For Better or Worse, Iron Overload by Superparamagnetic Iron Oxide

Nov 8, 2016 - addition, the challenge of the iron overload by SPIONs requires better designed SPIONs as MRI contrast agents for chronic liver diseases...
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For Better or Worse, Iron Overload by Superparamagnetic Iron Oxide Nanoparticles as a MRI Contrast Agent for Chronic Liver Diseases Qibing Zhou* and Yushuang Wei Department of Nanomedicine & Biopharmaceuticals, National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, Hubei, China ABSTRACT: Superparamagnetic iron oxide nanoparticles (SPIONs) have recently been used as an effective magnetic resonance imaging (MRI) contrast agent for the noninvasive diagnosis of chronic liver diseases including nonalcohol fatty liver diseases, nonalcohol steatohepatitis, and cirrhosis as well as liver tumors. However, the potential risk of the iron overload by SPIONs has been highly underestimated in chronic liver diseases. While most of SPIONs have been shown safe in the healthy group, significant toxicity potential by the iron overload has been revealed through immunotoxicity, lipid peroxidation, and fatty acid and cholesterol metabolism in cirrhosis as a high risk factor. As a result, the systems toxicology assessments of SPIONs are crucial in both healthy ones and chronic liver disease models to determine the margin of safety. In addition, the challenge of the iron overload by SPIONs requires better designed SPIONs as MRI contrast agents for chronic liver diseases such as the biodegradable nanocluster assembly with urine clearance.

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CONTENTS

Introduction SPIONs as an Effective MRI Contrast Agent for Chronic Liver Diseases Clinical Diagnosis of Liver Diseases Using SPIONs Investigational Diagnosis of Liver Diseases in Animal Models Using SPIONs Iron Overload by SPION in Chronic Liver Diseases Is a High Risk Factor Safety of Iron Overload in Healthy Patients and Animal Models Iron Overload as a High Risk Factor in the Cirrhosis Model Potential Strategies to Overcome the Iron Overload by SPIONs In Vivo Process of SPIONs Strategies with Nanocluster Assembly Author Information Corresponding Author Funding Notes Biographies Abbreviations References

nanoscale superparamagnetic property, high magnetism in an external magnetic field yet no residual magnetism when the external field is removed. Over the past two decades, several clinical SPION products have been approved by the US FDA and European Medicines Agency (EMA) including Feridex IV in 1996, Resovist (ferucarbotran) in 2001, and Feraheme (ferumoxytol) in 2009.3−5 Feridex IV and Resovist were both approved for the diagnosis of liver cancer. Significant enhancement has been demonstrated by Feridex IV and Resovist in the diagnosis of the liver tumors with cirrhosis, and even more with the inclusion of gadolinium contrast agent as a double contrast strategy to overcome the challenge of fibrotic tissues and dysplastic nodules.6,7 Ferumoxytol has been approved for the treatment of anemia in adults with chronic kidney disease through an intravenous infusion at a maximum dose of 510 mg elemental iron.5 SPIONs have been generally considered safe, and in most cases, the potential toxicity is related to coating materials.8 For instance, only mild inflammation in the lung tissue was found in the rat model with intratracheal instillation of SPIONs at 5 mg Fe/kg.9 The injection of oleate-coated SPIONs at 2.6 mg Fe/kg in rats resulted in a mild renal toxicity that was fully recovered within 4 weeks.10 The clinical injection doses of Feridex IV and Resovist were only 0.56 and 0.6 mg Fe/kg body weight, respectively.3,4 In addition, an overdose of Resovist at 2.4 mg Fe/kg has been proven safe in healthy volunteers. However, the production of Feridex IV was discontinued by the manufacturer in 2008 and so was Resovist in 2009. Recently, the off-label uses of

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INTRODUCTION Superparamagnetic iron oxide nanoparticles (SPIONs) have widely been used as a contrast agent for the enhancement of signal-to-noise ratio in medical magnetic resonance imaging (MRI), especially for early detection of cancer.1,2 The contrast enhancing capability of SPION is attributed to its unique © 2016 American Chemical Society

