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. 2021 Feb 24;24(3):102226. doi: 10.1016/j.isci.2021.102226

Regulation of Ce (Ⅲ) / Ce (Ⅳ) ratio of cerium oxide for antibacterial application

Haifeng Zhang 1,3,5, Jiajun Qiu 1,5, Bangcheng Yan 1,3, Lidan Liu 1,3, Dafu Chen 2,6,∗, Xuanyong Liu 1,3,4,6,∗∗
PMCID: PMC7944032  PMID: 33733075

Summary

Antibiotics have been considered as effective weapons against bacterial infections since they were discovered. However, antibiotic resistance caused by overuse and abuse of antibiotics is an emerging public health threat nowadays. Fully defeating bacterial infections has become a tough challenge. In this work, cerium oxide was fabricated on medical titanium by thermolysis of cerium-containing metal-organic framework (Ce-BTC). Regulation of Ce (Ⅲ)/Ce (Ⅳ) ratios was realized by pyrolysis of Ce-BTC in different gas environment, and the antibacterial properties were studied. The results indicated that, in acidic conditions, ceria with a high Ce (Ⅲ)/Ce (Ⅳ) ratio owned high oxidase-like activity which could produce reactive oxygen species. Moreover, ceria with high Ce (Ⅲ) content possessed strong ATP deprivation capacity which could cut off the energy supply of bacteria. Based on this, ceria with a high Ce (Ⅲ)/Ce (Ⅳ) ratio exhibited superior antibacterial activity

Subject areas: Microbiofilms, Surface Science

Graphical abstract

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Highlights

  • •

    Cerium oxide films were fabricated on the titanium surface by pyrolysis of Ce-BTC

  • •

    The valence states of cerium element on cerium oxide can be modulated flexibly

  • •

    CeO2-X with high Ce (Ⅲ)/Ce (Ⅳ) ratio possessed high antibacterial rate

  • •

    Antibacterial rate is related to the oxidase-like and ATP deprivation capacity

Microbiofilms; Surface Science

Introduction

There is no doubt that antibiotics are the most successful drugs developed over the past few centuries which not only save countless lives but also enable modern medical procedures (Wright, 2011). Unfortunately, due to the abuse of antibiotics, antibiotic resistance has become an emerging public health threat where antibacterial resistance brings about 700,000 deaths per year and the number of deaths will keep rising unless actions are taken. Fully defeating bacterial infections has become a tough challenge in various fields including biomedical implants and devices (Chen et al., 2018).

To address this problem, many works have been done to develop alternative antibacterial materials. For instance, metal or metal oxide nanoparticles such as Cu, Ag, and ZnO are used (Cao et al., 2018b; Li et al., 2016, 2019a; Sirelkhatim et al., 2015; Xia et al., 2020; Yang et al., 2020). Nevertheless, the dose-dependent cytotoxicity limits their applications. Currently, cerium oxide has attracted increasing attention for biological applications due to its catalytic activity which derives from the reversible switch between redox pairs Ce (Ⅲ)/Ce (Ⅳ) (Li et al., 2019b). In general, the catalytic activity of cerium oxide is closely related to the content of Ce (Ⅲ) and oxygen vacancies (Dong and Huang, 2019). Cerium oxide with a high content of Ce (Ⅲ) usually has abundant oxygen vacancies which are beneficial to easier oxygen exchange and redox reactions (Cao et al., 2018a). At basic physicochemical pH, Ce (Ⅲ)/Ce (Ⅳ) couple exhibits antioxidant properties by scavenging reactive oxygen species (ROS). At acidic pH, the Ce (Ⅲ)/Ce (Ⅳ) couple shows prooxidant properties by producing ROS (Mehmood et al., 2018). This unique redox potential of cerium oxide can be used to provide protection for normal cells and exhibit cytotoxic effects for bacteria in an acidic environment. Moreover, lanthanide-based materials such as cerium oxide usually possess high deprivation capacity toward ATP and cause cell death by cutting off the energy supply (Cao et al., 2018a).

