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A Contrast Examination of Proinflammatory Effects on Kidney Function for γ-Fe2O3 NP and Gadolinium Dimeglumine

Background Contrast-enhanced magnetic resonance imaging (MRI) is a powerful diagnostic tool for many diseases. In many situations, the contrasts are repeatedly administrated in order to monitor and assess the disease progression. Objective To investigate and compare the biological effects of γ-Fe2O3 nanoparticle (NP) and gadolinium dimeglumine (Gd-DTPA) with high and multiple doses on the kidney of healthy mice. Methods Polydextrose sorbitol carboxymethyl ether coated γ-Fe2O3 NP with hydrodynamic size of 68.2 nm and clinically applied Gd-DTPA were employed on healthy mice with the repeatedly intravenous administration of high doses. The cell viability of human umbilical vein endothelial cells (HUVEC) in high doses of these two contrast agents were measured using the xCELLigence Real-Time Cell Analysis (RTCA) S16 Instrument. The biological effects of γ-Fe2O3 NP and Gd-DTPA on the kidney were obtained using a biochemical automatic analyzer and multiple proinflammatory factor kit on the serum. Histopathological and immunohistochemistry analysis were taken on kidney tissues. Results It showed that the proinflammatory responses elicited by the γ-Fe2O3 NPs were weaker than that by Gd-DTPA, evidenced by the relatively much lower level of IL-1β, IL-6, IL-18, TNF-α, C-reactive protein (CRP) and Ferritin. At the same time, the γ-Fe2O3 NPs did not have the biochemical index elevated, while the Gd-DTPA did. Conclusion The γ-Fe2O3 NPs induced weaker proinflammatory effects in reference to the Gd-DTPA, indicating better renal safety. Therefore, it is suggested that γ-Fe2O3 NPs should be safer and optional choice when repeated contrast-enhanced MRI is necessary.


Introduction
Contrast-enhanced magnetic resonance imaging (MRI) is a common and necessary tool that has been used clinically for diagnosis of various organ diseases, such as cancer, infections, or bleeding. 1 In the process of imaging, contrast agents are usually administrated intravenously to enhance the visualization of illness lesions. Gadolinium (Gd)-based contrast agents (GBCAs) are currently the mainstream clinical MRI contrast agents. 2 However, there have been concerns on the increasing toxicological risk of nephrogenic systemic fibrosis (NSF) in patients with advanced renal dysfunction, 3 hence, GBCAs have been warned by the US Food and Drug Administration (FDA) to use in the patients with impaired renal function since 2010. 4 Therefore, for patients with renal dysfunction, an optional MRI contrast agent is necessary. Iron oxide nanoparticles (NPs) have attracted lots of research interests due to their influence on relaxation time, [5][6][7][8] and still being largely investigated in nanomedicine including the cell targeting, labeling and separation, drug or gene delivery system, and hyperthermia. [9][10][11][12][13][14] There are two products of iron oxide NPs, namely ferumoxsil (Lumirems/ Gastromarks) for oral administration and ferumoxide (Endorems/Feridexs) for intravenous injection received FDA approval previously, but both were discontinued for economic and safety issues reasons, [15][16][17][18] resulting from not adequate understanding of the drugs' action mechanisms. 19 So far there is one drug of Fe 3 O 4 NPs, namely Ferumoxytol approved by the FDA for the treatment of iron deficiency anemia in chronic kidney disease though a warning about the risk of allergic reaction of this drug. 20 It can be noticed that this drug is off-label used when MRI enhancing images are required for patients suffering from various tumors or kidney transplants, 21 implying valuable potentials of the iron oxide NPs in MRI imaging for patients with renal diseases. Of note, differences between GBCAs and iron oxide NPs are not investigated adequately yet, especially the biological effects on the kidney in the context of repeated administration that is often required in clinical practices. For examples, researchers compared the interactions of one single injection of GBCAs and PEG coated small-sized γ-Fe 2 O 3 NP in the healthy mice, examining the biodistribution and effects on the liver function, suggesting the iron oxide NP had a better safety profile than the GBCAs; 22 and another group studied the two contrasts in a renal failure rat model, showing the DSPE-PEG coated γ-Fe 2 O 3 NP potentially could serve as an alternative to GBCAs in patients with renal diseases. 23 It has been noticed that the repeat exposure with GBCAs demonstrated the accumulation in organs, alerting a potential threat by repeating administration, 24 however, effects of repeat administration of GBCAs and iron oxide NP on the function of kidney are still an open question. The aim of this work is to compare the influence of polydextrose sorbitol carboxymethyl ether coated γ-Fe 2 O 3 NP with hydrodynamic size of 68.2 nm and clinically applied GBCA gadolinium dimeglumine (Gd-DTPA), using the repeated administration regime and focusing on the longterm effects on the kidney from the aspect of inflammation in the level of both tissues and serum (Scheme 1).
This study was designed according to following reasons: first, as a regular diagnosis method, MRI may be conducted repeatedly every few months or weeks to assess the progress of disease or therapeutic efficacy for one patient. Second, although Gd-DTPA are considered safe for patients with normal kidney function, there are still several cases of Gd retention and fibrotic reactions reported, [25][26][27] strongly suggesting the Gd may trigger kidney injury in normal kidney for some populations. Here we showed that the serum kidney function indicators (BUN, Scr, and Cys-C) and blood inflammation factors (IL-1β, IL-6, IL-18, TNF-α, CRP and ferritin) of Gd-DTPA group were increased significantly when compared with those of γ-Fe 2 O 3 NP and the control group. The histopathological analysis of the mice kidneys also indicated that γ-Fe 2 O 3 NP was safer than Gd-DTPA with the repeatedly intravenous administration of high doses.