Special Issue: CRT30 Received: August 29, 2016 Published: November 8, 2016 73

DOI: 10.1021/acs.chemrestox.6b00298 Chem. Res. Toxicol. 2017, 30, 73−80

74

ferumoxytol

pediatric and young adults

P(VAc-MAA)/CS coated TEM: 20 nm HD: 180.4 nm dextran coated TEM, 12 nm; HD, 50 nm citrate coated TEM, 12 nm; HD, 30 nm Fe3O4−Au clusters TEM, 28 nm

high fat diet rat model

male Ob/Ob mice with HFHC diet

alcohol fatty liver, cirrhosis rat and HCC mouse models CCl4 cirrhosis rat model

CCl4 cirrhosis rat model

c(RGDyK)-poly(acrylic acid) coated TEM, 15 nm; HD, 38 nm Resovist

Resovist

MCD rat model (NASH)

CCl4 cirrhosis rat model

Resovist labeled BMSC

CCl4 cirrhosis rat model

1.68 mg Fe/kg

30 mg Fe/kg and 15 mg Au/ kg 5.6 mg Fe/kg

2.17 mg Fe/kg

2.17 mg Fe/kg

50 mmol/kg

mesentric vein: 0.72 mg Fe/ kg 0.45 mg Fe/kg

bolus: 3 mg Fe/kg

_c

serum biochemistry and hematology studies in healthy rats over 10 days not reported

blood compatibility

blood compatibility

not reported

_c

_c

blood pressure and heart rate changes, allergic reactions

off-label use

_c

_c

_

c

_c

in vivo toxicity study

23 (2010) 24 (2011) 25 (2012) 26 (2014) 27 (2014) 28 (2015) 29 (2016) 30 (2015)

18 (2010) 19 (2010) 20 (2011) 21 (2012) 13 (2015) 22 (2016)

ref no. (year)

Abbreviations: NAFLD, nonalcohol fatty liver disease; MCD, methionine-choline deficient diet; HCC, hepatocellular carcinoma; NASH, nonalcohol steatohepatitis; BMSC, bone marrow mesenchymal stem cells; P(VAc-MAA)/CS, poly(vinyl acetate-methylacrylic acid)/chitosan; TEM, transmission electronic microscopic diameter; HD, hydrodynamic diameter; Ob, obesity; HFHC, high fiber high cholesterol. bVia intravenous injection unless specified otherwise. cProduction discontinued in 2009.

a

10. MRI with SPION at 5 min indicates cirrhosis as pseudohyperintense signal. 11. MRI with SPION at 15 min indicates the presence of cirrhosis. 12. Dual CT-MRI shows well-defined difference of fatty liver, cirrhosis, and HCC from healthy ones. 13. MRI with SPION at 6 h shows signals correlate to the level of cirrhosis (integrin αvβ3). 14. MRI assessment of the effectiveness of pioglitazone treatment of the NAFLD model

7. MRI reveals cirrhosis with BMSC-SPION up to 12 days postinjection. 8. The rate of T2* signal reduction in the dynamic MRI from 3 to 15 min differentiates the progression of NASH. 9. MRI confirms the signal difference in the cirrhosis liver.

ferumoxytol

NASH and healthy patients

infusion: 3.6 mg Fe/kg

bolus: 39.2 mg Fe in 1.4 mL

Resovist

Resovist

nondiffuse fatty liver patients with cirrhosis HCC patients with cirrhosis

Resovist

injection doseb

Resovist

NAFLD patients and MCD rat model liver cirrhosis and HCC patients

1. MRI signal reduction with SPION is inversely proportional to the level of steatosis in NAFLD. 2. Fat-suppression T2-weighted MRI well defines benign nodules from HCC tumors. 3. MRI indicates the enhancement is reduced in fatty liver and much more in cirrhosis. 4. Dynamic MRI of the liver perfusion shows a slow flow in cirrhosis. 5. MRI with high dose at 72 h differentiates NASH from healthy and simple steatosis. 6. MRI of brain, abdomen/pelvis, cardiac, extremity, and spin