Based on the high oxidase-like activity and ATP deprivation ability, cerium oxide may show tremendous potential for antibacterial applications in the field of biomedical implants and devices. Many methods have been used to construct cerium oxide coatings on implant surface with different Ce (Ⅲ)/Ce (Ⅳ) ratio, such as plasma sprayed (Shao et al., 2020), magnetron sputtering (Hu et al., 2018), and atomic layer deposition (Gupta et al., 2019), but cerium oxides in these coatings tend to exist in aggregated states with limited catalytic activity. Currently, porous metal oxides derived from metal-organic frameworks (MOFs) with high surface areas and tunable porosity have been widely utilized as high-performance catalysts (Li et al., 2015). Moreover, the porous carbonaceous structures which transformed from organic linkers of MOFs during thermolysis process in inert atmosphere can avoid the potential aggregation from metal oxide nanocrystals (Cao et al., 2018a; Rahul et al., 2015; Xiao et al., 2018). As for cerium oxide, the fraction of Ce (III) is size dependent and generally increases with decreases in the particle size. Therefore, in this work, cerium oxide films were fabricated on medical titanium surface by thermolysis of cerium-containing MOFs, and the Ce (Ⅲ)/Ce (Ⅳ) ratio was regulated by conducting thermolysis process in the air or Ar atmosphere. Antibacterial activities of cerium oxide with different Ce (Ⅲ)/Ce (Ⅳ) ratios were systematically investigated.

Results

Surface characterization

Surface morphologies of Ti, Ce-BTC, CeO2, and CeO2-X are shown in Figure 1A. Ti presents a relatively flat surface with slight ups and downs after mixed acid cleaning. Ce-BTC exhibits rod-like morphology. After thermolysis in air, CeO2 inherits the rod-like morphology with slight deformation. Similarly, CeO2-X also shows a rod-like shape after pyrolysis in Ar atmosphere.

Figure 1.

Figure 1

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Material characterizations

(A) SEM surface morphology of Ti, Ce-BTC, CeO2, and CeO2-X. The scale bar is 2 μm.

(B) XRD patterns acquired from Ti, Ce-BTC, CeO2, and CeO2-X.

(C) FTIR spectra of Ti, Ce-BTC, CeO2, and CeO2-X.

(D) Raman spectra of Ti, Ce-BTC, CeO2, and CeO2-X.

Figure 1B shows the X-ray diffraction (XRD) patterns of Ti, Ce-BTC, CeO2, and CeO2-X. Peaks located at 35.1°, 38.4°, 40.2°, and 53.0° correspond to the (100), (002), (101), and (102) facets of titanium, respectively (JCPDS Card No. 44-1,294). A peak of TiO2 located at 62.8° appears which indicates that the TiO2 oxide layer exists on the titanium substrate due to the natural oxidation. The diffraction patterns located at 10.1° and 17.2° suggest the high quality crystalline of the prepared Ce-BTC (Luo et al., 2018). CeO2 sample shows a diffraction peak of (111) facet of ceria located at 28.5° indicating the thermolysis of organic ligands and change of valence states from Ce3+ to Ce4+ (He et al., 2020).

The Fourier transform infrared (FTIR) spectra of Ti, Ce-BTC, CeO2, and CeO2-X are presented in Figure 1C. No obvious peaks are observed from Ti. For Ce-BTC, characteristic peaks appear in the regions at 1,612 cm-1,550 cm−1 and 1,435 cm-1,369 cm−1 which correspond to the asymmetric vibrations (Vas(-COO-)) and symmetric vibrations (Vs(-COO-)), respectively (Zhang et al., 2018). After thermolysis in air, the feature peaks of Ce-BTC disappear in the CeO2 sample. However, for the CeO2-X sample with thermolysis in the Ar atmosphere, characteristic peaks in the regions 1,612 cm-1,550 cm−1 and 1,435 cm-1,369 cm−1 still remain, although the peak intensities decrease.

Figure 1D is the Raman spectra of Ti, Ce-BTC, CeO2, and CeO2-X. Ti sample exhibits a straight line with no peaks observed. For Ce-BTC, CeO2, and CeO2-X samples, a Raman peak at ∼462 cm−1 is detected which is attributed to the F2g vibration mode of O atoms around each Ce (Ⅳ) cation (Zhang et al., 2017).