Materials and Methods Reagents
Gd-DTPA was purchased from Bayer Pharma AG (Germany). Polydextrose sorbitol carboxymethyl ether coated iron oxide NPs (γ-Fe 2 O 3 NPs) were synthetized by alternating-current magnetic field inducing method following the reference. 28 Stroke-physiological saline solution was purchased from Peking Union Medical College Hospital.

Characterizations
The morphology of γ-Fe 2 O 3 NP was observed by transmission electron microscope (JEOL JEM-2100). Elemental analysis was performed with energy-dispersive X-ray (EDX) on field emission scanning electron microscope (FESEM, Hitachi S-4800, Tokyo, Japan). The hydrodynamic diameters and Zeta potential of γ-Fe 2 O 3 NP were detected by a Zetasizer Nano ZS90 analyzer (Malvern instruments).

Cell Culture
Primary human umbilical vein endothelial cells (HUVECs) and endothelial cell medium were all purchased from ScienCell Research Laboratories (San Diego, CA). Cells were grown on a cell culture plates and cultured at 37°C in a wet incubator with 5% CO 2 . submit your manuscript | www.dovepress.com

Measurement of Cytotoxicity
Experiments were carried out using the xCELLigence Real-Time Cell Analysis (RTCA) S16 Instrument (ACEA Biosciences, USA) which was placed into an incubator at 37 °C with 5% CO 2 . Cell cytotoxicity experiments were performed using gold microelectrodes embedded 16-well plates (E-plate 16 PET, ACEA Biosciences, USA). Cells were seeded at a density of 1×10 4 cells/well for HUVEC. The impedance was recorded at 15 min intervals. Different concentrations of nanomaterials were added to the culture at 5 h and recorded for 96 h.

Animal Experiments
Female Balb/c mice of 6-week old were maintained at the Experimental Animal Center at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China) under specific pathogen-free conditions. Mice were feed in temperature-controlled animal room on a 12: 12-h light dark cycle. All the mice had unlimited access to a standard commercial laboratory diet. All the mice were feed in sterile animal room for one week before the experiment. Animal experiments were carried out and approved in compliance with the regulations of Chinese Academy of Medical Sciences Standing Committee on animal experiments. On the first day, all the mice in the stratified according to body weight and then randomly allocated into groups, including control (saline), γ-Fe 2 O 3 NP and Gd-DTPA group (n = 3). The dose of γ-Fe 2 O 3 NP was 20 mg/kg per mouse (4 mg/mL), and the dose of Gd-DTPA was 234.5 mg/kg per mouse (46.9 mg/mL). The contrast agents were given on day 1-4, 7 and 10. All the agents were dissolved in saline. The administration volume for each injection was 100 μL.