SPION type and size human, 0.45 mg Fe/kg; rat, 0.56 and 2.8 mg Fe/kg 25.2 mg Fe (60 kg) bolus: 0.45 mg Fe/kg

human/animals

diseases investigated with SPIONs

Table 1. Recent Examples of SPIONs as a MRI Contrast Agent for Chronic Liver Diseasesa

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susceptibility contrast MRI, liver perfusion using Resovist revealed that there was a much slower hepatic blood flow in the cirrhosis liver than that in the healthy one, which could be potentially used in the diagnosis of cirrhosis patients (Table 1, entry 4).21 For clinically approved ferumoxytol, the off-label use in the MRI diagnosis of NASH patients revealed a significant MRI signal difference after 72 h as compared to the health ones and patients with simple steatosis (Table 1, entry 5).13 In addition, the safety of ferumoxytol as a MRI contrast agent was assessed in pediatric and young adults at one-third of the full 510 mg Fe dose (Table 1, entry 6).22 Among 86 patients with a variety of organs examined (3 on liver mass), no serious allergic reaction was observed, while significant changes of blood pressure and heart rate seemed to be related to the general anesthesia used. Thus, ferumoxytol was concluded to be well tolerated with a reduced dosage. Investigational Diagnosis of Liver Diseases in Animal Models Using SPIONs. In the animal models, more efforts have been focused on the development of effective diagnostic methodologies and functionalized SPIONs for chronic liver diseases. For instance, isolated bone marrow mesenchymal stem cells (BMSC) could be effectively labeled by Resovist with no obvious cytotoxicity (Table 1, entry 7).23 Subsequent local intravascular injection of SPION-labeled BMSC showed persistently reduced MRI signals in the cirrhotic rat liver over 12 days as compared to those with Resovist alone. Pathology staining analysis suggested that SPIONs delivered through BMSC were mainly located in the periportal and injured regions, different from that of SPION alone as in the liver Kupffer cells. The quantitative T2* relaxometry technique was reported to be able to differentiate the progression of NASH in the rat model through a dynamic MRI sequence with Resovist (Table 1, entry 8).24 Significant changes of the MR T2* rate over the time period from 3 to 10 min post-injection were observed as the NASH model progressed from 4, 7, to 10 weeks, possibly related to the increased dysfunctional level of Kupffer cells. Meanwhile, the development of more biocompatible and stable SPIONs for chronic liver diseases has been reported through modification of coating materials such as poly(vinyl acetate-methylacrylic acid)/chitosan using a layer by layer technique, dextran, and citrate (Table 1, entries 9− 11).25−27 In addition, the nanostructured assembly of SPIONs and gold nanoparticles enabled the dual imaging modularity of MR and computed tomography, which effectively differentiated the alcoholic fatty liver, cirrhosis liver, and HCC from the healthy one in the rat/mouse models (Table 1, entry 12).28 Furthermore, in the CCl4-induced cirrhosis model, the expression level of integrin αvβ3 was found to increase with the level of α-smooth muscle actin as the fibrosis progressed from week 3 to week 9.29 Thus, target-specific SPIONs conjugated with cyclic-RGD peptide for integrin αvβ3 led to enhanced MRI signals in the cirrhotic liver, which was attributed to increased accumulation of SPIONs in hepatic stellate cells (Table 1, entry 13). Finally, the effectiveness of pioglitazone treatment of NAFLD was assessed with MRI diagnosis using Resovist in an Ob/Ob mouse model (Table 1, entry 14).30 While the signal enhancement by SPIONs decreased as the NAFLD progressed from week 3 to week 9, the treatment of pioglitazone for 6 weeks showed a significantly improved enhancement by SPIONs in MRI, suggesting a partially recovered phagocytosis function of Kupffer cells.