The high-resolution X-ray photoelectron spectroscopy (XPS) spectra of Ce 3d acquired from Ce-BTC, CeO2, and CeO2-X are presented in Figures 2A–2C. The peaks located at 904.6 eV, 885.6 eV, and 880.8 eV are assigned to Ce (III) and the peaks at 917.0 eV, 907.5 eV, 901.1 eV, 898.4 eV, 889.3 eV, and 882.5 eV belong to Ce (IV) (Tavarez-Martínez et al., 2019). By computing the areas of high-resolution XPS spectra of Ce 3d, the percentage content of Ce (III) can be obtained with the values of 57.0%, 12.7%, and 52.1% for Ce-BTC, CeO2, and CeO2-X, respectively (Table 1). It indicates that a higher Ce (III)/Ce (IV) ratio can be acquired after thermolysis in Ar atmosphere. Figures 2D–2F shows the high-resolution XPS spectra of O 1s from the surfaces of Ce-BTC, CeO2, and CeO2-X. The O 1s peaks can be divided into three sub-peaks at the binding energies of 529.9 eV, 531.4 eV, and 532.4 eV, which correspond to lattice oxygen (OLatt), surface active oxygen (OSur), and adsorbed oxygen (OAds), respectively (He et al., 2020). The percent contents of OLatt, OSur, and OAds were calculated by integrating the XPS peaks areas, and the results are shown in Table 1. The contents of OLatt from Ce-BTC, CeO2, and CeO2-X are 0.6%, 74.1%, and 7.8%, respectively. OSur contents from Ce-BTC, CeO2, and CeO2-X are 58.4%, 15.5%, and 57.5%, respectively. For OAds contents, they are 41.0%, 10.4%, and 34.7% for Ce-BTC, CeO2, and CeO2-X, respectively. In general, oxygen vacancy is the indicator of the degree of lattice defects and it can be generated when high valent Ce ions can be converted into lower valent Ce ions. Accordingly, the increase of OSur contents can be ascribed to the formation of oxygen vacancies.

Figure 2.

Figure 2

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XPS high-resolution spectra of Ce 3d and O 1s

(A) XPS high-resolution spectrum of Ce 3d from Ce-BTC.

(B) XPS high-resolution spectrum of Ce 3d from CeO2.

(C) XPS high-resolution spectrum of Ce 3d from CeO2-X.

(D) XPS high-resolution spectrum of O 1s from Ce-BTC.

(E) XPS high-resolution spectrum of O 1s from CeO2.

(F) XPS high-resolution spectrum of O 1s from CeO2-X.

Table 1.

Percentage contents of Ce (Ⅲ) and Ce (Ⅳ) from Ce-BTC, CeO2, and CeO2-X and of OLatt, OSur, and OAds from Ce-BTC, CeO2, and CeO2-X

Samples Ce valence concentration (%)
 O 1s concentration (%)
Ce (III) Ce (IV) OLatt OSur OAds
Ce-BTC 57.0 43.0 0.6 58.4 41.0
CeO2 12.7 87.3 74.1 15.5 10.4
CeO2-x 52.1 47.9 7.8 57.5 34.7
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Oxidase-like activity at different pH values

