(b)). The signal intensity was determined post-iv injection of the SPIO nanoclusters. The consistently reduced to approximately 50% post-injection clearance of excess iron was consistently observed with all of the SPIO solutions, confirming the reduction of the T2 these SPIOs at 24 h post injection (Fig. 4(f)). Therefore, relaxivity in the MRI. Thus, negatively enhanced MRI conrapid clearance of iron was observed in the lung tissue trast for liver tumor diagnosis was confirmed with SPIO as early as 3 h post-injection, followed by the spleen and nanoclusters at a low dose of 0.5 mg Fe/kg. In addition, serum within 24 h. In addition, it is indicated that SPIO it was observed that the negative contrast enhancement by with 3.5 g/mL PLL had the highest bioclearance rate the SPIO clusters with 3.5 g/mL PLL seemed to decrease in the lung and spleen tissues among these three SPIO the most at 24 h post-injection, implying a possible fastest solutions. metabolic profile among these three SPIO solutions. To verify the bioclearance results, pathological staining of organ tissues was carried out with Prussian blue stainIncreasing SPIO Nanocluster Size Facilitates the ing, which is specific for iron deposits in tissues. RepreRapid Bioclearance of Excess Iron sentative staining images at 3 h post-iv injection of these three SPIO nanoclusters are shown in Figure 5 with PBS Urine excretion of excess iron post-iv injection of as the control. In the liver, SPIOs were confirmed to accuSPIO nanoclusters was first confirmed with ICP atomic mulate in Kupffer cells (inserted images of the liver tissues absorbance spectroscopic analysis. The iron level in the in Fig. 5), consistent with the reported study using the liver urine of normal mice increased significantly within 24 h contrast agent Feridex21 and our iron level in Figure 4(b). post-iv injection of the SPIO nanoclusters compared with Interestingly, compared to other tissue samples, only the that prior to injection (Fig. 4(a)). The urine samples were Prior injection

SPIO at 5.0 mg Fe/kg

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Figure 3. MR images of orthotopic liver tumors in mice with SPIO nanocluster solutions as contrast agents. (a) MR images of the liver tumor at 0.5, 3 and 24 h post-intravenous of SPIO (0.5 mg Fe/kg body Delft weight) containing 0, 2.0, and Delivered by Publishinginjection Technology to:solutions Technische Universiteit 3.5 g/mL PLL representing 35, 80 and nm sizes, respectively, mice 18:52:17 were euthanized after MRI at 24 h and the liver IP:120 145.94.2.95 On: Fri, 10where Jul 2015 tumors were exposed for verification (right panels). American (b) Plots of Scientific MRI signalPublishers intensity of the muscle and normal liver tissues of Copyright: the same mouse pre- and post-injection of SPIO solutions, where the MRI signal intensity is the average of four independent measurements with standard deviation (∗ P < 001).

Figure 4. Bio-clearance of SPIO nanoclusters in normal mice post-iv injection. (a) Profile of iron excretion in urine over 11 days post-injection. Urine samples were collected from urine collection cages for 24 h, and urine samples at time 0 were obtained prior to the injection of SPIO solutions (0.5 mg Fe/kg body weight, average 11.5 g iron per mouse, 6 mice per group). AUCs (areas under the curve) were calculated with GraphPad prism software. (b)–(f) Profiles of iron concentrations in tissues and blood including the liver, spleen, kidney, lung and serum prior to or at 0.5, 3 and 24 h post iv-injection of SPIO solutions (3 mice per group per time point). Iron concentrations were determined by ICP atomic absorbance spectrometer and calculated based on the volume or weight of samples (∗ P < 005).

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Figure 5. Images of Prussian blue/eosin staining of mouse tissues including the liver, spleen, kidney and lung post-iv injection of SPIO nanocluster solutions. Images were representative for each tissue section obtained at 3 h post-iv injection of SPIO solutions (0.5 mg Fe/kg body weight, 3 mice per group) with PBS as a control. Prussian blue staining exhibited a blue color with iron particles in tissue as shown in the spleen samples. Inserts in the liver samples indicates the accumulation of SPIOs in liver Kupffer cells.