ferumoxytol have been reported as a contrast agent in MRI with an excessively high iron overload dose. For example, ferumoxytol was used for the delineation of resectable pancreatic tumor in MRI at an injection dose of 6 mg Fe/kg body weight to a maximum of 510 mg iron.11 In the enhancement of the MRI diagnosis of central nerve system lymphoma in the brain, the full dose of ferumoxytol was also used in patients.12 Similarly, ferumoxytol was reported in the MRI diagnosis of nonalcohol steatohepatitis (NASH) patients at a dose of 3.6 mg Fe/kg body weight.13 The safety of these ferumoxytol off-label uses in MRI diagnosis was most likely based on the fact that the adverse reactions were limited to the hypersensitive and allergic patients with anemia.5 In March 2015, the US FDA issued a safety warning on ferumoxytol for “serious, potentially fatal allergic reactions” and emphasized that “Feraheme (Ferumoxytol) is specifically approved for use only in adults with iron deficiency anemia in patients with chronic kidney disease”.14 More critically, iron overload is a serious safety concern in patients with chronic liver diseases such as nonalcohol fatty liver disease (NAFLD), NASH, and cirrhosis.15−17 Considering these points, we would like to present our personal perspective on SPIONs as a MRI contrast agent for chronic liver diseases as follows: (1) SPIONs are effective MRI contrast agents for the noninvasive diagnosis of the progression of chronic liver diseases; (2) however, iron overload by SPIONs is a high risk factor in chronic liver diseases that should be addressed as a high priority; and (3) challenge of the iron overload by SPIONs requires better designed SPIONs as a MRI contrast agent for chronic liver diseases such as the biodegradable nanocluster assembly with urine clearance.

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SPIONS AS AN EFFECTIVE MRI CONTRAST AGENT FOR CHRONIC LIVER DISEASES Clinical Diagnosis of Liver Diseases Using SPIONs. The application of SPIONs as a contrast agent for chronic liver diseases is based on the fact that injected SPIONs are mainly taken up by liver macrophage Kupffer cells and that in the case of chronic liver diseases, partially impaired Kupffer cells have much less uptake of SPIONs as compared to the healthy ones resulting in decreased enhancement of MRI signals. In contrast, there are few Kupffer cells in hepatocellular carcinoma (HCC). There have been numerous reported studies of MRI with SPIONs for chronic liver diseases including NAFLD, NASH, and cirrhosis. Some recent representative examples are listed in Table 1. For the clinically approved SPION Resovist, Ono and co-workers showed that the reduced uptake of Resovist in the NASH rat liver was not related to the number of Kupffer cells but was rather due to the partially impaired function of liver macrophages (Table 1, entry 1).18 More importantly, the enhancement by Resovist in NAFLD was much less in patients with severe steatosis than mild ones. In addition, there were also differences in the enhancement by Resovist between NAFLD and chronic hepatitis C patients. In the HCC patients with liver cirrhosis, Chou et al. reported that the T2-weighted fat suppression MRI with Resovist could well differentiate the chronic liver diseases such as hemangioma, dysplastic nodules, and focal nodular hyperplasia from HCC tumors due to a partial uptake of SPIONs by Kupffer cells (Table 1, entry 2).19 Resovist was also effective to distinguish between fatty liver tissues and cirrhosis in the nondiffuse fatty infiltrated patients with a much lower uptake of SPIONs by Kupffer cells in cirrhosis (Table 1, entry 3).20 With the technique of dynamic 75

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Figure 1. Impact of iron overload by SPIONs is recoverable in the normal liver but induces significantly different responses in the cirrhosis liver. Serum biochemistry profiles of cirrhosis and normal mice were determined over 14 days post-SPION injection. Correlation scatter plots were obtained at 24 h post-SPION injection by RT2 PCR array analysis of the expression levels of 370 genes of toxicity pathways. Abbreviation for serum markers: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AKP, alkaline phosphatase; TBIL, total bilirubin; CHOL, cholesterol; HDL, high-density lipoprotein cholesterol; LDL, low density lipoprotein cholesterol; TG, triglycerides; CREA, creatinine. This figure was adapted with permission from ref 41. Copyright 2016 Nature Publishing Group.