Many research studies have confirmed that ceria showed enzyme-mimicking activities, which are attributed to the valence states of Ce3+ and Ce4+, as well as oxygen vacancies (Artiglia et al., 2014; Tian et al., 2015; Wei and Wang, 2013). To investigate the oxidase-like activity of Ce-BTC, CeO2, and CeO2-X at different pH values, TMB (3,3′,5,5′-tetramethylbenzidine) was used. TMB is a typical oxidase chromogenic substrate that can be oxidized to its colored product (oxidized TMB, TMBox) which has characteristic absorption peaks at 370 and 652 nm (Huang et al., 2017). Adsorption spectra from Ti, Ce-BTC, CeO2, and CeO2-X (pH = 6.0) are shown in Figure 3A. No peaks are observed from Ti, while characteristic peaks of TMBox at 370 and 652 nm are detected from Ce-BTC, which indicates that Ce-BTC has oxidase-like activity in acidic conditions. After pyrolysis in air, characteristic peaks of TMBox at 370 and 652 nm can still be obtained from CeO2 while the adsorption intensities decrease. It suggests that the oxidase-like activity of CeO2 is lower than that of Ce-BTC. However, adsorption intensities of TMBox at 370 and 652 nm from CeO2-X significantly increase indicating the enhanced oxidase-like activity. The highest oxidase-like activity of CeO2-X had also been confirmed in acetate buffer, PBS, and bacterial culture media (Figure S1). As presented in Figure 3B, when the reaction was carried out in an alkaline environment, the absorption spectra from Ce-BTC, CeO2, and CeO2-X do not show any obvious peaks at 370 and 652 nm. It indicates that TMBox is not produced, which confirms that catalytic reaction does not occur because of low oxidase-like activity in alkaline conditions.

Figure 3.

Figure 3

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Oxidase-like activity, ATP hydrolysis ability, and ROS level assessment

(A) Catalytic activity of Ti, Ce-BTC, CeO2, and CeO2-X toward TMB at pH of 6.0.

(B) Catalytic activity of Ti, Ce-BTC, CeO2, and CeO2-X toward TMB at pH of 7.4.

(C) ATP hydrolysis ability of Ti, Ce-BTC, CeO2, and CeO2-X. ∗∗∗p < 0.001 versus Ti; &&p < 0.01 versus CeO2; $$p < 0.01 versus Ce-BTC.

(D) ROS levels produced from Ti, Ce-BTC, CeO2, and CeO2-X. ∗∗∗p < 0.001 versus Ti; &&&p < 0.001 versus Ce-BTC; $$$p < 0.001 versus CeO2;###p <0.001 versus Ti.

See also Figures S1 and S2.

ATP hydrolysis assay

Compounds based on lanthanide series including Ce-containing materials own high ATP deprivation ability which can hydrolyze ATP to release phosphate and adenosine leading to serious cell death. The ATP deprivation abilities of Ti, Ce-BTC, CeO2, and CeO2-X were investigated, and the corresponding results are shown in Figure 3C. The ATP deprivation contents of Ti, Ce-BTC, CeO2, and CeO2-X are 7.5%, 88.2%, 59.5%, and 97.7%, respectively. CeO2-X has the highest ATP deprivation ability, followed by Ce-BTC and CeO2. It indicates that, compared with Ce-BTC, the ATP deprivation ability of CeO2 decreases while it increases for CeO2-X. Moreover, the high ATP deprivation capacity of CeO2-X was also verified in the bacterial growth medium and PBS (Figure S2).

ROS levels

To examine the ROS levels produced by Ti, Ce-BTC, CeO2, and CeO2-X, 2',7'-dichlorodihydrofluorescein (DCFH) assay was used, and the results are presented in Figure 3D. At pH = 6.0, the ROS level of Ce-BTC is about 19 times than that of the black group. Compared with Ce-BTC, the ROS level of CeO2 decreases which is about 6 times than that of the black group. However, CeO2-X shows the highest ROS level which is approximately 62 times than that of the black group. At pH = 7.4, the abilities of ROS production of Ce-BTC, CeO2, and CeO2-X significantly decrease. It indicates that, in alkaline conditions, the catalytic activities of Ce-BTC, CeO2, and CeO2-X decline, especially for CeO2-X.

