Preparation, Characterization and Multiple Biological Properties of Peptide-Modified Cerium Oxide Nanoparticles

Although cerium oxide nanoparticles are attracting much attention in the biomedical field due to their unique physicochemical and biological functions, the cerium oxide nanoparticles greatly suffer from several unmet physicochemical challenges, including loss of enzymatic activity during the storage, non-specific cellular uptake, off-target toxicities, etc. Herein, in order to improve the targeting property of cerium oxide nanoparticles, we first modified cerium oxide nanoparticles (CeO2) with polyacrylic acid (PAA) and then conjugated with an endothelium-targeting peptide glycine-arginine-aspartic acid (cRGD) to construct CeO2@PAA@RGD. The physiochemical characterization results showed that the surface modifications did not impact the intrinsic enzymatic properties of CeO2, including catalase-like (CAT) and superoxide dismutase-like (SOD) activities. Moreover, the cellular assay data showed that CeO2@PAA@RGD exhibited a good biocompatibility and a higher cellular uptake due to the presence of RGD targeting peptide on its surface. CeO2@PAA@RGD effectively scavenged reactive oxygen species (ROS) to protect cells from oxidative-stress-induced damage. Additionally, it was found that the CeO2@PAA@RGD converted the phenotype of macrophages from proinflammatory (M1) to anti-inflammatory (M2) phenotype, inhibiting the occurrence of inflammation. Furthermore, the CeO2@PAA@RGD also promoted endothelial cell-mediated migration and angiogenesis. Collectively, our results successfully demonstrate the promising application of CeO2@PAA@RGD in the future biomedical field.

Despite much recent progress made in the biomedical application of cerium oxide nanoparticles, the further biomedical application of cerium oxide nanoparticles is severely hindered by some unmet physicochemical challenges, including loss of enzymatic activity during the storage, non-specific cellular uptake, off-target toxicities, etc. Among them, smaller cerium oxide particles (less than 5 nm) have been shown to have higher enzymatic high-glucose culture solution, RPMI.1640 Incomplete Medium (1X), fetal bovine serum (FBS), trypsin EDTA digestion solution, and phosphate buffer PBS (1X) were purchased from Jiangsu KeyGEN Biotechnology Co., Ltd. (Nanjing, China). CD86-APC antibody and CD206-Alexa Fluor 488 antibody were purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Matrigel Basement Membrane Matrix was obtained from Corning Life Sciences Co., Ltd. (Wujiang, China).

Synthesis and Characterizations of CeO 2 @PAA and CeO 2 @PAA@RGD
Synthesis: The CeO 2 @PAA was prepared according to the previously reported protocol developed by Asati et al. with minor modifications ( Figure S1 in the Supporting Information) [29]. Briefly, a total of 2.17 g (Ce(NO 3 ) 3 ·6H 2 O) was dissolved in 5 mL of deionized water at room temperature. The solution was then mixed with 10 mL of PAA (2.5 M). Then, the mixture was added dropwise into 30 mL of NH 3 ·H 2 O under stirring at room temperature for 24 h. Subsequently, the CeO 2 @PAA solution was then concentrated using a 30 KDa ultrafiltration centrifuge tube to remove any free PAA.
The modification of the c(RGDyK) peptide (M = 619.69) on CeO 2 @PAA was based on the amide condensation reaction [38]. The obtained CeO 2 @PAA (30 mg, Ce element mass in CeO 2 NP S ) was dispersed in the 10 mL of MES buffer (pH = 5.50). EDC (30 mg) and NHS (20 mg) were added to the above solution and stirred for 30 min. Then, the mixture was purified using a 10 K ultrafiltration centrifuge tube to remove the extra EDC and NHS. Subsequently, 10 mg of c(RGDyK) peptides was dissolved in borate buffer (pH = 8.20) and added to the above reactants to react for 24 h under shaking conditions. After the reaction, the CeO 2 @PAA@RGD solution was obtained by centrifugation and ultrafiltration using a 10 KDa ultrafiltration tube.

CAT Mimetic Activity Assay
The CAT activity of cerium oxide nanoparticles was measured by monitoring the generated O 2 content from the decomposition of H 2 O 2 using a dissolved oxygen electrode (JPSJ-605F, Lei ci, Shanghai, China) according to the previously reported literature [39,40]. Briefly, nanoparticles (500 µL, 10 mg/mL, calculated as Ce) were added into 8 mL of Tris-Cl buffer (pH = 7.48), and then 500 µL of H 2 O 2 (30%) solution was added into the solution. The ability of CeO 2 @PAA@RGD to catalyze the decomposition of H 2 O 2 to produce O 2 was detected by the dissolved oxygen electrode in 30 min according to the manufacturer's instruction [39].

