The Feasibility of Targeted Magnetic Iron Oxide Nanoagent for Noninvasive IgA Nephropathy Diagnosis

IgA nephropathy is the most common glomerular disease in the world and has become a serious threat to human health. Accurate and non-invasive molecular imaging to detect and recognize the IgA nephropathy is critical for the subsequent timely treatment; otherwise, it may progress to end-stage renal disease and lead to glomerular dysfunction. In this study, we have developed a sensitive, specific, and biocompatible integrin αvβ3-targeted superparamagnetic Fe3O4 nanoparticles (NPs) for the noninvasive magnetic resonance imaging (MRI) of integrin αvβ3, which is overexpressed in glomerular mesangial region of IgA nephropathy. The rat model of IgA nephropathy was successfully established and verified by biochemical tests and histological staining. Meanwhile, the clinical 18F-AlF-NOTA-PRGD2 probe molecule was utilized to visualize and further confirmed the IgA nephropathy in vivo via positron emission computed tomography. Subsequently, the Fe3O4 NPs were conjugated with arginine–glycine–aspartic acid (RGD) molecules (Fe3O4-RGD), and their integrin αvβ3-targeted T2-weighted imaging (T2WI) potential has been carefully evaluated. The Fe3O4-RGD demonstrated great relaxation in vivo. The T2WI signal of renal layers in the targeted group at 3 h after intravenous injection of Fe3O4-RGD was distinctly lower than baseline, indicating MRI signal decreased in the established IgA nephropathy rat model. Moreover, the TEM characterization and Prussian blue staining confirmed that the Fe3O4-RGD was located at the region of glomerulus and tubular interstitium. Moreover, no obvious signal decreased was detected in the untargeted Fe3O4 treated and normal groups. Collectively, our results establish the possibility of Fe3O4-RGD serving as a feasible MRI agent for the noninvasive diagnosis of IgA nephropathy.


TEM investigation:
To investigate the immune complex deposition in rat kidney, the renal sections were fixed in 2.5% glutaraldehyde overnight at 4℃, and fixed in 1% Russian acid solution for 1 hour. After that, samples washed with PBS for three times. Renal tissues were dehydrated with gradient ethanol solutions (including 30%, 50%, 70%, 80%, 90%, 95% and 100%) for 15-20min, and treated with pure acetone for 20 min. The mixture of Spurr embedding agent and acetone (1:1), the mixture of Spurr embedding agent and acetone (3:1), as well as pure Spurr embedding agent were treated samples for 1 h, 3 h and overnight respectively. After heating at 70 ℃ overnight, the samples were sliced and obtained 70-90 nm slices. The slices were stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10 min. Finally, the samples were observed by TEM (H-7650, Hitachi, Tokyo, Japan).

Synthesis of 18 F-AlF-NOTA-PRGD2
Synthesis of 18 F-AlF-NOTA-PRGD2 was based on the previous literature [Lang et al., 2011] with some adjustments. 4 mg NOTA-PRGD2 was accurately weighed and dissolved in 640uL of pure water in a 2mL centrifuge tube and mixed well. Then, 40 uL of NOTA-PRGD2 solution, 6uL of 0.01mM AlCl3 solution, 5 μL of glacial acetic acid, as well as 324 uL of DMF was added into a 2 mL centrifuge tube and mixed sufficiently. 0.5mL 18 F fluoride (about 30mCi) was added into the mixture, and then heat it at 100 o C for 10 min. After cooling, for purifying the mixture, it was transferred to C18 column with 4 mL water, washed with 20 mL water for 3 times, and finally eluted with 1 mL ethanol to acquire product. A little of product was taken for HPLC analysis to detect the radiochemical yield and purity.

Stability of 18 F-AlF-NOTA-PRGD2
The stability of 18 F-ALF-NOTA-PRGD2 was performed by paper chromatography. Firstly, 1 mL of fetal bovine serum and 1mL of PBS buffer were added into two 2 mL centrifuge tubes respectively.
Then, 400 μL product was added and mixed at room temperature. At 0, 1, 2, 4, 6 h, a little of mixture were pointed on two pieces of filter papers respectively, and hanged in chromatography cylinder with acetone solution. Finished that, the filter papers were taken out, dried and divided into 10 parts. Finally, radioactivity was measured by gamma counter.

Hemolysis assay
To evaluate the hemocompatibility of the Fe3O4-RGD through hemolysis assays, 2mL of SD rat blood was collected and centrifuged at 1500 rpm for 15 minutes. The plasma was removed. The erythrocytes were washed and diluted with PBS. 800 μL of Fe3O4-RGD solutions (6.25, 12.5, 25, 50, 100, 200 μg/mL) as samples were added to 200 μL of diluted erythrocytes. For positive control, 800 μL of distilled water was added into erythrocytes solution. And, 800 μL of PBS was added into erythrocytes solution as negative control. These tubes were incubated at 37 °C for 1 h. Each tube was centrifuged at 12000 rpm for 15 minutes. The supernatant was collected for measuring the absorbance at 540 nm with a microplate reader (Epoch 2, BioTek, Winooski, VT, USA). The hemolysis ratio was calculated with following equation:

Blood circulation of Fe3O4-RGD
3 Male SD rats were bought from Zhejiang Academy of Medical Sciences and fed with 22.8°C room temperature and 59.6% relative humidity level in the SPF degree animal laboratory of the First Affiliated Hospital, College of Medicine, Zhejiang University. The model SD rats (n=3) were anesthetized with 4% chloral hydrate at a dose of 0.8 ml/kg by intraperitoneal injection and then were administrated with Fe3O4-RGD at a dose of 15 mg/kg via intravenously injection. After that, blood sample from rat eyes were collected at preset time points (0, 0.5, 1, 2, 4, 6, 12, 24 h). ICP-MS (Agilent 7700, Agilent Technologies, California, USA) was used for measuring the Fe content of blood samples. And the ratio of Fe content to per gram blood samples were calculated.

Renal biodistribution of Fe3O4-RGD
The model SD rat (n=1) was anesthetized with 4% chloral hydrate at a dose of 0.8 ml/kg by intraperitoneal injection and then was injected with Fe3O4-RGD (15mg/kg) as administration group. After injection for 3 h, the SD rat was sacrificed through cervical dislocation and the kidney were collected. Besides, a health SD rat was not accepted any management and took cervical dislocation to collect kidneys as normal group. The Fe content deposition of these kidneys were detected by ICP-MS (Agilent 7700, Agilent Technologies, California, USA). The ratio of Fe content to per gram kidney was calculated.

Statistical Analysis
Statistical analysis was performed by using SPSS 26 (IBM Corp, Armonk, NY). T test was applied for the comparison in intragroup when the data followed normal distribution, otherwise, Mann-Whitney U test was performed. One Way ANOVA test was used to evaluate the intergroups correlations. P<0.05 were considered significant.

Figure S5
The hemolysis assay of Fe3O4-RGD samples to SD rat blood. The above figure showed that positive control group presented red liquid comparing with faint-yellow liquid of Fe3O4-RGD samples (6.25, 12.5, 25, 50, 100, 200μg/mL) and negative control group. All the hemolysis rate for Fe3O4-RGD samples were lower than 5%.

Figure S6
IgA Immunofluorescence of renal tissue with different treatment. There were a lot of IgA molecule expression in glomerular mesangial area for model group. A small amount of IgA molecule was found in the glomerular mesangial area of the normal group.

Figure S8
The 24 h pharmacokinetic curves of Fe3O4-RGD in SD rats through intravenous injection.