Antibacterial activity assessment

Gram-negative E. coli and gram-positive S. aureus were applied to assess the antibacterial activities of Ti, Ce-BTC, CeO2, and CeO2-X. Plate colony counting and scanning electronic microscopy (SEM) cell morphology observation were carried out, and the results are presented in Figure 4. From Figure 4A, a lot of bacteria colonies of S. aureus can be seen from Ti. Compared with Ti, numbers of bacteria colonies from Ce-BTC, CeO2, and CeO2-X reduce, especially for CeO2-X where almost no colonies are observed. Based on the plate colony counting results, antibacterial rates are obtained and presented in Figure 4B. Antibacterial rates of Ce-BTC, CeO2, and CeO2-X are 46.2%, 9.7%, and 100%, respectively. It suggests that CeO2-X exhibits the highest antibacterial rate, followed by Ce-BTC and CeO2. Figure 4C shows the SEM morphologies of S. aureus from Ti, Ce-BTC, CeO2, and CeO2-X. S. aureus with round and intact morphology is observed from Ti. No significant change of cell morphology of S. aureus can be seen from Ce-BTC and CeO2. However, S. aureus on CeO2-X is dead with deformed and wizened cell morphology as indicated in red arrows. A similar trend can be obtained from E. coli on Ti, Ce-BTC, CeO2, and CeO2-X. Lots of bacteria colonies of E. coli can be seen from Ti, whereas bacteria numbers on Ce-BTC, CeO2, and CeO2-X decrease (Figure 4D). As shown in Figure 4E, the antibacterial rates of Ce-BTC, CeO2, and CeO2-X are 60.8%, 23.8%, and 91.6%, respectively. It indicates that CeO2-X exhibits the highest antibacterial activity. SEM cell morphologies of E. coli from Ti, Ce-BTC, CeO2, and CeO2-X are presented in Figure 4F. Bacterial flagella of E. coli can be clearly seen from Ti which suggests that E. coli grow well on Ti. However, some bacteria with slight deformation can be observed from Ce-BTC and CeO2 as shown in red arrows. For E. coli on CeO2-X, they are badly wizened as presented in red arrows.

Figure 4.

Figure 4

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Antibacterial activity assessment

(A) Photographs of agar culture plates cultured with S. aureus suspension detached from Ti, Ce-BTC, CeO2, and CeO2-X.

(B) Antibacterial rates of Ti, Ce-BTC, CeO2, and CeO2-X against S. aureus based on the plate colony counting method. ∗∗∗p < 0.001 versus Ti; &&&p < 0.001 versus CeO2; $$$p < 0.001 versus Ce-BTC.

(C) SEM cell morphology of S. aureus on Ti, Ce-BTC, CeO2, and CeO2-X. The scale bar is 1 μm.

(D) Photographs of agar culture plates cultured with E. coli suspension detached from Ti, Ce-BTC, CeO2, and CeO2-X.

(E) Antibacterial rates of Ti, Ce-BTC, CeO2, and CeO2-X against E. coli based on the plate colony counting method. ∗∗p < 0.01 and ∗∗∗p < 0.001 versus Ti; &&&p < 0.001 versus CeO2; $$$p < 0.001 versus Ce-BTC.

(F) SEM cell morphology of E. coli on Ti, Ce-BTC, CeO2, and CeO2-X. The scale bar is 1 μm.

See also Figures S3 and S4.

Cytocompatibility evaluation

MC3T3-E1 cells were used to evaluate the biocompatibility of Ti, Ce-BTC, CeO2, and CeO2-X. Figure 5A shows the cell live/dead staining fluorescence images of Ti, Ce-BTC, CeO2, and CeO2-X. Green fluorescence represents the live cells while red fluorescence indicates the dead cells. Lots of green fluorescence can be observed from Ti, Ce-BTC, CeO2, and CeO2-X while almost no red fluorescence can be seen, which indicates that Ti, Ce-BTC, CeO2, and CeO2-X have no cytotoxicity. AlamarBlue assay was used to assess the cell proliferation of Ti, Ce-BTC, CeO2, and CeO2-X, and the results are presented in Figure 5B. On day 1, there is no significant difference of cell proliferation among Ti, Ce-BTC, CeO2, and CeO2-X. With the extension of culture time, cell viabilities of Ti, Ce-BTC, CeO2, and CeO2-X increase. On day 7, Ce-BTC, CeO2, and CeO2-X present a higher cell proliferation rate than that of Ti (p < 0.01). Figure 5C shows the SEM cell morphologies of Ti, Ce-BTC, CeO2, and CeO2-X after culturing for 1, 4, and 7 days. Cells with the whole contour can be observed on Ti, Ce-BTC, CeO2, and CeO2-X after culturing for 1 day. As the culture time increases, cells grow and gradually cover the sample surface. On day 7, the cells almost cover the whole surfaces of Ti, Ce-BTC, CeO2, and CeO2-X. Based on the results of cell live/dead staining, cell proliferation, and cell morphology observation, it can be concluded that Ce-BTC, CeO2, and CeO2-X show good biocompatibility against MC3T3-E1 cells.

