Hollow cerium nanoparticles synthesized by one-step method for multienzyme activity to reduce colitis in mice

Abstract

BACKGROUND

Inflammatory bowel disease (IBD) is a common chronic intestinal inflammatory disease. High oxidative stress is a treatment target for IBD. Cerium oxide (CeO₂) nanomaterials as nanozymes with antioxidant activity are potential drugs for the treatment of colitis.

AIM

To synthesize hollow cerium (H-CeO₂) nanoparticles by one-step method and to validate the therapeutic efficacy of H-CeO₂ in IBD.

METHODS

H-CeO₂ was synthesized by one-step method and examined its characterization and nanoenzymatic activity. Subsequently, we constructed dextran sulfate sodium (DSS)-induced colitis in mice to observe the effects of H-CeO₂ on colonic inflammation. The effects of H-CeO₂ on colon inflammation and reactive oxygen species (ROS) levels in IBD mice were detected by hematoxylin and eosin staining and dichlorofluorescein diacetate staining, respectively. Finally, the biological safety of H-CeO₂ on mice was evaluated by hematoxylin and eosin staining, blood routine, and blood biochemistry.

RESULTS

H-CeO₂ nanoparticles prepared by the one-step method were uniform, monodisperse and hollow. H-CeO₂ had a good ability to scavenge ROS, ∙OH and ∙OOH. H-CeO₂ reduced DSS-induced decreases in body weight and colon length, colonic epithelial damage, inflammatory infiltration, and ROS accumulation. H-CeO₂ administration reduced the disease activity index of DSS-induced animals from about 8 to 5. H-CeO₂ had no significant effect on body weight, total platelet count, hemoglobin, white blood cell, and red blood cell counts in healthy mice. No significant damage to major organs was observed in healthy mice following H-CeO₂ administration.

CONCLUSION

The one-step synthesis of H-CeO₂ nanomaterials had good antioxidant activity, biosafety, and inhibited development of DSS-induced IBD in mice by scavenging ROS.

Key Words: Inflammatory bowel disease; Nanozymes; Hollow cerium nanoparticles; One-step method; Cerium oxide biosafety

Core Tip: Inflammatory bowel disease (IBD) is a common chronic intestinal inflammatory disease. High oxidative stress is a treatment target for IBD. Cerium oxide nanomaterials as nanozymes with antioxidant activity are potential drugs for the treatment of colitis. To synthesize hollow cerium (H-CeO₂) nanoparticles by one-step method and to study the physicochemical properties, biosafety, and therapeutic efficacy of H-CeO₂ in IBD. In this study, we found that a one-step synthesis of H-CeO₂ nanomaterials has good antioxidant activity, biosafety, and can inhibit the development of dextran sulphate sodium-induced IBD in mice by scavenging reactive oxygen species.

INTRODUCTION

Inflammatory bowel disease (IBD) is a common disease characterized by chronic inflammation of the intestine. IBD includes two subtypes: Crohn’s disease (CD) and ulcerative colitis (UC). IBD can present with bloody or non-bloody diarrhea, abdominal pain, weight loss, or extraintestinal symptoms such as a rash or arthritis[1]. Due to the damage of intestinal mucosal barrier and the increase of inflammatory cells, the content of fecal calprotectin is increased in IBD patients. Currently, there are at least three main classes of drugs for the treatment of IBD: Anti-inflammatory drugs, immunosuppressants/immunomodulators, and biologics/biosimilars (e.g., anti-tumor necrosis factor-α antibodies, anti-integrins, etc.). However, these drugs have the defects of non-specific distribution and short retention time. Patients taking these drugs for a long time may suffer from side effects, and the symptoms of a considerable number of patients are not relieved. Safe and effective drugs for IBD treatment remain to be discovered and developed[2].

Although the exact etiology of IBD remains uncertain, hyperoxidative stress caused by excess production of reactive oxygen species (ROS) is a major key to the progression of colitis and is considered a therapeutic target for the treatment of colitis. Oxidative stress generated by inflammatory activity is induced by the accumulation of ROS beyond the regulation of mechanisms of antioxidant protection. Oxidative stress causes damage to proteins, lipids, cell membranes, organelles, etc., making cell function and cell signaling difficult. In addition, the cell damage caused by the accumulation of ROS induces the production of proinflammatory mediator molecules, which further amplifies inflammation and aggravates the symptoms of IBD. Unfortunately, traditional reductive drugs are not sufficient to clear a large amount of ROS induced by acute inflammation[3-5].