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IRON OVERLOAD BY SPION IN CHRONIC LIVER DISEASES IS A HIGH RISK FACTOR Iron overload has been identified as a high risk factor in the progression of chronic liver diseases from NAFLD, NASH, to cirrhosis, and eventually to HCC.15−17,31,32 The excess iron oxide deposits in the liver with chronic diseases contributing to two types of risks, lipid peroxidation via oxidative stress33 and increased level of the iron homeostasis regulator hepcidin. Increased level of hepcidin inhibits the release of iron from enterocytes and liver macrophages to reduce the serum iron level, and thus, lipid peroxidation via oxidative stress persists.15−17 It has been shown that liver samples with iron deposits in the Kupffer cells had more severe steatosis and advanced level of NAFLD.34 On the other hand, SPIONs are generally considered safe, and the potential toxicity has been reported to be mostly related to coating materials.8 Only mild inflammation in the lung and mild renal toxicity were found in rats post-injection of SPIONs at 5 mg Fe/kg and oleate-coated SPIONs at 2.6 mg Fe/kg, respectively.9,10 In contrast to dextran-coated Feridex IV, long circulating PEG-phospholipid coated SPIONs with the iron oxide core size of 5, 15, or 30 nm had no impact on the blood cell count at 5 mg Fe/kg injection dose but resulted in increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in healthy mice after 30 days, suggesting potential impact on liver functions.35 Most importantly, one common phenomenon that has been consistently found with various

types of SPIONs post-injection is that iron overload persisted in the liver and spleen as excess iron deposits over a period of 20 days and even months.1,2,35−37 Considering the high risk of iron overload in chronic liver diseases, the potential toxicity of SPIONs is a great safety concern as a contrast agent for NAFLD, NASH, and cirrhosis. Safety of Iron Overload in Healthy Patients and Animal Models. Unfortunately, the safety assessment of the iron overload by SPIONs such as Feridex IV and Resovist had been mainly conducted in healthy volunteers. Because Feridex IV was nonrenal excretable, the increased serum iron level only returned to normal after 7 days in the healthy volunteers at the dose of 0.56 mg Fe/kg body weight with no other reported side effects.3 On the contrary, there were more reported back pain in patients with cirrhosis (18 out of 144, 12.5%) than those without cirrhosis (10 out 545, 1.8%). Unfortunately, no further data were available on adverse events in chronic liver diseases with Feridex IV. For Resovist, a high injection dose at 2.24 mg Fe/kg was reported to be safe in healthy volunteers.4 Similarly to Feridex IV, the serum iron level increased to the maximum after 24 h; and the iron from SPIONs was not excreted but taken up by body iron homeostasis. The reported adverse events with Resovist did not list chronic liver diseases as a potential factor. In 2008 and 2009, Feridex IV and Resovist were discontinued by the manufacturers, respectively. Meanwhile, the off-label use of ferumoxytol as a contrast agent for chronic liver diseases will be most likely restricted due to the excess amount of iron used and is shown by the FDA recent 76