Sample Collection and Analysis
On Day 17 post the first injection, mice were sacrificed. Serum samples were collected by centrifugation at 4 °C. Blood urea nitrogen (BUN), serum creatinine (Scr), cystatin-c (Cys-C), C-reactive protein (CRP) and ferritin were determined by using a biochemical automatic analyzer (AU5800, Beckman Coulter, USA). The serum level of IL-6, IL-1β and TNF-α was determined by using a mouse multi-factor detection kit (MCYTOMAG-70, Merck Millipore). IL-18 was measured using mouse ELISA kit (EMC011, eBioscience).

Histopathological and Immunohistochemistry Analysis
Kidneys of the mice were harvested, weighed and fixed in 10% neutral-buffered formalin. Paraffinated sections were stained with hematoxylin and eosin (HE) and Masson's trichrome stain (BA-4079A, BASO, China).

Statistics
All the experiments were repeated at least three times unless otherwise stated, and the quantitative data are expressed as mean ± standard deviation (SD). Statistical significance was ascertained using the SPSS software (SPPS 20.0) and indicated in the corresponding figure legends. As presented in the figures, p-values below 0.05 were considered to be statistically significant.

Characterizations of MRI Contrast Agents
In this study, two MRI contrast agents were investigated. The molecular formula of Gd-DTPA was provided in Figure 1A. For the γ-Fe 2 O 3 NP, Fe atom was detected in the EDX measurement ( Figure 1B) and the inserts showed the transmission electron microscopy (TEM) image of γ-Fe 2 O 3 NP with the crystal core about 10 nm in diameter. Results obtained from the dynamic light scattering (DLS) exhibited a narrow size distribution of particle diameter both in ddH 2 O and in the CM, and the average hydrodynamic diameter of γ-Fe 2 O 3 NP was 68.2 ± 1.3 nm and 55.1 ± 0.9 nm in ddH 2 O and the CM, respectively. The Zeta potential of γ-Fe 2 O 3 NP was -26.4 ± 1.2 mV in ddH 2 O to -7.9 ± 0.6 mV in the CM (Figure 1C), the decrease of the negative charge was attributable to the protein adsorption occurring in the CM.

Cell Viability of Human Umbilical Vein Endothelial Cells (HUVEC) in High Dose of Contrast Agents
Firstly, to evaluate the cytotoxicity of these two contrast agents in vitro, the growth curves of HUVEC exposed to high dose of Gd-DTPA or γ-Fe 2 O 3 NP were measured by RTCA. The dimensionless parameter (cell index) corresponding to the relative change in measured electrical impedance represents cell status (number of attached  DovePress cells) in RTCA system. 29 The values obtained from RTCA system indicated that these two contrast agent observed no significant difference of cytotoxicity over a long incubation time of 96 h ( Figure 2).

Histopathological Images of Kidney
The effects in vivo were investigated on female balb/c mice of 6-weeks old, the doses of the contrast agents were equivalent to five times of the dose of clinical routine prescription (1-7 mg/Kg for γ-Fe 2 O 3 and 46.9 mg/Kg for Gd-DTPA). 30,31 Histological analysis with the renal tissues of mice was performed with different specific histochemistry staining after 7 days of the last administration. When stained with HE, there were a large number of cytoplasmic vacuoles observed in the renal tubular cells of Gd-DTPA group. By statistically counting the cytoplasmic vacuoles in 10 fields of vision, we showed that there were 45, 48, and 126 cytoplasmic vacuoles in the control, γ-Fe 2 O 3 NP and Gd-DTPA group respectively. This indicated the occurrence of kidney injury resulted from the injection of Gd-DTPA, while few cytoplasmic vacuoles were observed in the control group and γ-Fe 2 O 3 NP group (Figure 3).
Because organ injury usually leads to the tissue fibrosis and chronic organ dysfunction, 32 the kidney tissue samples from different groups were stained with Masson to examine whether the tissue fibrosis occurred. It was shown that the tissue was darker stained with Masson staining in the Gd-DTPA group, the area was pointed with yellow arrow heads in Figure 4. Differently, there was only a small amount of the area stained in blue in the γ-Fe 2 O 3 NP group, not showing significant difference when compared with that in the control group. These indicated that the administration of Gd-DTPA induced the clear signs of tissue fibrosis. We next verified the tissue fibrosis by using anti-mice α-SMA antibody ( Figure 5) and antimice TGF-β antibody upon the kidney tissues ( Figure 6). Results showed that the tissues of Gd-DTPA group exhibited stronger brown stains than that of the other two groups, indicating the occurrence of fibrosis; while no significant difference was observed in the control group and γ-Fe 2 O 3 NP group. The statistical analysis result for the histology on the renal was summarized in Table 1, which was performed by two experienced pathologist semi-quantitatively. The staining images and histochemistry statics analysis consistently showed that the administration of Gd-DTPA induced significant kidney injury, while that of γ-Fe 2 O 3 NP did not.