With the development of nanomaterials, nanomaterials such as Prussian blue, selenium, and cerium oxide (CeO₂) have been explored and synthesized for the treatment of IBD. These nanomaterials have reductive properties, good biocompatibility, few side effects, and easy surface modification for enhanced targeting capabilities[6-8]. In addition, microenvironmental changes such as decreased intestinal tight junctions and increased permeability due to intestinal inflammation lead to reduced effectiveness of traditional therapeutic drugs. Nanomaterials with appropriate sizes are passively enriched in the area of inflammatory lesions[9,10]. Therefore, compared with traditional drugs, nanomaterials show potential in the treatment of IBD. CeO₂ belongs to the series of lanthanide metal oxides and has multienzyme activities, including superoxide-dismutase-like activity, hydroxyl radical scavenging activity, and catalase-like activity, which can effectively scavenge oxygen free radicals such as ROS, and has anti-inflammatory and immune adjustment function. A study has found that gold nanoparticles-embedded cerium nanoparticles with enhanced antioxidant activity can treat IBD by scavenging excessive ROS production and blocking inflammatory responses. Coated with negatively charged hyaluronic acid, gold nanoparticles-embedded cerium nanoparticles coated with negatively charged hyaluronic acid can effectively alleviate colon injury in mice by promoting the accumulation of inflammatory colon tissue and reducing proinflammatory cytokines through oral administration[11]. One study modified Cu single-site onto CeO₂ nanoparticles with a variety of catalytic activities, such as peroxidase (POD) activity and hydroxyl radical antioxidant capacity. Density functional theory calculations further confirmed that the synergistic interaction between Cu and CeO₂ substrate enhanced POD while inhibiting hydroxyl radical antioxidant capacity[12]. A number of studies have been done to develop CeO₂ for IBD, and the therapeutic strategies for colitis based on CeO₂ nanomaterials are promising[13,14].

However, previously well-designed materials have limited applications due to uncertain biocompatibility and complex fabrication processes. Various synthetic methods such as electrochemical deposition, hydrothermal/solvothermal treatment and thermal decomposition of complex sources in solution phase have been developed for the preparation of shape-controllable CeO₂ nanomaterials. The traditional synthesis method of CeO₂ nanomaterials involves more experimental steps. For example, the steps of the hydrothermal method include: (1) Dissolving Ce(NO₃)₃∙6H₂O and NaOH in deionized water; (2) The two solutions are combined and stirred continuously to form a milky white mixture; (3) The mixture is treated under hydrothermal conditions for 6-72 hours; and (4) Centrifugation to separate the white precipitate, wash with deionized water, and dry to obtain a yellow powder[15]. To enhance feasibility, in this study, we synthesized homogeneous, monodisperse hollow CeO₂ nanomaterials (H-CeO₂) by a simple and economical one-step method (Figure 1), and validated their multienzyme activity to effectively treat murine colitis by scavenging ROS.

Figure 1  Hollow cerium nanoparticles was synthesized by the one-step method.

MATERIALS AND METHODS

Synthesis of H-CeO₂

The H-CeO₂ were synthesized by a one-step method as follows: Dissolve cerium nitrate (1.5 mg/mL), urea (0.6 mg/mL), citric acid (0.2 mg/mL) and cetyltrimethyl ammonium bromide (2 mg/mL) in a mixture of 5 mL water and 5 mL absolute ethanol to form a transparent solution. Add 1 mL cyclohexane into the solution and stir rapidly to form a white microemulsion. The mixture was heated at 80 °C for 24 hours. The pellet was obtained by centrifugation and washed. Finally, the precipitate was calcined at 650 °C for 3 hours.

Characterization

Scanning electron microscopy pictures were obtained using a Hitachi S-4800 high-resolution scanning electron microscope (accelerating voltage 1.0 kV). Transmission electron microscopy pictures were obtained using a JEOL JEM-2100 F high-resolution transmission electron microscope (acceleration voltage 200 kV). Preparation of samples: Ultrasonically disperse the material powder in ethanol, then drop it on copper mesh. The dried samples on the copper mesh were directly used for testing.