Figure 5.

Figure 5

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Biocompatibility evaluation

(A) Live/dead cell staining of MC3TC-E1 cells on Ti, Ce-BTC, CeO2, and CeO2-X. Green fluorescence represents live cells while red fluorescence indicates dead cells. The scale bar is 100 μm.

(B) Cell proliferation of MC3TC-E1 cells on Ti, Ce-BTC, CeO2, and CeO2-X after culturing for 1, 4, and 7 days. ∗∗p < 0.01.

(C) SEM cell morphologies of MC3TC-E1 cells on Ti, Ce-BTC, CeO2, and CeO2-X after culturing for 1, 4, and 7 days. The scale bar is 50 μm.

Discussion

Ce-containing MOF pyrolysis strategy was used to obtain cerium oxide in the present study. The Raman peak at ∼462 cm−1 was detected in Ce-BTC, CeO2, and CeO2-X, which indicated the existence of cerium oxygen clusters or cerium oxide nanocrystalline in those samples (Figure 1D). The characteristic peaks appear in the regions at 1,612 cm−1,550 cm−1 and 1,435 cm−1,369 cm−1 which corresponded to the vibrational modes of -COO- presented in the FTIR spectra of Ce-BTC nearly total vanished after thermolysis in air (Figure 1C). However, the characteristic peaks still remained although the peak intensities decreased after thermolysis in the Ar atmosphere, which indicated that the organic frameworks still existed after the incomplete carbonization of Ce-BTC. The XRD results showed that Ce-BTC could be converted into CeO2 with high crystallinity after thermolysis in air, which may be originated from the aggregation of cerium oxide nanocrystals without the restriction of carbonaceous frameworks. On the contrary, due to the existence of carbonaceous frameworks and potential loose structure, cerium oxide nanocrystals were restricted to aggregate and exhibited relative weakly crystallized form (Cao et al., 2018a).

What's more, the proportion of Ce (III) in ceria nanoparticles is size dependent and generally increases with decreases in the particle size. Hence, modulation of valence states of cerium could be realized by thermolysis of Ce-containing MOFs in different gas environment by controlling the aggregation of cerium oxide nanocrystals. As shown in Figures 2 A–2C and Table 1, CeO2-X, obtained by pyrolysis of Ce-BTC in Ar atmosphere, has the percentage content of Ce (Ⅲ) with 52.1% whereas the percentage content of Ce (Ⅲ) from CeO2 with thermolysis of Ce-BTC in air is 12.7%. It indicates that Ce (Ⅲ)/Ce (Ⅳ) ratio from CeO2-X is much higher than that from CeO2. Cerium oxide with a high Ce (Ⅲ)/Ce (Ⅳ) ratio usually has abundant oxygen vacancies (Cao et al., 2018a; Vernekar et al., 2016). As presented in Figures 2D–2F and Table 1, O 1s high-resolution XPS spectra can be divided into three sub-peaks which correspond to lattice oxygen, surface active oxygen, and adsorbed oxygen. Low content of lattice oxygen and high content of surface active oxygen indicates a high concentration of oxygen vacancies. Thus, CeO2-X has higher oxygen vacancy concentration than that of CeO2 which can adsorb more O2. Therefore, CeO2-X has high percentage content of adsorbed oxygen with 34.7%.

