Cerium nitrate (Ce(NO3)3·6H2O) was obtained from Sinopharm Group Chemical Reagents Co., Ltd; Ammonia (NH3·H2O, 30%) was purchased from Shanghai Macklin Biochemical Technology Company; Dextran T-10 was obtained from Xi’an Ruixi Biological Technology Co., Ltd; 5,5-dimethyl-1-pyrroline N-oxide (DMPO), L-methionine (L-met), nitrogen blue tetrazolium (NBT), and riboflavin were purchased from Aladdin Industrial Inc. Hydrogen peroxide (H2O2, 30%) was obtained from Tianjin Damao Chemical Reagent Co., Ltd; 2,7-dichlorofluorescein diacetate (DCFH-DA) was purchased from Beyotime Biotechnology Co., Ltd.
Synthesis of cerium oxide (CeO2) and dextran-coated cerium oxide (D-CeO2)
Cerium oxide (CeO2) was synthesized by the precipitation method. Briefly, 1 mL of 1 M Ce(NO3)3·6H2O was added to the 6 mL of 30% ammonia drop by drop and stirred for 24 h. Finally, the solution was alternately washed with deionized water and ethanol three times at 9000 rpm for 5 min each. D-CeO2 were synthesized by precipitation based on the previously reported protocol [31, 32]. Briefly, 1 mL of 1 M Ce(NO3)3·6H2O was added dropwise to 2 mL of 0.2 M Dextran T-10 solution. Then, this mixture was added to 30% ammonia (6 mL) and stirred for 24 h, the color of the solution could be observed to change from yellow to dark brown gradually. The suspension obtained was first centrifuged twice to extract the larger precipitates, then transferred to an ultrafiltration tube (MWCO 100 kDa) and centrifuged three times at 4500g for 15 min each.
Preparation of chitosan/alginate hydrogel
Chitosan/alginate hydrogel was prepared according to our reported protocol [57, 58]. Firstly, dissolved chitosan with an appropriate amount of acetic acid, then adjusted the pH of the solution to 7 and ensured that the ultimate concentration is 0.6% (wt/vol). The sodium alginate solution (1.4%) was prepared by dissolving the sodium alginate in 0.15 mol/L NaCl and stirring overnight. The polysaccharide solution was made by mixing the chitosan solution with the sodium alginate in a 1:1 ratio. The chelating solution was made by mixing 30 mM Na2SO4 and 70 mM CaCl2 in a 1:1 ratio. Finally, the hydrogel was prepared by mixing the polysaccharide and the chelating solution in a 2:1 volume. D-CeO2 was dissolved in the polysaccharide solution before gavage. For gavage, mice were given 100 μL of polysaccharide solution followed by 50 μL of chelated solution.
The morphology of the synthesized CeO2 and D-CeO2 was characterized using transmission electron microscopy (TEM, JEM-2100). The hydrodynamic particle size and zeta potential were taken with a Malvern laser particle size analyzer (Malvern Instruments, UK). Fourier Transform infrared spectroscopy (FT-IR, Thermo Fisher Nicolet 5700) was performed to observe the characteristic peaks of CeO2, D-CeO2, and Dextran. X-ray diffraction (XRD, Bruker D8 Advance) and X-ray photoelectron spectroscopy (XPS, Thermo Fisher ESCALAB Xi+) were determined by the Analytical Testing Centre of Xi’an Jiaotong University.
The SOD-mimicking activity of D-CeO2
The SOD-mimicking activity was detected by electron spin resonance (ESR) and nitrogen blue tetrazolium (NBT) assays. Under light conditions, L-methionine (L-met) and riboflavin can react to produce superoxide anion (·O2−), and the DMPO can capture ·O2−. 50 µL of PBS (25 mM, pH 7.4),10 µL of L-met (130 mM), riboflavin (200 µM), EDTA (100 µM), and DMPO (250 mM) were added sequentially to the reaction system. Then added 10 µL of D-CeO2 (100 μg/mL) to the above reaction system. After 20 min of light, analysis was performed by Bruker A300-9.5/12 spectrometer.