POD assay

POD is an ROS scavenging enzyme. Using 3,3’,5,5’-tetramethylbenzidine (TMB) as the detection substrate, the POD-like activity of H-CeO₂ was detected in an HAc-NaAc buffer solution of 0.2 M H₂O₂ at room temperature. We measured the absorbance of the sample at 650 nm with a microplate reader. The reaction system was 30% H₂O₂, 10 mg/mL TMB, 10 μg/mL H-CeO₂ solution and buffer solution.

Catalytic decomposition of H₂O₂

We added 0.6 mL of H₂O₂ solution (0.6 M) to the pH = 7.4 phosphate buffered saline, then 0.2 mL of H-CeO₂ solution (100 μg/mL) and mixed well. The oxygen content of H₂O₂ catalyzed by H-CeO₂ was measured on a multiparameter analyzer with a specific oxygen electrode.

Effects of H-CeO₂ on hydroxyl radicals

Various concentrations of H-CeO₂ (0, 5, 10, 20, and 40 μg/mL) were added to solutions containing 0.1 mg/mL TiO₂ and 50 mmol/L 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). The mixture was placed in a quartz capillary and exposed to 300 nm UV for 10 minutes. Electron spin resonance and Bruker EMX Plus were used for recording.

Effect of H-CeO₂ on superoxide anions

The xanthine/xanthine oxidase system was used as the substrate to generate superoxide anion, and BMPO was used as the superoxide anion detector. Electron spin resonance spectra were obtained after adding different concentrations of H-CeO₂ to the xanthine/xanthine oxidase system.

ROS determination in cells

To examine the effect of H-CeO₂ on ROS elimination at the cellular level, we used H₂O₂ to induce excessive ROS production in HCT116 cells. 1 × 10⁵ cells/well were cultured in a six-well culture dish, treated with 100 μM H₂O₂ for 2 hours, then added 5 μg/mL H-CeO₂ was added for another 2 hours, and stained with 10 μM DCFH-DA at 37 °C for 15 minutes. The ROS concentration in the stained cells was observed using a confocal laser microscope.

Colitis mouse model and H-CeO₂ administration

BALB/c mice (8-10 weeks, 22-25 g, male, n = 21) were purchased from Shanghai Animal Experiment Center and raised in a specific pathogen-free facility. Mice were randomly divided into four groups: Control group, dextran sulfate sodium (DSS)-induced group, DSS + H-CeO2 group, DSS + sulfasalazine (SASP) positive control group. The mice were fed with 3% (weight%) DSS solution instead of water for 1 week to construct a DSS-induced colitis mouse model. The colitis group and the treatment group were injected with normal saline and H-CeO₂ (20 mg/kg) intravenously on days 3-10. The positive control group was given SASP (100 mg/kg) by gavage. During the treatment period, the body weight of each mouse was determined. On day 11, mice were anesthetized with isoflurane, and disease activity index (DAI) was measured. The length of colon samples was measured and stored.

Hematoxylin and eosin staining

Colon tissue samples were paraffin-embedded and sectioned. Paraffin tissue sections were rehydrated and stained with aqueous hematoxylin solution for several minutes. After the samples were dehydrated with alcohol, they were incubated in alcohol-eosin staining solution for 2-3 minutes. Finally, the tissue sections were dehydrated with alcohol, mounted and observed under a microscope.

ROS determination in tissue sections

Fresh colon tissue of control, DSS and treatment groups was taken for frozen sectioning. Tissue sections were stained with DCFH-DA and developed using a chromogenic reagent for 15 minutes at 37 °C in the dark. Finally, tissue sections were incubated with stop solution and observed under a microscope.

Biosecurity

Healthy BALB/c mice were injected with 100 μL normal saline and different concentrations of H-CeO₂ (0.01 mg/mL, 1 mg/mL, and 2 mg/mL) via the tail vein. The main organs (heart, liver, spleen, lung and kidney) of nude mice were collected on 7 and 30 days after injection, and the damage to specific organs by nanomaterials was observed by hematoxylin and eosin staining. The effects of H-CeO₂ on blood and liver function in mice were detected by a blood routine detector and biochemical analyzer, respectively.