SOD Mimetic Activity Assay
The SOD activity of cerium oxide nanoparticles was measured with an SOD assay kit. The amount of formazan produced by the (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) (WST-8) with O 2 − was directly correlated with the number of O 2 − . The substances with SOD enzyme activity can catalyze the disproportionation of O 2 − , thus decreasing the production of formazan dye. The quantitative determination of SOD mimetic activity was obtained by recording the absorbance of formazan at 450 nm. The concentration of nanoparticles used in the experiment was 10 mg/mL (calculated as Ce). The specific reaction system is shown in Table S1. The percentage inhibition of O 2 − was calculated by Equation (1).

H 2 O 2 Scavenging
The detection method of H 2 O 2 refers to the previously reported literature using the Amplex-Red reagent assay to detect the absorbance at 570 nm [40]. Briefly, 5 µL of cerium oxide dispersions (10 mg/mL, Ce element mass in CeO 2 NP S ) was added into 100 µL of Tris-HCl buffer (pH = 7.50) and mixed with 10 µL of an H 2 O 2 (120 mg/mL) solution. The reaction was performed for 10 min at room temperature. Then, 10 µL of Amplex Red (10 mM) reagent and 5 µL of horseradish peroxidase (400 µg/mL) were added and quickly transferred to a multifunctional enzyme labeler (Infinite M200, Männedorf, Switzerland to measure the absorbance at 570 nm.

Hydroxyl Radicals (·OH) Scavenging
The hydroxyl radicals (·OH) scavenging assay was performed according to the report by Zhou et al. [41]. ·OH was produced by the Fenton reaction: H 2 O 2 + Fe 2+ = ·OH + H 2 O + Fe 3+ . Salicylic acid can react with ·OH to produce 2,3-dihydroxybenzoic acid, which has a specific absorption at 510 nm. When an ·OH scavenger is added to the reaction system, the production of colored compounds would be reduced accordingly. All reagents were sequentially added to the 96-well plates according to Table S2 and reacted at 37 • C for 30 min. The absorbance at 510 nm was measured using a multifunctional enzyme standard (Infinite M200, Männedorf, Switzerland). The scavenging rate of ·OH (X) was calculated by Equation (2).

Cytotoxicity
A Cell Counting Kit-8 (CCK8) was used to evaluate the cytotoxicity of nanoparticles according to the previously reported method by Du et al. [42]. The cells (Raw264.7 or HUVEC) were seeded into 96-well plates with an equal density of 5 × 10 4 cells and cultured in 100 µL of DMEM (Raw264.7) or RPMI.1640 (HUVEC) medium supplemented with 10% FBS prior to the assay, respectively. Then, cells were treated with a medium containing different concentration of nanoparticles (CeO 2 @PAA or CeO 2 @PAA@RGD) varying from 0-100 µg/mL (calculated based on the amount of Ce content in CeO 2 ) and incubated for an additional 12 h or 24 h, respectively. Untreated cells were chosen as a control group. At the end of the treatment, the cells were rinsed with PBS three times and incubated in a serum-free medium containing 10% CCK8 for 2 h. Finally, the absorbance was measured at 450 nm using a multifunctional enzyme marker (Infinite M200, USA). All samples were measured in triplicate. Relative cell viability was calculated by Equation (3).
Relative viability = [(absorbance value of experimental group − absorbance value of blank group)/ (absorbance value of control group − absorbance value of blank group)] * 100 (3)

Cellular Uptake
The cellular uptake of the different nanoparticles by HUVEC cells was examined according to the method previously reported by Yu et al. [43]. HUVEC cells were seeded in the laser confocal Petri dishes and incubated overnight. Then, the cells were treated with CeO 2 @PAA or CeO 2 @PAA@RGD containing RPMI-1640 culture medium for 4 h or 8 h, respectively. After rinsing with PBS buffer, the cells were fixed with 1 mL of 4% formaldehyde for 15 min at 4 • C condition, followed by another round of PBS washing. The cell nuclear staining was performed using Hoechst33342. The cells were imaged using a CLSM imaging system (TI2-S-HU, Nikon, Tokyo, Japan).