Biochemical and Inflammation Factors Measurement
The renal function related biochemical parameters and inflammation factors were measurement by using the biochemical automatic analyzer, multi-factor detection and ELISA kits. The biochemical parameters in renal function include BUN, serum creatinine (Scr) and cystatin C (Cys-C). Results showed that the administration of the two contrast agents did not affect the level of BUN compared with the control. The level of Scr and Cys-C was significantly increased in the Gd-DTPA group while not changed in the γ-Fe 2 O 3 NP group when compared with that in the control group (Figure 7). These two are accurate indicators of the kidney function and sensitive to the kidney injury, 33,34 therefore, these results strongly suggested that DovePress the multiple administration at the high doses of Gd-DTPA was able to cause mild to moderate kidney injury in the healthy mice. With the same injection regime, γ-Fe 2 O 3 NP did not affect the renal function. These results were consistent with the histopathological observations.
The inflammation factors are commonly used to indicate the occurrence of acute inflammation. Here we examined IL-1β, IL-6, IL-18, TNF-α, and ferritin by using ELISA kits, and CRP in the blood by biochemical automatic analyzer, all of which are the most common inflammation factor in clinic. 35,36 It was shown that the Gd-DTPA group elicited the highest level of the inflammation factors (IL-1β, IL-6, IL-18, TNF-α, and ferritin). The level of IL-1β in γ-Fe 2 O 3 NP group was lower than Gd-DTPA group, although it was also increased compared with the control group. There was no difference detected between the control and γ-Fe 2 O 3 NP group for the other inflammation factors (IL-6, IL-18, TNF-α, and ferritin) (Figures 8 and 9). At the same time, Gd-DTPA group showed the highest CRP level (Figure 9), though the differences were not significant in the groups. These results suggested that the administration of γ-Fe 2 O 3 NP induced clearly weaker inflammatory responses than that of Gd-DTPA.