Statistical analysis

The experimental data were expressed as mean ± SD. The results were analyzed by one-way analysis of variance (ANOVA) with the statistical software SPSS version 16.0. The t-test was used for comparison between the two groups, and one-way ANOVA was used for the comparison of the means of multiple samples. P < 0.05 represented a statistically significant difference.

RESULTS

Synthesis and characterization of H-CeO₂ nanoparticles

The morphology of the nanoparticles was observed by transmission and scanning electron microscopy (Figure 2A and B). It can be seen that the prepared H-CeO₂ nanoparticles had a uniform, monodisperse and hollow morphology. The cavity of H-CeO₂ was well maintained without collapse. Dynamic light scattering showed that the average particle size of H-CeO2 was about 140 nm (Figure 2C).

Figure 2 Characterization of hollow cerium nanoparticles. A: Transmission electron microscopy image of hollow cerium (H-CeO₂) nanoparticles; B: Scanning electron microscope image of H-CeO₂; C: Dynamic light scattering image of H-CeO₂.

H-CeO₂ exhibits good elimination of ∙OH, ∙OOH and ROS in vitro

We investigated the scavenging effect of H-CeO₂ on ROS in the TiO₂/UV system generated by hydroxyl radicals in vitro. On the TiO₂/UV system without H-CeO₂ (control, 0 μm/mL), the signal intensity of BMPO/∙OH was the strongest. When the concentration of H-CeO₂ increased from 0 to 10 μm/mL, the signal intensity of BMPO/∙OH had a sharp drop to 30.8% of the control group. When the concentration of H-CeO₂ increased from 10 μg/mL to 40 μg/mL, the signal intensity of BMPO/∙OH still showed a decreasing trend, and the signal intensity at 40 μg/mL was only 12.9% of the control group. The results show that H-CeO₂ had good ability to scavenge ∙OH (Figure 3A).

Figure 3 Hollow cerium nanoparticles exhibits good elimination of ∙OH, ∙OOH and reactive oxygen species in vitro. A: The effect of hollow cerium (H-CeO₂) nanoparticles on the formation of ∙OH in the TiO2/UV system; B: Peroxidase activity of H-CeO₂; C: Effects of H-CeO₂ on ∙OOH produced by the xanthine/xanthine oxidase system; D: Dichlorofluorescein Diacetate probe to detect the ability of H-CeO₂ to scavenge reactive oxygen species in HCT116 cells. H-CeO₂: Hollow cerium.

The POD-like activity of H-CeO₂ was investigated with TMB; a natural POD substrate. After treatment with H-CeO₂ nanoparticles, the absorbance of TMB at 650 nm rapidly and significantly increased from 0 to 0.9 within 13 minutes, indicating that H-CeO₂ possessed POD-like activity (Figure 3B). In addition, the signal intensity of BMPO/∙OOH was significantly decreased in a H-CeO₂ concentration-dependent manner, suggesting that H-CeO₂ has SOD-like enzymatic activity for scavenging ∙OOH (Figure 3C).

Finally, we examined the scavenging ability of H-CeO₂ on ROS in HCT116 cells. Cells were incubated with 100 μM H₂O₂ to generate excess ROS intracellularly. The results of DCFH-DA probe staining showed that H₂O₂-induced ROS production was high, while H-CeO₂ nanoparticles could effectively scavenge ROS. These results indicated that H-CeO₂ had a good ROS scavenging ability (Figure 3D).