Intracellular ROS Scavenging in Raw264.7 Cells
The intracellular ROS-scavenging ability of different nanoparticles were compared. Briefly, Raw264.7 cells were seeded in the laser confocal Petri dishes and stimulated with H 2 O 2 (100 µM) for 12 h to establish an oxidative stress cell model, followed by treat-ment with different nanoparticles for 24 h, including CeO 2 @PAA or CeO 2 @PAA@RGD (100 µg/mL, calculated based on the amount of Ce content). Cells without H 2 O 2 treatment were used as a negative control group. After the treatment was completed, the cells were washed with PBS three times and stained with DCFH-DA (10 µM) for 30 min. Finally, intracellular fluorescence levels were observed and measured using a laser confocal microscope (TI2-S-HU, Japan, excitation wavelength: 488 nm, emission wavelength: 525 nm) and a Becton Dickinson FACScan analyzer (Franklin Lakes, NJ, USA).

Assessment of Macrophages Polarization In Vitro
The cellular expression of CD86 (a M1 macrophage marker) and CD206 (a M2 macrophage marker) were detected by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) to assess the extent of macrophage polarization according to the previous reported [44]. Briefly, Raw264.7 cells were seeded into the 6-well plate and cultured in DMEM medium supplemented with 10% FBS overnight, followed by being treated with LPS (2 µg/mL) for another 24 h. The cells treated with LPS were subsequently treated with CeO 2 @PAA@RGD or CeO 2 @PAA (100 µg/mL) for 24 h, respectively. At the end of the study, the cells were incubated with CD86-APC diluted with 1% BSA/PBS for 30 min at room temperature. Then, the cells were washed with PBS and were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 1% (v/v) PBS/Trixton-100 for 5 min, followed by incubation with CD206-Alexa Fluor 488 diluted with 1% BSA/PBS for 30 min at room temperature. The value of the average fluorescence intensities of positive CD206 and CD86 was found using flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Qrt-PCR Detection of Anti-Inflammatory and Proinflammatory Cytokines
The expression levels of proinflammatory markers (CD86, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)) in M1 cells and anti-inflammatory markers (CD206, vascular endothelial growth factor (VEGF), and interleukin-10 (IL-10)) in M2 cells were further analyzed by quantitative RT-PCR analysis. Raw264.7 cells were treated with the same method as 2.10 above. Total RNA was extracted using Trizol reagent and quantified by the fluorescence quantitative PCR instrument (Bio-rad CFX). The Cdna was then synthesized by reverse transcription according to the reaction system in Table S3. The sequences of the primers are listed in Table S4 of the Supporting Information. The expression gene levels were normalized to GAPDH used as a housekeeping gene, and the relative levels were measured by the 2 −(∆∆CT) method [45].

In Vitro Tube Formation of HUVEC
The HUVEC tube information was examined using Matrigel (Corning 356234, Nanjing, China) precoated in 96-well plates to assess the effect of the CeO 2 @PAA@RGD on the tube formation of HUVEC. The detailed procedure referred to that previously reported by Liu et al. [46]. HUVEC cells were cultured in a starving condition one day before the experiment. Then, HUVEC cells treated with different cerium oxide nanoparticles, including CeO 2 @PAA or CeO 2 @PAA@RGD (25 µg/Ml, calculated based on the amount of Ce content), were seeded on the polymerized Matrigel plates. After incubation for 2 h, 4 h, and 8 h, tube formation was observed using the microscope (CKX53, Tokyo, Japan), respectively. The node points and branches' lengths were also calculated using ImageJ software.

Promotion of Cell Migration
Cell migration was recorded by microscopy at different time points to assess the effect of CeO 2 @PAA@RGD on HUVEC cell migration. According to the previously reported method [47], HUVEC cells were seeded into 6-well plates. After overnight incubation, a straight scratch was created with a pipette tip (200 µL), and the cells were gently washed with PBS three times. The fresh medium containing different cerium oxide nanoparticles, including CeO 2 @PAA or CeO 2 @PAA@RGD (25 µg/mL, calculated based on the amount of Ce content), was added. Images were taken at different time points with a microscope. The gap of the wound scratch was then determined using ImageJ software, and the percentage of wound healing was calculated according to Equation (4).

Statistical Analysis
Statistical analysis of the experimental data was calculated using GraphPad and Origin software. The images were analyzed by ImageJ. One-way ANOVA was conducted during the statistical analysis; p-values represent significant differences. A p > 0.05 indicates no significant difference (ns) in experimental data. All * p < 0.05, ** p < 0.01, or *** p < 0.001 were considered statistically significant.