The catalytic activity of cerium oxide is closely related to the content of Ce (Ⅲ) and oxygen vacancies. At acidic pH of 6.0, Ce-BTC, CeO2, and CeO2-X can oxidize TMB to TMBOX which show absorption peaks at 370 nm and 652 nm. It suggests the oxidase-like activity of Ce-BTC, CeO2, and CeO2-X. Due to the higher content of Ce (Ⅲ) and oxygen vacancies, CeO2-X exhibits higher adsorption intensity at 370 nm and 652 nm. Cheng et al.(Cheng et al., 2016) have investigated the reaction mechanism of oxidase-like activity. From their perspective, under acidic condition, O2, adsorbed onto the cerium oxide surface, would be converted to O2·- by Ce (Ⅲ) and TMB would be oxidized to TMBOX by O2·-. Meanwhile, Ce (Ⅳ) would be reduced to Ce (Ⅲ) with the oxidation of TMB to TMBOX and the reduced Ce (Ⅲ) could be re-oxidized to Ce (Ⅳ) by O2·- in situ. However, at basic physiological pH of 7.4, oxidation of TMB did not occur (Figure 3B) and MC3TC-E1 cells grew well on Ce-BTC, CeO2, and CeO2-X (Figure 5). Based on this, it can be concluded that, at acidic pH, Ce (Ⅲ)/Ce (Ⅳ) couple serves as prooxidant for the production of ROS and the effect would be enhanced with the increase of Ce (Ⅲ)/Ce (Ⅳ) ratio. ROS levels produced by Ti, Ce-BTC, CeO2, and CeO2-X further confirm this view (Figure 3D). Besides, compounds based on lanthanide series including Ce-containing materials own high ATP deprivation ability by hydrolyzing ATP to release phosphate and adenosine which can cut off the energy supply and lead to serious cell death. As shown in Figure 3C, CeO2-X shows the highest ATP deprivation ability, followed by Ce-BTC and CeO2.

The antibacterial mechanisms of CeO2 nanoparticles proposed in most literatures are the electrostatic attraction between CeO2 nanoparticles and bacteria where CeO2 nanoparticles are positively charged and bacteria are negatively charged, leading to oxidative stress and interfering with the nutrient transport functions (Nadeem et al., 2020; Qi et al., 2020; Zhang et al., 2019). Moreover, the antibacterial effects of CeO2 nanoparticles against S. aureus and E. coli are different, which is closely related to cell membrane structures. In general, gram-positive bacteria are composed of thicker, waxy cell wall, making them more resistant to the CeO2 nanoparticles than gram-negative bacteria. For example, the gram-positive Bacillus cereus has a cell wall of 55.4 nm, while the gram-negative S. typhimurium has a cell wall of only 2.4 nm (Kalantari et al., 2020; Pop et al., 2020). However, in this study, bacteria are living well on CeO2, meaning that the electrostatic attraction between CeO2 and bacteria is not the main antibacterial factor. Besides, antibacterial rate of CeO2-X against S. aureus is higher than that to E. coli, which further confirms that the antibacterial mechanism mentioned above is not suitable here. Results of agar diffusion assay (Figure S3) showed that no inhibition zone could be seen around the samples which suggested that CeO2 species leached into the growth medium during the bacterial growth were negligible to the antibacterial effects. The pH of bacterial culture media (Luria-Bertani medium and Nutrient Broth No.2 medium) used in this study was weakly acidic (Figure S4), and the oxidase-like activity of Ce-BTC, CeO2, and CeO2-X had been confirmed in acetate buffer, PBS, and bacterial culture media with the similar pH (Figure S1). Moreover, the high ATP deprivation capacity of CeO2-X was also verified in the bacterial growth medium and PBS, and the ATP deprivation capacity of CeO2-X had no significant difference at pH of 6.0 and 7.4 which were similar to the pH of bacterial culture medium and cell culture medium in this study, respectively (Figure S2). Compared with Ce-BTC and CeO2, the oxidase-like activity and ATP deprivation ability of CeO2-X were the highest, which corresponded to the highest antibacterial rate against S. aureus and E. coli. Therefore, we believe that CeO2-X shows antibacterial activity which is ascribed to the both oxidase-like activity by producing ROS in the acidic bacterial environment and ATP deprivation ability. Based on the results in this study, it can be seen that the antibacterial activity of cerium oxide can be tailed by regulating of Ce (Ⅲ)/Ce (Ⅳ) ratio which higher Ce (Ⅲ)/Ce (Ⅳ) ratio indicates higher ROS levels and ATP deprivation capacity leading to higher antibacterial activity. Although the percentage content of Ce (Ⅲ) from Ce-BTC is slightly higher than that from CeO2-X, the central Ce atom on Ce-BTC is nine coordinated by six oxygen atoms from water molecule and three oxygen atoms from the carboxylate groups of 1,3,5-H3BTC ligands, leading to less active sites (Zhang et al., 2018). Therefore, the oxidase-like activity and ATP deprivation capacity of Ce-BTC are lower than those of CeO2-X.