In the presence of L-Met and riboflavin, NBT undergoes a photochemical reduction reaction to produce blue methyl hydrazone after illumination, which has the maximum absorption at 560 nm. SOD enzyme can inhibit the reduction of NBT under light. PBS, riboflavin, L-Met, and EDTA were added sequentially in the reaction plate, then added 10 µL of NBT (750 μM) and 10 µL of D-CeO2 nanoparticles with different concentrations. After illumination for 10 min, the absorption value at 560 nm was measured by enzyme standard (Bio Tek NEO2), and SOD-mimicking activity was calculated.
The CAT-mimicking activity of D-CeO2
The CAT-mimicking activity of D-CeO2 nanoparticles was assessed by analyzing the amount of O2 generation from the catalytic decomposition of H2O2 using the dissolved oxygen electrode. Briefly, H2O2 and different concentrations of D-CeO2 nanoparticles were added to the reaction bottles, then dissolved oxygen generated during 900 s was recorded with 30 s intervals. The final concentration of H2O2 was maintained at 120 mM.
Raw 264.7 and NIH 3T3 cells were cultured in DMEM medium (Gibco), and Colon-26 cells were cultured in RPMI Medium 1640 (Gibco) medium. The complete medium contains 10% fetal bovine serum and 1% penicillin/streptomycin. All the cells were grown at 37 °C in 5% CO2 humidified incubator.
Raw 264.7, Colon-26, and NIH 3T3 cells were chosen to perform the cellular uptake assay. Firstly, DiL fluorescent dye was added during the synthesis of D-CeO2 to make it monitorable under fluorescence microscopy. Then cells were incubated with D-CeO2 for 6 h and performed by fluorescent microscopy to observe the cellular uptake.
Cell viability assay
Raw 264.7 cells and Colon-26 cells were seeded in cell culture plates, respectively. After cell adherence, different concentrations of nanoparticles were added and co-incubated with cells for 24 h and 48 h. Then the culture was terminated, and MTT was incubated for 4 h. Aspirated medium and added 150 μL DMSO to each well. The absorbance at 490 nm was detected by enzyme standard, and cell viability was calculated.
Intracellular ROS scavenging ability of D-CeO2
The ability of D-CeO2 to scavenge ROS was assayed using DCFH-DA. Firstly, different concentrations of D-CeO2 (100, 500 μg/mL) were used to treat Raw 264.7 cells for 6 h. Next, a fresh medium containing 1 mM H2O2 was used to stimulate all groups for 3 h except the negative group. After washing with PBS, DCFH-DA (10 μM) was added and co-incubated with cells at 37 °C for 1 h. Then intracellular fluorescence was observed by fluorescence microscopy. Alternatively, the cells were washed with PBS and collected for detection by flow cytometry.
The protective ability of D-CeO2 against oxidative stress damage
We selected Raw 264.7 and Colon-26 cells to investigate the protection of D-CeO2 against H2O2-induced oxidative stress damage. Briefly, cells were seeded to each well of 96-well plates. After 12 h, added 1 mM H2O2 to induce the cells for 1 h in addition to the negative control group. The D-CeO2 treatment group was incubated with H2O2 in conjunction with different concentrations of nanoparticles (100, 200, 500 μg/mL). The cells were then washed with PBS and further cultured in a complete medium for 24 h. MTT assay was used to measure cell viability.
In vitro anti-inflammatory assay
Firstly, Raw 264.7 cells were seed into a 6-well plate at 1 × 104 cells/well. After 12 h, washed cells three times with PBS and then incubated with D-CeO2 nanoparticles dispersed in completed media for 6 h. The final concentration of D-CeO2 was 500 μg/mL. Lipopolysaccharide (LPS) had a final concentration of 100 ng/mL was used to stimulate cells for 24 h. Cells were collected for RNA analysis by centrifugation at 2000 rpm for 3 min.