In Vitro Physicochemical Characterization, Enzymatic Activity, and ROS-Scavenging Ability of CeO 2 @PAA@RGD
The CeO 2 @PAA nanoparticles were conjugated with RGD peptides to form CeO 2 @PAA @RGD. The DLS analyzer data showed that after the RGD modification, the hydrodynamic diameter of CeO 2 @PAA@RGD increased to 14.93 ± 1.52 nm from 6.84 ± 0.15 nm of CeO 2 @PAA ( Figure 1a). The zeta potentials of CeO 2 @PAA and CeO 2 @PAA@RGD nanoparticles in a neutral aqueous solution were −23.80 ± 1.38 mV and −11.63 ± 1.41 mV, respectively ( Figure 1b). The change in zeta potentials after conjugation was due to the RGD occupying part of the free carboxyl group on CeO 2 @PAA. TEM imaging indicated that CeO 2 @PAA@RGD presented a regular special shape with a uniform distribution and an average diameter of 3.35 ± 0.04 nm (Figure 1c), which was lower than the hydrodynamic size, due to the increased hydration layer caused by the PAA and RGD peptide modification on the particle surface. Meanwhile, the UV-Vis spectra showed that the CeO 2 @PAA@RGD had a characteristic absorbance peak similar to the CeO 2 @PAA at around 280 nm (Figure 1d), which is consistent with that previous reported by Lei et al. [48]. X-ray photoelectron spectroscopy (XPS) analyses were used to determine the surface Ce 3+ /Ce 4+ ratio on the surface of the CeO 2 @PAA@RGD nanoparticles. Two main peaks, including Ce 3d 3/2 and Ce 3d 5/2, in the Ce 3d spectrum of CeO 2 @PAA@RGD are presented in Figure 1e. The Ce 3d spectrum was decomposed into six peaks [49], including four peaks of Ce 4+ and two peaks of Ce 3+ [41]. The peaks of Ce 3+ are 881.90 and 900.60 eV, while the peaks of Ce 4+ at 885.30, 904.00, 887.10, and 906.30 eV. The ratio of the total integrated area under the fitted peaks of Ce 3+ (or Ce 4+ ) to the total area of all peaks was used to calculate the relative concentration of the oxidized state of Ce 3+ (or Ce 4+ ) on the surface of cerium oxide nanoparticles, and it was found that the concentration of Ce 4+ (63.05%) was higher than that of the Ce 3+ (36.95%) oxidation state. Overall, these results implied the successful formation of CeO 2 @PAA@RGD.
We further determined the O 2 generation ability via a time-dependent H 2 O 2 decomposition assay to evaluate the catalase mimic enzyme (CAT) of CeO 2 @PAA@RGD. As shown in Figure S3, a large number of O 2 bubbles were observed, and the solution showed a significant yellow color in the presence of nanoparticles. The color generated could be ascribed to the oxidation of Ce 3+ to Ce 4+ [40]. In contrast, almost no oxygen bubbles were observed in the PBS, and the solution appeared clear and colorless. The dynamic monitoring further showed that the oxygen concentration in the PBS group was almost unchanged for 30 min continuously. However, the dissolved O 2 produced by the decomposition of H 2 O 2 increased with the extended reaction time in the presence of nanoparticles (Figure 1f). The results indicated that CeO 2 @PAA@RGD had catalase-like catalytic activity. The RGD peptide modification did not affect its catalytic activity. could be ascribed to the oxidation of Ce to Ce [40]. In contrast, almost no oxygen bubbles were observed in the PBS, and the solution appeared clear and colorless. The dynamic monitoring further showed that the oxygen concentration in the PBS group was almost unchanged for 30 min continuously. However, the dissolved O2 produced by the decomposition of H2O2 increased with the extended reaction time in the presence of nanoparticles (Figure 1f). The results indicated that CeO2@PAA@RGD had catalase-like catalytic activity. The RGD peptide modification did not affect its catalytic activity. and Ce 4+ are indicated by different colors; (f) the generation of oxygen was measured by a dissolved oxygen electrode to assess the catalase activity of CeO2@PAA@RGD; (g) the SOD-like activ- Figure 1. In vitro physicochemical characterizations, enzymatic activity, and ROS-scavenging ability of CeO 2 @PAA@RGD nanoparticles. (a) Hydrodynamic size distribution of CeO 2 @PAA (red line) and CeO 2 @PAA@RGD (green line) nanoparticles; (b) zeta potential analysis of CeO 2 @PAA (red) and CeO 2 @PAA@RGD (green) nanoparticles; (c) transmission electron microscopy (TEM) morphology characterization of CeO 2 @PAA@RGD nanoparticles. Scale bar = 20 nm (inset image: the TEM size distribution of CeO 2 @PAA@RGD nanoparticles); (d) UV-Vis spectra of CeO 2 @PAA and CeO 2 @PAA@RGD nanoparticles; (e) the X-ray photoelectron spectra of CeO 2 @PAA@RGD show the variation of spin-orbit double peaks of 3d 3/2 and 3d 5/2. The characteristic peaks of Ce 3+ and Ce 4+ are indicated by different colors; (f) the generation of oxygen was measured by a dissolved oxygen electrode to assess the catalase activity of CeO 2 @PAA@RGD; (g) the SOD-like activity of CeO 2 @PAA@RGD was assessed by calculating the percentage of superoxide anion inhibition; (h) the H 2 O 2 scavenging ability of CeO 2 @PAA and CeO 2 @PAA@RGD was assessed by measuring the absorbance at 570 nm; (i) the scavenging percent of ·OH by CeO 2 @PAA and CeO 2 @PAA@RGD. Error bars = mean ± standard deviation (SD) (n = 3); ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, using a one-way ANOVA (and nonparametric or mixed).
Superoxide dismutase (SOD) is a critical antioxidant enzyme in cells; it can catalyze the decomposition of O 2 − into O 2 and H 2 O 2 [50]. The SOD mimetic activity of the CeO 2 @PAA@RGD was evaluated using the WST-8 method. Figure 1g indicates that compared to the control PBS (0.46%), the O 2 ·− inhibition rate of CeO 2 @PAA and CeO 2 @PAA@RGD reached 21.67% (p < 0.01) and 34.26% (p < 0.001), respectively. In addition, the RGD peptide modification further increased the inhibition rate of CeO 2 @PAA@RGD by 14.59% (p < 0.05) compared to that of CeO 2 @PAA (at the same Ce concentration). The enhanced SOD-mimicking activity of CeO 2 @PAA@RGD may be due to the improved surface affinity resulting from RGD peptide modification.
The ROS-scavenging activity of CeO 2 @PAA@RGD was further demonstrated. Two representative ROS, including H 2 O 2 and ·OH, were selected for this assay. The results suggested that both nanoparticles were effective in scavenging H 2 O 2 and ·OH compared to the control group. Moreover, there was no significant difference between CeO 2 @PAA and CeO 2 @PAA@RGD for H 2 O 2 scavenging (Figure 1h), which proved that the surface modification of nanoparticles has almost no effect on H 2 O 2 scavenging. In contrast, the inhibition of ·OH by CeO 2 @PAA@RGD was 12.72% higher than that of CeO 2 @PAA, indicated by the significantly decreased absorbance (p < 0.001) at 510 nm (Figure 1i), which demonstrated the effective removal of ·OH. These results suggest that the CeO 2 @PAA@RGD exhibits multienzyme mimetic activities and can significantly scavenge ROS.