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warning that it “is specifically approved for use only in adults with iron deficiency anemia in patients with chronic kidney disease”.14 Perhaps more alarmingly, intravenous iron treatment in the nondialyzed patients with chronic kidney disease and anemia was terminated early due to serious adverse events such as infections and cardiovascular events.38 In the development of specialized SPIONs for chronic liver diseases, most of studies showed the in vitro SPION toxicity in liver cells was low, below 1.0 mg/mL, yet few studies reported the in vivo toxicity except for the dual SPION-Au contrast agent (Table 1, entries 9−13). However, the safety profile of the SPION-Au contrast agent was obtained in the healthy ones over time at the same dose of 30 mg Fe/kg for imaging (Table 1, entry 12).28 According to the US FDA guidelines on the drug development, the 30 mg Fe/kg dose in the mouse and rat is equal to the human equivalent dose of 2.4 mg Fe/kg (the conversion factor is 0.08) and 4.8 mg Fe/kg (the conversion factor is 0.16) based on the body surface area, respectively.39 In addition, the FDA guidance on the drug development requires that “in general, one should consider using a safety factor of at least 10” for the dosage.39 More specifically, the FDA guidance for the development of contrast agents recommends that “the no-observed-adverse-effect level (NOAEL) in expanded-acute, single-dose toxicity studies in suitable animal species be at least one hundred times (100×) greater than the maximal mass dose to be used in human studies.”40 Thus, a safety study should be ideally carried out at a dose of 300 mg Fe/kg based on the 10fold imaging dose in animals or minimally at 240 mg Fe/kg according to human equivalent dose, which is clearly not feasible in the healthy animals, let alone in the chronic liver disease models. Iron Overload as a High Risk Factor in the Cirrhosis Model. Recently, iron overload by a biocompatible SPION was revealed to be a high risk factor at a dose of 5 mg Fe/kg in the cirrhosis mouse model as compared to the healthy ones by a systems toxicology assessment (Figure 1).41 The SPIONs studied had low retention in the liver and spleen tissues, and the excess iron from SPIONs was excreted in urine within 9 days.42 In the cirrhosis group, injection of SPIONs at 5 mg Fe/ kg induced significantly elevated levels of ALT, AST, alkaline phosphatase (AKP), total bilirubin (TBL), triglycerides (TG), and creatinine (CREA) and decreased levels of cholesterol (CHOL), high-density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), and serum iron at 24 h post-injection.41 While ALT, AST, TBL, and TG returned to normal by day 7, levels of CHOL, HDL, and LDL increased significantly on day 3 and remained higher than those of control after 14 days. In addition, a persistently decreased serum iron level was observed over a period of 14 days postSPION injection. In contrast, in the healthy control, the same dose of SPION injection only induced elevated levels of TBIL, TG, ALT, and AST and decreased iron level, which were fully recovered within 14 days (Figure 1). Further PCR array analysis indicated that SPION injection resulted in a distinct expression pattern of genes of the toxicity pathways in the cirrhosis group from that of the normal group (Figure 1).41 Predominantly up-regulated gene expressions were found in the toxicity pathways including immunotoxicity such as elevated colony-stimulating factor and IL−4, TNF induced cell death, oxidative stress and lipid metabolism including fatty acid metabolism, phospholipidosis, steatosis, and cholestasis in the cirrhosis liver tissue post-SPION injection. All of these results were consistent with the reported impact by iron overload with

elevated lipid peroxidation in chronic liver diseases.32,33,36,43 Besides SPIONs, high toxicity potentials have also been reported with Au nanoparticle and nanorods in the NASH and cirrhosis mouse models via oxidative stress, whereas low toxicity was observed in the healthy control with the same injection dose.44 On the contrary, minimal impact was found with a low injection dose of SPIONs at 0.5 mg Fe/kg in the cirrhosis group as compared to those of 5 mg Fe/kg injection dose.41 Of note, the biocompatible SPIONs at the 0.5 mg Fe/ kg injection dose were able to effectively increase the MRI contrast to reveal the presence of liver tumors below the 5 mm size.42 Thus, although SPIONs at the 5 mg Fe/kg injection dose (10 times the dose used for MRI imaging) exhibit no potential toxicity in healthy animals, the potential toxicity still needs to be independently assessed in the chronic liver disease models. Clearly, iron overload by SPIONs as a MRI contrast agent at a high dose will be a high risk factor in chronic liver diseases.

