Synthesis of Small-Size PEGylated Superparamagnetic Iron Oxide Nanocrystal ([email protected])
Small size [email protected] were prepared as previously reported method  . In brief, hydrophobic SPIO nanocrystals were synthesized by high-temperature thermal decomposition of iron acetylacetonate, and the small crystal size was composed by controlling the reaction heating conditions. After that, SPIO nanocrystals were transferred to aqueous phase by ligand exchange reaction with the sodium citrate. Subsequently, PEG-DA was coated on the surface of SPIO nanocrystals in an aqueous environment. The crystal size and dispersity were examined using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The composition changes of SPIO nanocrystals were characterized by Fourier transform infrared spectroscopy (FTIR). The concentration of iron was determined by using an atomic absorption spectrometer (AAS).
In Vitro MRI Study of Small-Size [email protected] Nanoparticles
Small-size [email protected] aqueous samples with different iron concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mmol/L) were prepared and placed in 2 mL test bottles. Longitudinal and transverse relaxation times (T1 and T2) were measured at room temperature at 0.5 T (Magnetic resonance developer relaxation rate analyzer, Niumag, Shanghai, China), 1.5 T (Minispec Mq60 NMR Analyzer, Bruker, Beijing, China), and 3.0 T (Discovery MR750, GE Medical System, Milwaukee, WI), respectively. Then, the T1 and T2 relaxivities (1/T1 and 1/T2, s− 1) were determined by curve fitting of the longitudinal or transverse relaxation rate (r1 or r2, s− 1) versus of the Fe concentration. MR imaging of samples was performed under a clinical 3.0 T MR scanner (Discovery MR750, GE Medical System, Milwaukee, WI) with a head coil. Axial images of the phantoms were acquired by T1-weighted spin-echo sequence (repetition time = 200 ms, echo time = 9 ms, matrix = 320 × 320, field of vision = 18 × 18 mm, slice thickness = 3 mm, flip angle = 90°) and T2 weighted spin echo sequence (repetition time = 2500 ms, echo time = 100 ms, matrix = 320 × 320, field of vision = 18 × 18 mm, slice thickness = 3 mm, flip angle = 90°).
[email protected] nanoparticles were tested for cytotoxicity on hepatic cancer cells (Hep G2). Hep G2 was provided by the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose (4.5 g/L glucose) containing 10% (v/v) fetal bovine serum (Biological Industries, Israel) and 1% (v/v) penicillin-streptomycin (Hyclone, Logan, UT) at 37°C, 5% CO2, and saturated humidity. Hep G2 cells were plated in 96-well plates at 4×103 cells per well (100 µL) of complete media. The cells were added with varying concentrations (5, 10, 15, and 20 µg/ml) of [email protected] nanoparticles and with a followed incubation for 24 h. Afterward, each well was added with a 10 µL Cell Counting Kit-8 (CCK-8) solution (Boster Biological Technology, Wuhan, China) and incubated at 37°C for 1 h in the following. The absorbance of the samples was measured using a microplate reader (Thermo Scientific). Cell viability was calculated according to the equation: Cell viability (%) = (NS / NC) × 100%, where NS and NC are the absorbances of living cells treated with or without [email protected] nanoparticles. And the absorbance measurements were used and corrected as blank control group.
Animal Liver Fibrosis Model
This study has been approved by the Ethics Committee of our research institution (NSMC-2021-79). All experimental procedures were followed in strict accordance with the proposal in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Healthy male Sprague-Dawley (SD) rats (180-200g and 6–7 weeks old) were obtained from the Experimental Animal Center of our institute. Twenty-eight rats were randomly divided into the control group and four experimental groups. The control group rats (n = 7) were fed a standard commercial diet. The four experimental groups were fed a 0.1% 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) supplemented diet (Tokyo Chemical Industry, Co, LTD, Tokyo, Japan) for 1 week, 4 weeks, 8 weeks and 12 weeks, respectively, to build different stages of biliary liver fibrotic model . All animals were raised at 23 ± 3°C with a 12:12h light/dark cycle and tap water ad libitum.
In Vivo MR Imaging
All MR imaging was performed at a 3.0 T MRI scanner (GE healthcare, MR750 Medical System, Milwaukee, WI). The rats were inhalation anesthetized by isoflurane (RWD Life Science, Shenzhen, China) with a face mask (Anesthetic Conc.: 1.0-1.5%; 20–30 mL O2/kg) and transferred to the MR scanner. A custom-made animal receiver coil was used to obtain the MR images. MRI scans were carried out before (pre) and immediately after (post) [email protected] intravenous injection at a dose of 2.5 mg (Fe) /kg body weight through the tail vein of the rats. Conventional T1 and T2 weighted MR imaging were also performed pre- and post-contrast injection, and the detail parameter of T1 and T2-weighted sequences were as follows: T1 weighted imaging (GRE sequence, repetition time (TR) = 9 ms, echo time (TE) = 3 ms, flip angle (FA) = 30°, field of view (FOV) = 80 × 80 mm); T2 weighted imaging (fast spin-echo sequence, TR = 4000 ms, TE = 76 ms, FA = 90°), FOV = 80 × 80 mm and slice thickness = 2.0 mm).