Discussion
In this work, γ-Fe 2 O 3 NP was coated with polydextrose sorbitol carboxymethyl ether. The average hydrodynamic diameter of γ-Fe 2 O 3 NP was a little different with that in TEM image due to the highly hydrophilic coating which increased the hydrodynamic layer on the surface of NPs. The Zeta potential of γ-Fe 2 O 3 NP in ddH 2 O was -26.4 ± 1.2 mV, indicating a high dispersing stability. Upon the interaction with proteins in the CM, the size decreased a little and Zeta potential changed to close to that of the pure CM (-6.8 ± 0.5 mV), indicating a dispersing stability in the biological liquids. Here, we have shown that Gd-DTPA was able to cause moderate harmful changes on the kidney even in healthy mice by multiple administrations with the high doses. On the contrary, γ-Fe 2 O 3 NP showed a better compatibility than Gd-DTPA from the view of proinflammatory effects. These results indicated that although no significant cytotoxicity was detected with both contrasts in the endothelial cells experiment for a long incubation time, the two contrasts exhibited significantly different effects in vivo, which strongly suggested that in this situation, the conventional cytotoxicity assay cannot well reflect the real situation in vivo. Furthermore, even in the healthy mice, repeated administration of Gd-DTPA could induce kidney injury, tissue fibrosis and inflammation, on the contrary, γ-Fe 2 O 3 NP did not cause less changes.
It has been recognized that NP only less than 6 nm in diameter are able to go renal clearance, and 30-99% of administered NP will accumulate and sequester in the liver after administration into the body. 37 Given the average diameter of 68.2 ± 1.3 nm, γ-Fe 2 O 3 NPs were likely go through hepatobiliary excretion pathway and may interact with hepatic cells. This is the reason its lower kidney toxicity was exhibited in reference to that of GBCAs. On the other hands, this feature may bring potential risk in liver. In our previous investigations, the γ-Fe 2 O 3 NPs were observed in the liver tissue of mice receiving the intravenous injection once a day for 7 days with the same high dose of this work. 38 We found out that the injection up regulated the expression of CD31 and α-SMA in the liver, indicating an oxidative stress occurred, and which could be rescued by the use of reactive oxygen species scavenger mannitol or ascorbic acid. In addition, iron oxide-based nanomaterials were reported to undergo slow degradation in Kupffer cells and become sequestered in the mononuclear phagocyte system organs in the non-toxic form. 39,40 From above aspects, it is possible to take prevention and treatment to overcome the limitation in clinic applications.
For patients, many factors were able to cause kidney damage, regardless the disease itself, ages, hypertension, diabetes, and drugs or ways to treat the disease were involved. Due to above reasons, chronic kidney diseases are highly prevalent in all over the world and most of them were silent and unknown without obvious indicators. 41 This may explain that serious side effects of Gd-DTPA could happen sometimes in certain patients with "healthy" Notes: Scores 0, normal histology; 1, slight injury; 2, mild injury; 3, moderate injury; 4, severe injury.

Figure 7
Measurement of the biochemical factors (BUN, Scr and Cys-C) from serum of mice. Data are presented from a representative experiment (mean ± SD, n = 3). Statistical analysis was performed with one-way ANOVA followed by leastsignificant-difference (LSD) post hoc test for BUN, Scr. Statistical analysis was performed with one-way ANOVA followed by Dunnett-T3 post hoc test for Cys-C. *P < 0.05, compared with control, # P < 0.05 compared with γ-Fe 2 O 3 NP group.
International Journal of Nanomedicine 2021:16 submit your manuscript | www.dovepress.com DovePress kidney. 26,42 Given above circumstances, if any potential kidney injury risk factors are pre-existed, the patients even with normal kidney function should be not given Gd-DTPA as MRI imaging contrast. Therefore, when repeated MRI enhancement imaging scanning was required for one patient, such as cancer and vascular disease with multilesions, γ-Fe 2 O 3 NP should be a safer and important option of MRI contrast by clinicians. Due to the possible potential toxicity in the liver, prevention and treatment can be taken to overcome this possible limitation in clinic applications.

Conclusion
In summary, when administrated repeatedly with high doses, Gd-DTPA could induce moderate kidney injury through eliciting inflammatory effects leading to the fibrosis while γ-Fe 2 O 3 NP did not induce obvious difference compared with the control group. It is important for both healthcare practitioners and patients to avoid side effects and following problems of each measure as far as possible during the diagnosis, and the results in this study provided an extremely important hint for clinicians.

Ethics Approval
This study was performed in accordance with the regulations of Chinese Academy of Medical Sciences Standing Committee on animal experiments and was approved by the Institutional Animal Care and Use committee (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and Peking Union Medical College, Beijing, China). Statistical analysis was performed with one-way ANOVA followed by leastsignificant-difference (LSD) post hoc test *P < 0.05, **P < 0.01 compared with control, # P < 0.05 compared with γ-Fe 2 O 3 NP group.

Figure 9
Measurement of TNF-α, CRP and Ferritin from serum of mice. Data are presented from a representative experiment (mean ± SD, n = 3). Statistical analysis was performed with one-way ANOVA followed by least-significant-difference (LSD) post hoc test for CRP. Statistical analysis was performed with one-way ANOVA followed by Dunnett-T3 post hoc test for TNF-α and Ferritin. **P < 0.01, compared with control, # P < 0.05, ## P < 0.01 compared with γ-Fe 2 O 3 NP group.