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POTENTIAL STRATEGIES TO OVERCOME THE IRON OVERLOAD BY SPIONS Obviously, one of possible ways to reduce the risk of iron overload by SPIONs is to decrease the injection dose in the MRI diagnosis. The US Office of Dietary Supplements at National Institute Health recommends the dietary intake of iron is 8 mg for men and 18 mg for women, and the upper limit of iron intake is 45 mg for healthy adults.45 Therefore, an injection of 8 mg of Fe in total or less would be a safe margin for SPIONs as a contrast agent for chronic liver diseases. This equals to a dose of 0.13 mg Fe/kg for the body weight of 60 kg, four times less than those of Feridex and Resovist. Also, according to the FDA’s recommendation for the development of contrast agents, the animal safety study would require a dose of 13 mg Fe/kg as 100 times the human dose,40 which may still be a safety concern in cirrhosis based on the results describe above in the mouse model.41 Clearly, de novo designed SPIONs are needed as a MRI contrast agent for chronic liver diseases. In Vivo Process of SPIONs. The reported studies of protein corona of SPIONs in blood serum and the uptake of leukocytes as well as subsequent metabolism have provided significant insights for better designs. The composition of the protein corona of SPIONs had been thoroughly investigated in serum with organic, inorganic, and metallic shells as well as charges.46 It was found that SPIONs with a negatively charged poly(vinyl alcohol) (PVA) shell had the least number and types of proteins adsorbed, namely, α-2-HS-glycoprotein, complement C3, serum albumin, α-1-antiproteinase, and heat shock protein 90α.46 In addition, the adsorbed proteins were tightly bound and significantly more than those on the dextran-coated SPIONs, resulting in a longer circulation time in blood.47 Moreover, the coating of protein corona also modulated the magnetism property of SPIONs.8,46,47 In blood, the dextrancoated worm-shaped SPIONs were mainly taken up by neutrophils among leukocytes through a complement C3 dependent pathway.48 On the contrary, the covalent cross-link of the coating dextran to form poly(2-hydyoxypropyl ether) significantly reduced the uptake of SPION by leukocytes by two-thirds.48,49 Because the hydrodynamic diameter (HD) of most SPIONs used as the MRI contrast agent is larger than 8 nm, SPIONs are not renal clearable and eventually accumulate in the liver and spleen.50,51 The coating materials of SPIONs have been mostly reported to be extruded from the liver 77

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Figure 2. Potential strategies for the development of SPIONs as an effective MRI contrast agent for chronic liver diseases with minimal iron overload.

nanocluster should be negative and of organic polymeric nature to reduce the adsorption of serum proteins.46 If polysaccharides are used, further covalent cross-linking is recommended to decrease the recognition by leukocytes via the complementary C3 pathway.48,49 Finally, target-specific conjugation will be used for the enhanced delivery of SPIONs to each specific chronic disease based on different pathologies as given in the examples above (Table 1, entries 7 and 13).23,29 In summary, the challenge for SPIONs as a contrast agent for chronic liver diseases is that while the contrast enhancement in MRI is effective for the noninvasive diagnosis of liver diseases, the iron overload is potentially a high risk factor for the progression of the diseases. One of the effective ways to overcome this challenge is to reduce the amount of iron used with better designed SPIONs through the assembly of SPIONs as biodegradable nanoclusters with urine clearance. For the animal safety study, the assessment of SPIONs with a 10-fold dosage or 100 times human dosage in healthy ones is not sufficient to predict the potential risks. It is crucial to independently carry out the systems toxicology assessment in the chronic disease models. Because of the different conversion factors based on the body surface area among different animals, multiple injections with scaled-up doses are necessary to define the margin of safety for clinical usage. In addition, the systems toxicology analysis is essential to define biomarkers for the iron overload induced toxicity pathways such as oxidative stress, lipid and cholesterol metabolism, and immunotoxicity in chronic diseases. Finally, the concern of the potential risk of SPIONs should not be limited to the noninvasive MRI diagnosis of chronic liver diseases but all of the applications of SPIONs in clinical settings with complications of liver diseases.