The biocompatibility of Ce-BTC, CeO2, and CeO2-X was also investigated in this study. Interestingly, the oxidase-like activity of Ce-BTC, CeO2 and CeO2-X is pH dependent, and the oxidation of TMB by Ce-BTC, CeO2, and CeO2-X can only occur under acidic conditions. Thus, in the weak alkaline environment of cell culture medium, the generation of ROS can be negligible. Moreover, the CeO2-based coating can directly contact the bacterial membrane and cut off the energy supply of bacteria. However, mammalian-based cells are much bigger than bacteria cells, and the mitochondria serve as the main organelles in the production of ATP for the mammalian-based cells which is membrane bounded into a more complex endomembrane system (Cao et al., 2011). It is difficult for CeO2-based coating to interfere with the ATP synthesized in mammalian-based cells. Therefore, Ce-BTC, CeO2, and CeO2-X showed negligible side effects to MC3T3-E1 cells.

In conclusion, cerium oxide was fabricated on the medical titanium surface by pyrolysis of Ce-BTC, and regulation of Ce (Ⅲ)/Ce (Ⅳ) ratio was realized by thermolysis of Ce-BTC in different gas environment. CeO2-X, obtained by thermolysis of Ce-BTC in Ar atmosphere, has higher Ce (Ⅲ) content and oxygen vacancies than those of CeO2 with thermolysis of Ce-BTC in air. Therefore, CeO2-X shows a higher ROS level and ATP deprivation capacity which exhibits superior antibacterial activity.

Limitations of the study

In this study, cerium oxide with alterable ratios of Ce (III)/Ce (IV) was constructed on titanium surface by pyrolysis of Ce-BTC in different gas atmosphere. However, the cerium-oxygen clusters in Ce-BTC will inevitably agglomerate together to form ceria nanoparticles during the pyrolysis process. The enzyme-like catalytic activity of cerium oxide will be partially inhibited when the content of surface oxygen vacancies decreases as the nanoparticles grow up. Moreover, only S. aureus and E. coli were used to evaluate the antibacterial properties in this study, and broad-spectrum antibacterial effect should be further investigated.

Resource availability

Lead contact

Dafu Chen: chendafujst@126.com Xuanyong Liu: xyliu@mail.sic.ac.cn.

Material availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate data sets/code.

Methods

All methods can be found in the accompanying Transparent methods supplemental file.

Acknowledgments

This study acknowledged financial support from the National Natural Science Foundation of China (51831011, 31670980), National Science Fund for Distinguished Young Scholars of China (51525207), Science and Technology Commission of Shanghai Municipality (19JC1415500, 20ZR1465100), and Beijing Municipal Health Commission (Grant No. BMHC-2019-9；BMHC-2018-4；PXM2020_026275_000002).

Author contributions

X.L., J.Q., and H.Z. participated in the conception and design of the research. H.Z., B.Y., and L.L. performed the experiments. J.Q. and H.Z. prepared the manuscript. D.C. and X.L. revised the manuscript.

Declaration of interests

The authors declare no competing financial interests.

Published: March 19, 2021

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102226.

Contributor Information

Dafu Chen, Email: chendafujst@126.com.

Xuanyong Liu, Email: xyliu@mail.sic.ac.cn.

Supplemental information

Document S1. Transparent methods and Figures S1–S4
mmc1.pdf (705KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent methods and Figures S1–S4
mmc1.pdf (705KB, pdf)

Data Availability Statement

This study did not generate data sets/code.

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