Firstly, pre-adhered the scratch inserts to the bottom of 12-well plates. Next, NIH 3T3 cells were plated uniformly into the cell culture plate. After cells reached the monolayer state, the scratch insert was removed. Suspended cells were washed with PBS. Next serum-free medium containing 10 ng/mL TGF-β1 and different concentrations of material was added to all groups except the negative group. Cells were future-cultured for 24 h and then observed by using a microscope.
NIH 3T3 cells were seeded in cell crawling sheets and incubated with D-CeO2 for 6 h. Then added TGF-β1 (10 ng/mL) to each well. After 24 h stimulating, 4% paraformaldehyde was used to fix cells for 15 min, 0.1% TritonX-100 was used to permeabilize for 10 min, and 2% BSA was used to bloke for 60 min. For staining of α-SMA (14395-1-AP, Proteintech) and Collagen 1 (14695-1-AP, Proteintech), the cells were incubated with primary antibody at 4 °C overnight. The next day, cells were incubated with fluorescent secondary antibodies (A1108, Invitrogen) for 60 min at 37 °C, followed by washing for 10 min three times. Images were captured by fluorescence microscopy.
RNA extraction and qRT-PCR
According to the instructions, total RNA was isolated from cells or tissues using the Total RNA Extraction Kit (R0027, Beyotime). The total RNA concentration was determined by NanoDrop spectrophotometer. 1.0 μg of isolated RNA was used to prepare cDNA with cDNA Synthesis Premix (D7185M, Beyotime). qRT-PCR was performed on a BIOER Quant Gene 9600 real-time PCR system using Green Master (Roche) with 20 μL reaction mixture. The primer sequences were presented in Additional file 1: Table S1. Relative mRNA levels were quantified by the 2−ΔΔCt method.
NIH 3T3 cells were treated as described previously. At the end of incubation, cells were collected and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail. The concentration of protein was assessed by NanoDrop spectrophotometer. Then loaded the protein (200 μg) on SDS-PAGE (10% or 12%) and blotted onto NC membranes. After blocking, the primary antibodies were incubated with membranes overnight at 4 °C to detect the specific protein. Anti-α-SMA (14395-1-AP, Proteintech), anti-Collagen 1 (14695-1-AP, Proteintech), and anti-GADPH (GB12002, Servicebio) antibodies used in this part were configured at a concentration of 1:1000. The HRP-conjugated secondary antibodies against mouse (1:3000, GB23301, Servicebio) or HRP-conjugated secondary antibodies against rabbit (1:3000, GB23303, Servicebio) were used to detected appropriate primary antibodies. Bands were visualized with ECL-system, and images were captured using the chemiluminescence instrument.
Female BALB/c mice were purchased from Xi'an Keao Biotechnology Co., Ltd. Female C57BL/6 mice were obtained from the Experimental Animal Center of Xi'an Jiaotong University. The animals were kept under 22–25 °C, 65 ± 5% humidity with a 12 h light–dark cycle, and fed regular and free drinking water. All experiments complied with the Institutional Animal Care and Use Committee at Xi’an Jiaotong University.
Since CeO2 is the main component of D-CeO2 that exerts a CT imaging effect, we quantified the Ce content in D-CeO2 by ICP-MS and calculated the CeO2 content in it. The D-CeO2 concentrations covered in this section are representative of the CeO2 concentrations. Iohexol (an FDA-approved CT contrast agent) was used as a control to study the CT imaging properties of D-CeO2. Firstly, different concentrations of Iohexol solution (0.55, 1.1, 2.25, 4.5, 9.0, 18.0 mg/mL) and D-CeO2 solution (0.6, 1.2, 2.4, 220.127.116.11, 18.9 mg/mL) were measured in vitro to compare their CT imaging ability. Next, a DSS-induced acute colitis mouse model was established to explore the CT imaging ability of D-CeO2 in vivo, referencing the previous literature . Briefly, C57BL/6 mice were given water containing 2% (w/v) DSS for 7 consecutive days. The healthy group was given the same DSS-free drinking water. Then mice were gavaged with iohexol or D-CeO2 at a dose of 38 mg/kg. After administration, in vivo CT imaging was performed at 5 min, 30 min, 60 min, 120 min, and 24 h.