Kupffer cells without the iron oxide core and thus are considered as loosely bounded.3,4,35−37 For instance, the dextran coating of ultrasmall SPION ferumoxtran-10 was mainly excreted in urine, whereas the iron of SPIONs remained in the reticulum endothelial system and was slowly incorporated into the iron homeostasis over months with only 20% excreted through bile.52 Recently, urine excretion of excess iron has been reported with the hydroxyethyl starchcoated SPIONs with the iron oxide core diameter between 5 and 10 nm,42 suggesting that rapid bioclearance of SPIONs is possible. Strategies with Nanocluster Assembly. On the basis of the discussion above, we propose the following strategies for SPIONs as an effective MRI contrast for chronic liver diseases with minimal iron overload (Figure 2). The first three are to enhance the potential renal clearance of SPIONs, while the next three are to maximize the MRI contrast effect. They are (1) ultrasmall iron oxide core below 8 nm diameter; (2) tightly bound coating materials of the ultrasmall iron oxide core; (3) zwitterionic surface of the ultrasmall iron oxide core; (4) assembly of the ultrasmall SPION clusters for high MRI relaxivity; (5) covalent modification of the nanocluster surface such as the negatively charged organic polymer to minimize serum protein binding and complement the C3 pathway; (6) target-specific conjugation of nanocluster surface for NAFLD, NASH, or cirrhosis; (7) the nanoclusters can be metabolically degraded into the ultrasmall SPIONs for renal clearance after MRI. The assembly of ultrasmall SPIONs into the nanocluster is essential to have a sensitive contrast capability in MRI and at the same time an effective renal clearance. It has been shown that SPIONs have a reduced magnetization when the iron oxide core size decreased,53 which means that a higher injection dose of SPIONs would be needed with a smaller iron oxide core size in MRI. However, this can be overcome by forming SPION nanoclusters. We recently reported that using the same amount of SPIONs of 5−10 nm size, the assembled nanoclusters had a proportionally increased magnetization capacity (measured as T2 relaxivity) with the increased diameter of the nanocluster.42 Therefore, if the assembled nanocluster were metabolically degradable, an effective contrast agent with rapid renal clearance could be achieved as demostrated.42 In addition, the ultrasmall iron oxide core would be tightly bound by coating materials such as PVA46,47 or nitrodopamine anchors54 to facilitate the extrusion of SPIONs from the Kupffer cells to minimize the breakdown of the coating materials resulting in the formation of iron deposits in the macrophage cells. Moreover, the coating materials of the ultrasmall iron oxide core may be further modified with zwitterions such as glutathione to enhance renal clearance as demonstrated with gold nanoparticles in vivo.55 For MRI, the overall charge of the

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-27-87792147. Fax: 86-27-87794517. E-mail: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (81671812 and 81372403). Notes

The authors declare no competing financial interest. Biographies Dr. Qibing Zhou is the chair of Department of Nanomedicine & Biopharmaceuticals (from 2012 to 2016) and full professor at the College of Life Science and Technology, Huazhong University of Science and Technology in Wuhan, China. Dr. Zhou received his Ph.D. degree in the US from University of Arkansas, Fayetteville. He had his postdoctoral training at University of Maryland, College Park and initiated his own research group in the US at Virginia 78

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Chemical Research in Toxicology

Perspective

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Commonwealth University, Richmond. His current research includes the development of quinoxaline conjugates as an anticancer agent and nanomaterials as a safe contrast agent for cancer detection. Yushuang Wei is a Ph.D. candidate at the College of Life Science and Technology, Huazhong University of Science and Technology in Wuhan, China. He received his B.Sc. in 2011 from Huazhong University of Science and Technology. He joined Dr. Qibing Zhou’s research group in 2011, and his research interests include superparamagnetic iron oxide nanoparticles, MRI contrast agents for cancer detection, and systems toxicology assessment.

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ABBREVIATIONS AKP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMSC, bone marrow mesenchymal stem cells; CHOL, cholesterol; CREA, creatinine; EMA, European Medicines Agency; HCC, hepatocellular carcinoma; HD, hydrodynamic diameter; HDL, highdensity lipoprotein cholesterol; LDL, low density lipoprotein cholesterol; MRI, magnetic resonance imaging; NAFLD, nonalcohol fatty liver disease; NASH, nonalcohol steatohepatitis; PVA, poly(vinyl alcohol); SPIONs, superparamagnetic iron oxide nanoparticles; TBL, total bilirubin; TG, triglycerides

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REFERENCES

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