bysignificant Publishingdifference Technologyepithelial to: Technische Universiteit cells,39 which spleen samples showedDelivered a clear and might Delft stimulate cell growth at low IP: 145.94.2.95 Julbut 2015 18:52:17at high levels.40 Thus, the low injeclevels be cytotoxic among these SPIO nanoclusters. Consistent with the On: ironFri, 10 Copyright: American Scientific tion dosePublishers and rapid bioclearance of SPIO contrast would level in the spleen samples (Fig. 4(d)), the SPIO nanoclusbe critical factors in minimizing the potential long-term ters with 3.5 g/mL PLL have the least blue staining while cytotoxic effects. SPIOs with no PLL had the most extensive staining. Prussian blue staining of the samples at 0.5 h post-injection showed a similar difference with a much more pronounced DISCUSSION staining among these three SPIO nanoclusters than those The presence of HES was critical in the formulation at 3 h (Fig. 6(a)). At 24 h post-injection, no significant of water-soluble SPIOs that had particle sizes between staining was observed in any of these spleen samples 5–10 nm to produce a stable and clear solution in PBS. (Fig. 6(a)), which was fully consistent with the complete We found that the use of citric acid alone only led to clearance of excess iron in the spleen in Figure 4(d). precipitation in the PBS solution. HES (200/0.5) is a modIn contrast, no significant staining was found in the kidney ified starch with an average molecular weight of 200 kDa or lung tissues at 3 h post-injection (Fig. 5) or even at the containing 2-hydroxyethyoxyglucose unit at 50% and is early time point of 0.5 h. Therefore, SPIO nanoclusters used for blood volume expansion in clinics. We recently with 3.5 g/mL PLL (125.30 nm) have the most rapid bioreported that the addition of HES significantly enhanced clearance of excess iron in the lung, spleen and serum and the stability and distribution of nanobubbles in a PBS the most efficient urine excretion compared with SPIOs buffer.41 Our results in this study showed that the hydrodywith 2.0 g/mL PLL (82.57 nm) and SPIOs with no PLL namic size of water-soluble SPIOs was 33.65 nm (Table I), (33.65 nm). Finally, the potential cell cytotoxicity of SPIO while the TEM image showed well-separated particles nanoclusters was assessed on the normal liver-derived cell with sizes ranging from 5–10 nm (Fig. 1(d)). The large difline HL-7702. Our results showed that upon the addition of ference in particle size may suggest that highly branched 0.2 mg/mL SPIO, the cell viability was increased by 20% HES molecules could lead to well dispersed SPIO particompared with cells alone after incubation for 72 h, while cles to prevent aggregation as amorphous clusters and thus there was no significant difference among the nanoclusters result in high stability in PBS. with or without PLL (Fig. 6(b)). However, decreasing cell SPIOs with enhanced bioclearance and urine excretion viability was observed when the concentrations of SPIOs for MRI were achieved through the formation of nanwere more than 0.5 mg/mL. It has been shown that iron oclusters with polycationic PLL. The hydrodynamic size oxide nanoparticles can induce reactive oxygen species in of the nanoclusters fully depends on the amount of PLL J. Biomed. Nanotechnol. 11, 854–864, 2015

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Figure 6. Images of the mouse spleen at 0.5 and 24 h post-iv injection and cellular toxicity of SPIO nanoclusters. (a) Prussian blue staining of the spleen tissue with blue color indicating iron deposits. The images were representative for each tissue section post-iv injection of SPIO solutions (0.5 mg Fe/kg body weight, 3 mice per group). (b) Plot of cell cytotoxicity on a normal liver cell line HL-7702 by MTT assay after incubation of SPIO nanoclusters for 72 h. Percent cell viability was calculated relative to the cell alone control as 100%.