H-CeO₂ inhibits occurrence and development of colitis in mice by scavenging ROS

Based on the good POD-like and SOD-like activities of H-CeO₂ nanoparticles, it is speculated that H-CeO₂ scavenges ROS in inflammation-related diseases. DSS-induced mice exhibited weight loss, indicating the establishment of colitis. We found that H-CeO₂ nanoparticle treatment inhibited the DSS-induced substantial decrease in body weight and colon length (Figure 4A-C). At the same time, the colon was collected for histological analysis, and the changes in colon structure after different treatments were observed. Histological analysis showed that the mucosal area of the colitis mice was severely damaged, a large number of inflammatory cells were infiltrated, and the colonic epithelium was severely damaged. However, after H-CeO₂ treatment, only mild damage and inflammatory infiltration were observed in the mucosa or muscle. As a positive control, SASP, like H-CeO₂, reduced DSS-induced colon tissue damage and inflammatory infiltration (Figure 4D and E). The DAI value of DSS-induced mice was about 8, while H-CeO₂ treatment alleviated the DAI value to about 5, which means that H-CeO₂ is effective in treating colitis (Figure 4F). Finally, we found that ROS was significantly increased in the tissues of the DSS-induced group compared with the control group, while ROS levels were significantly decreased after H-CeO₂ treatment. This indicates that H-CeO₂ has a good therapeutic effect on colitis by effectively scavenging ROS (Figure 4G). Compared with the control group, the levels of inflammatory factors tumor necrosis factor-α, interleukin 6, monocyte chemoattractant protein-1, and myeloperoxidase were significantly increased in the DSS group (Figure 5). These results suggested that H-CeO₂ alleviated inflammation by eliminating ROS, although the effect was as good as that of the positive control.

Figure 4 Hollow cerium inhibits occurrence and development of colitis in mice by scavenging reactive oxygen species. A-C: Hollow cerium nanoparticles (H-CeO₂) treatment reduced the changes in weight (A) and colon length (B and C) of dextran sulfate sodium (DSS)-induced colitis mice; D and E: Hematoxylin and eosin detected the effect of H-CeO₂ treatment on the histopathology of the colon in DSS-induced mice; F: H-CeO₂ treatment reduces disease activity index values in DSS-induced mice; G: H-CeO₂ treatment reduced reactive oxygen species levels in DSS-induced mouse tissues. Cell nuclei stained with 4’,6-diamidino-2-phenylindole (blue), and intracellular reactive oxygen species levels were quantitated in dihydroethidium (red). ^aP < 0.001 vs control group; ^bP < 0.01 vs dextran sulfate sodium group; ^cP < 0.001 vs dextran sulfate sodium group; ^dP < 0.01 vs dextran sulfate sodium + hollow cerium nanoparticles group; ^eP < 0.001 vs dextran sulfate sodium + hollow cerium nanoparticles group; NS indicates statistical difference. DSS: Dextran sulfate sodium; H-CeO₂: Hollow cerium; SASP: Sulfasalazine; NS: Not significant.

Figure 5 Hollow cerium inhibits inflammatory factors. A-C: The expression levels of pro-inflammatory factors tumor necrosis factor-α (A), interleukin-6 (B), and monocyte chemoattractantprotein-1 (C) in serum were detected by enzyme-linked immunosorbent assay; D: Myeloperoxidase activity in serum. ^aP < 0.01; ^bP < 0.001. DSS: Dextran sulfate sodium; H-CeO₂: Hollow cerium; SASP: Sulfasalazine; TNF: Tumor necrosis factor; IL: Interleukin; MCP: Monocyte chemoattractantprotein; MPO: Myeloperoxidase.

H-CeO₂ has good biological safety

We randomly divided 20 BALB/c mice (6-8 weeks, female) into four groups of five, and injected 100 μL saline, or 0.01 mg/mL, 1 mg/mL or 2 mg/mL H-CeO₂ intravenously. Thirty days after the first drug injection, serum was collected to measure blood biochemical and blood routine indexes. The total number of platelets, hemoglobin, white blood cells and red blood cells were not affected by H-CeO₂, and there were no significant changes in liver cell injury markers, alanine aminotransferase and aspartate aminotransferase after H-CeO₂ administration. In addition, no significant change in body weight was found (Figure 6). Finally, we took the main organs (heart, liver, spleen, lung and kidney) of one mouse on 1, 7, and 30 days, and the histopathological changes of the main organs were observed by hematoxylin and eosin staining. The results showed no obvious damage to the main organs of the H-CeO₂ administration groups (Figure 7). These results confirmed the negligible toxicity of H-CeO₂ after administration and demonstrated that H-CeO₂ has good biocompatibility and biosafety.

Figure 6 Hollow cerium has good biological safety. A-F: The effect of hollow cerium nanoparticles on the levels of blood routine (A-D) and liver function indexes alanine aminotransferase (E) and aspartate aminotransferase (F) in the serum of healthy mice; G: Hollow cerium nanoparticles did not affect the bodyweight of healthy mice. H-CeO₂: Hollow cerium; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.