In vivo biocompatibility evaluation
To evaluate the biocompatibility of D-CeO2 in vivo, C57BL/6 mice were orally administered D-CeO2 (30 mg/kg in hydrogel) for 7 consecutive days and sacrificed one month later. At the end of the experiment, vital organs and the gastrointestinal tract were taken for H&E staining. Blood samples were analyzed using a hematology analyzer and compared with the control group.
TNBS-induced chronic colitis mouse model
Six to eight-week-old female BALB/c mice, weighing 18–20 g, were used to establish a TNBS-induced chronic colitis model reference to previous studies [59,60,61]. The specific experimental steps were as follows: Firstly, randomly divide the mice into 4 groups (Control, EtOH, TNBS, TNBS + D-CeO2) and fasted overnight before each enema. The enema needle was inserted into the anus for 4 cm to inject 100 μL of enema solution after mice were anesthetized with isoflurane. Then mice were immediately inverted for 1–2 min to ensure that the solution was retained in the entire colon. The control group was fed a normal diet without special treatment, the EtOH group was administered 50% ethanol solution via enema, and the TNBS group and TNBS + D-CeO2 group were given increasing concentrations of TNBS via enema once per week for 7 weeks consecutively (1.5 mg/0.1 mL for 1st and 2nd weeks, 2.0 mg/0.1 mL for 3rd and 4th weeks, and 2.5 mg/0.1 mL for the last three weeks). Mice in the TNBS + D-CeO2 group were orally administrated D-CeO2 (30 mg/kg in hydrogel) every 2 days in addition to weekly TNBS. Mice were executed after 2 days of the last enema. The body weight was recorded during the experimental period, and the intestinal condition was observed through a multifunctional small animal soft endoscope (MiniScope 2V, SHINOVA) on the day after the enema. After mice were sacrificed, recorded the colon length and collected colon tissues for RNA analysis, histopathological analysis, and immunofluorescent staining.
DSS-induced chronic colitis mouse model
Female C57BL/6 mice aged 6–8 weeks, weighing 18–20 g, were used to set up a DSS-induced chronic colitis model according to the previous study . First, we divided the mice into 3 groups (Control, DSS, DSS + D-CeO2). The mice were given 1.5–2% DSS in drinking water for 4 cycles except for the control group. On days 1–6 and 11–16, mice received drinking water containing 2% DSS. On days 21–26 and 31–36, mice received drinking water containing 1.5% DSS. On the remaining days, mice received regular water. Mice in the DSS + D-CeO2 group were orally administrated D-CeO2 (30 mg/kg in hydrogel) every two days starting from day 0. After mice were sacrificed, the colon lengths were measured and recorded, and colonic tissues were collected for analysis.
Colon tissues were histologically analyzed by hematoxylin, eosin staining (H&E), and Masson staining. Briefly, 4% paraformaldehyde was used to fix the obtained colonic tissue, followed by paraffin embedding and cutting to 5 μm layer thickness. The sections were placed on slides for drying and subsequent staining. Masson staining was performed using a kit (G1346, Solarbio) according to the instructions.
Immunofluorescence assay of the colon tissues
Colon tissue was embedded with OCT, cut into 4–6 μm sections. After permeabilizing and blocking, they were incubated with primary antibodies overnight at 4 °C, fluorescent secondary antibodies at 37 °C, and cell nuclei were stained with DAPI. Fluorescence microscopy was used to capture images.
MPO activity assay
MPO activity was analyzed by colorimetric method, and the MPO assay kit (A044-1-1, Nanjing Jiancheng Institute of Biological Engineering) was used to quantify MPO activity according to the manufacturing instructions.
GraphPad Prism 7 software was used for statistical analysis. One-way of variance (ANOVA) and t-tests were performed for statistical comparison. Statistically significant was indicated as *p < 0.05, **p < 0.01, ***p < 0.001.