T 1 mapping images were acquired by using the variable flip angle (VFA) technique  which contains a series of rat liver acquisition volume acceleration (LAVA) with five different flip angles (3, 6, 9, 12, and 15°). The followings are the MR imaging parameters: TR = 5.0 ms, TE = 2.4 ms, number of excitations = 4, FOV = 80 × 80 mm, matrix = 512 × 512, reconstruction matrix = 400 × 400, bandwidth = 83.33 Hz per pixel, slice thickness = 0.8 mm. T2 mapping images were acquired by using free-breath fast spin echo (FES) sequences, the detail parameters were as follows: TR = 1200 ms, TE = 8, 16, 24, 32, 40, 48, 56, and 64 ms, number of excitations = 2, FA = 90°, FOV = 80 × 80 mm, matrix = 512 × 512, reconstruction matrix = 400 × 400, bandwidth = 166.67 Hz per pixel, thickness = 2.0 mm, slice space = 0.6 mm.
Two experienced radiologists performed image analyses with three and fourteen diagnosis experiences who were blinded to both the animal groups and pathologic results of this study. The conventional T1 and T2 weighted MR images, including per- and post-contrasted images, were presented on a picture archiving and communication system (PACS; GE Advantage Workstation Version 4.4-09, Sun Microsystems, Palo Alto, CA, USA) with an optimal window setting adjustment. Subsequently, we measured each experimental rat’s signal to noise ratios (SNR = mean signal intensity of the liver / background noise). Furthermore, based on the SNR measurements, the contrast-to-noise ratio (ΔCNR = abs (SNRpre - SNRpost)) as described by Siedek F et al.  was calculated for each rat include T1 and T2 weighted MR images pre and post [email protected] enhanced (ΔCNR-T1 and ΔCNR-T2, respectively).
T 1 mapping and T2 mapping images, including per- and post-contrasted images, were transferred to matrix laboratory (MATLAB) for the next analysis. T1 relaxation times were measured by the VFA method, which has been demonstrated by Deoni SC et al. . Three regions of interest (ROIs) of one section were respectively drawn within the liver parenchyma and constantly measured on five liver sections of each rat, avoiding confounding factors like biliary structures, vessels, and organ boundaries. The size of ROI was controlled within 0.8-1.2cm2. The ROIs were first placed into the pre-contrast T1 and T2 mapping. And then, the ROIs were copied and placed on the same area on post-contrast relaxation maps for each rat. Mean T1 and T2 values were used for further analyses. The decreasing rate of T1 relaxation time (ΔT1%) and T2 relaxation time (ΔT2%) were calculated as ΔT1% = (T1pre-T1post)/T1pre ×100% and ΔT2% = (T2pre-T2post)/T2pre ×100%. Where T1pre (or T2pre) and T1post (or T2 post) are relaxation times of pre- and post- the small size [email protected] injection via the tail vein of rat .
Based on the calculated relaxation time of T1 and T2, we adapted the T1 mapping and T2 mapping sequences for the T1-T2 dual-mode image fusion. Firstly, we associated the T1 mapping and T2 mapping images with image registration. Secondly, the pixel of ROIs in these two sequences was dividing and coloring via a “logic gate”. The logic gate was briefly depicted in Fig. 8. Third, the number of positive pixels and all the pixels in ROIs were measured to calculate the PPR (PPR = the number of positive pixel / all the pixel in ROI). It is worth noting that, the threshold value of the logic gate was defined as subtracting the mean reduction value of T1 and T2 values from corresponding pre-contrasted enhanced T1 and T2 values. All the image fusion analyses were performed by using MATLAB (version 9.7 (R2019b), Mathworks, USA).
All the quantitative data were shown as mean ± stander deviation. All the statistical analyses were performed by using commercial software SPSS (SPSS version 26.0, SPSS Inc., Chicago, IL). Spearman’s ranked correlation test was used to investigate the correlation between the ΔCNR (ΔCNR-T1 and ΔCNR-T2), relaxation time parameters (T1 values, T2 values, ΔT1%, and ΔT2%), PPR, and pathological liver fibrosis stages. Unpaired student’s t-test or one-way analysis of variance (ANOVA) were used to compare the mean-variance between different fibrosis groups. Intraclass correlation coefficient (ICC) was used to test the consistency of signal intensity and relaxation time measurements of two observers, ICC values less than 0.5, between 0.5 and 0.75, between 0.75 and 0.90, and greater than 0.90 represent poor, moderate, good, and excellent repeatability, respectively . All tests were two-tail with a P value less than 0.05 was considered statistically significant.
After MRI examination, animals were sacrificed by cervical dislocation under deep anesthesia with isoflurane, and the livers were removed and immediately fixed in 10% formalin. All the liver samples were stained with hematoxylin-eosin stain (HE), Masson’s trichrome stain, and Prussian blue stain. Pathologic analysis was regarded as the reference standard of staging rat liver fibrosis according to the METAIVR classification score system , in which S0 = no fibrosis, S1 = portal fibrosis without septa, S2 = portal fibrosis and a few septa, S3 = numerous septa without cirrhosis, and S4 = cirrhosis. A pathologist with five years’ experience in the diagnosis of liver pathology, has evaluated the stage of liver fibrosis (S0-S4) based on the standard classification mentioned before.