Figure 7 Hematoxylin and eosin detected the effect of different doses of hollow cerium nanoparticles administration on the main organs of healthy mice. A-D: Hematoxylin and eosin staining of the main organs from mice receiving 2 mg/mL (A), 1 mg/mL (B), 0.01 mg/mL (C), and saline (D) injection. H-CeO₂: Hollow cerium.

DISCUSSION

CeO₂ nanoparticles are potential nanomedicines for the treatment of colitis[16]. In this study, we reported that the one-step synthesis technology of H-CeO₂; clarification that the H-CeO₂ particles synthesized by the one-step method had the characteristics of uniform particle size, monodispersity, and hollowness; H-CeO₂ synthesized by the one-step method had antioxidant activity in vitro, and effectively scavenged ROS in colon tissue of DSS-induced mice in vivo, inhibit colonic inflammatory injury, reduce DAI index, and showed good biocompatibility and security.

CeO₂ nanomaterials are widely used in biology, catalysis, gas sensors, and other fields. Through different methods and parameter adjustment, CeO₂ nanomaterials with different structures and shapes such as rods, flowers and tubes can be obtained[17-20]. The morphology of CeO₂ nanomaterials is closely related to their catalytic activity and physicochemical properties. It has been reported that adjusting the temperature parameters of the hydrothermal method can control the formation of spherical or cubic morphologies of nanoparticles, and the morphology of nanoparticles affects their antibacterial biological activities[21]. Other researchers have synthesized CeO₂ nanomaterials with different morphologies and sizes such as nanospheres, nanorods, and flower-like microspheres by hydrothermal and solvothermal methods[22]. We constructed uniform, monodisperse, and H-CeO₂ nanomaterials by a one-step method. The simplification of the synthesis method of medical nanomaterials is beneficial to the control of large-scale production costs and reduces the economic burden on patients. The one-step synthesis scheme in this study can provide technical support for the application of H-CeO₂.

Several studies have shown that CeO₂ has good antioxidant and anti-inflammatory effects, and can relieve the inflammation and clinical symptoms of IBD in mice. Yang et al[16] reported that H-CeO₂ synthesized by hydrothermal method as an ROS scavenger reduced the level of proinflammatory cytokines and slowed DSS-induced colitis. Zhang et al[7] found that CeO₂ modified by polydopamine and polyethylene glycol could scavenge ROS and regulate macrophage polarization, with antioxidant and anti-inflammatory properties, thus treating colitis. Zeng et al[23] developed a drug-free CeO₂ nanoparticles that can inhibit the coactivation of nuclear factor-kappaB and Janus kinase 2/signal transducer and activator of transcription 3 pathways, thereby continuously scavenging ROS, downregulating the levels of a variety of proinflammatory cytokines, inhibiting the proinflammatory profile of macrophages and T helper 1/T helper 17 response, and improving the proinflammatory microenvironment. These studies suggest that changes in the material process may alter its physicochemical properties and catalytic activity, which in turn may alter its therapeutic effect on diseases. Our study demonstrates that the H-CeO₂ constructed by the one-step method still has good nanoenzyme activity and can inhibit DSS-induced colonic inflammation in IBD mice. We also proved that H-CeO₂ has good biocompatibility and safety.

Although the physicochemical properties and antioxidative and anti-inflammatory effects of H-CeO₂in vitro and in vivo were studied in this study, the oral acceptability of the drug needs to be assessed to realize the clinical transformation of H-CeO₂[10]. There is currently no evidence that cerium is naturally present in the human body, and no studies have reported how the human body clears CeO₂ nanoparticles, leaving doubts about the safety of CeO₂ for use in medicine[24]. Therefore, we will continue to explore the oral acceptability and in vivo metabolic pathways of CeO₂ nanoparticles in future studies.

CONCLUSION

H-CeO₂ nanoparticles prepared by the one-step method are homogeneous, monodisperse, hollow, and have good antioxidant activity. In an IBD mouse model, we demonstrated that H-CeO₂ nanoparticles have good biosafety and inhibits the development of colonic inflammation by effectively scavenging ROS.