added, which is proportionally translated into the extent The addition of HES in the synthesis of SPIO clearly of reduced relaxivity and enhanced inverse relaxivity in facilitated the urine excretion of excess iron over 9 days T2-weighted MRI. This is in significant contrast with the post-iv injection. In contrast, no significant urine excretion reported result that the addition of PLL to resovist at a of excess iron has been reported with other SPIO contrast ratio of 0.05:1 to 0.5:1 had no impact on the hydrodynamic agents for liver imaging such as Feridex using dextran as size of SPIOs (60 nm).42 It has been shown that the inverse the excipient.21 Compared to dextran, HES 200/0.5 has a relaxivity increases when the size of iron oxide cores of higher molecular weight (average of 200 kDa vs. 40 kDa SPIO particles increases from 5 to 15 nm.21 Our result was for dextran) and additional modification of half of the hydroxyl groups to hydroxyethyl ether groups. One posconsistent with that the increase of the inverse relaxivity sible rationale for the facilitated excretion of excess iron could be alternatively achieved via cluster formation by Delivered Publishing Technology is to:that Technische 29 HES acts Universiteit as a carrier Delft for SPIOs and traps SPIOs tightly controlling the size of SPIObyparticles. IP: 145.94.2.95 On: Fri, 10 Jul 2015 18:52:17 in the highly branched polymeric structure via hydrogen Our results demonstrated that iv injection of the SPIO Copyright: American Scientific Publishers bonding between hydroxylethoxyl residues of HES and the nanoclusters at a low dose of 0.5 mg Fe/kg body weight, SPIO surface. Therefore, the bioclearance profile of the only one-tenth of the amount used in the reported mouse resulting nanoclusters would resemble that of HES. It has and rat studies,17 19 21 was sufficient to clearly reveal the been shown that infusion of HES 200/0.5 solution has an presence of 2 mm orthotopic liver tumors. The signal elimination half-time of 8.66 h in humans, and 52% of reduction of normal liver tissue in T2-weighted MRI is the HES was directly excreted in urine within 24 h.43 This consistent with SPIOs as a negative MRI contrast agent with reduced T2 relaxivity to reveal the presence of liver rationale is supported by our TEM imaging results of the tumors.33–35 More importantly, the rapid bioclearance of the nanoclusters showing that SPIO nanoparticles (dark spots) were well separated from each other and evenly dispersed SPIO nanoclusters was found to correlate with nanoclusin the clusters (Fig. 1(d), A2–D2). In addition, the gray ter size in an incremental manner in the lung, spleen and spherical shades observed in the TEM images (Fig. 1(d), serum. Urine excretion of excess iron similarly increased B2–D2) were the result of positive phosphotungstic acid with the nanocluster size over 9 days post-injection. Based staining, suggesting the presence of polysaccharides, and on the iron level of the tissue samples, it is possible that in our case, HES in the nanoclusters. The addition of PLL SPIOs remained mainly in the liver and kidney at 24 h postfurther resulted in morphological change from amorphous iv injection. In contrast, significant Prussian blue staining clusters to spheres with increasing size, leading to a more was observed in the spleen samples at 0.5 or 3 h postHES-like profile and thus, an overall increased excretion injection but not after 24 h. This result was supported by of excess iron (Fig. 4(a)). the iron level measured in the spleen tissue (Fig. 4(d)). It is Overall, the rapid bioclearance of SPIOs and full urine possible that the positive staining in the spleen may not be excretion of excess iron provide an effective solution to of SPIOs, as only 11.5 g of iron was injected per mouse minimize the concern of potential cytotoxicity. Labhaseton average and was distributed among the liver, kidneys, war and coworkers showed that injection of oleic acid spleen, lungs and blood post-injection. Rather, the positive pluronic-coated SPIOs in rats led to a significant retention staining in the spleen tissue could be iron deposits recyof excess iron in spleen and liver tissues after 21 days, cled by the spleen in response to elevated serum iron levresulting in a significant increase in lipid hydroperoxide els, which were then effectively cleared within 24 h. The production in the spleen and kidney compared to the liver detailed mechanism of the biodegradation of SPIO nandue to excess iron overload.17 The other liver imaging oclusters is currently under investigation. 862

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contrast agent Feridex exhibited an even higher accumulation of excess iron in the spleen after 30 days post-iv injection in mice.21 Our data also showed that SPIOs stimulated cell growth at 0.2 and 0.5 mg/mL and were cytotoxic at 1.0 and 2.0 mg/mL after incubation for 72 hours, which is consistent with the induction of reactive oxygen species in cells as a result of excess iron by SPIOs.39 40 44 Thus, the rapid bioclearance of excess iron and low retention in the spleen, as demonstrated by our SPIO nanoclusters, is imperative for SPIO contrast agents to address potential safety concerns for MRI. In conclusion, the addition of PLL to SPIO HES solutions produced tightly controlled monodispersed nanoparticles as an effective contrast agent for MRI. High MRI contrast was demonstrated with an orthotopic liver tumor model at a low injection dose. More importantly, the rapid bioclearance of excess iron was observed in the lung, spleen and blood serum. The full excretion of excess iron through the urine indicates the effective clearance of SPIOs, suggesting that acute iron overload can be minimized with the use of our SPIO nanoclusters as a contrast agent for MRI.

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