Cytotoxicity Tests
Raw264.7 cells and HUVEC cells were employed to evaluate cytotoxicity upon interaction with CeO 2 @PAA and CeO 2 @PAA@RGD particles. Cell toxicity was determined using Cell Counting Kit-8 (CCK8) proliferation assay under different concentration of CeO 2 @PAA and CeO 2 @PAA@RGD (25, 50, and 100 µg/mL) treating for 12 h or 24 h, respectively. No notable toxicity was observed for both CeO 2 @PAA and CeO 2 @PAA@RGD on two types of cells under all tested concentrations during the different incubation period, suggesting the good biocompatibility of modified CeO 2 nanoparticles (Figure 2a,b).

ROS-Scavenging Effects
The ROS-scavenging effects of CeO2@PAA@RGD on Raw264.7 cell were evaluated under a cellular oxidative microenvironment induced by 100 µm of H2O2. Intracellular ROS content was indicated via the ROS probe DCFH-DA (green color). As depicted in Figure 3b, H2O2 (100 µM) triggered a remarkably higher ROS in Raw264.7 than untreated Moreover, we further assessed the RGD-mediated targeted uptake of CeO 2 @PAA@RGD by HUVEC cells with significant levels of integrin expression by confocal laser scanning microscopy (CLSM). CLSM images showed that iFluor-CeO 2 @PAA was not apparently taken up by HUVEC cells (Figure 2c). In contrast, iFluor-CeO 2 @PAA@RGD was notably internalized, as evidenced by the cytoplasmic distribution of red fluorescence noted. The cellular uptake level of CeO 2 @PAA@RGD was notably higher than CeO 2 @PAA, and the cellular uptake of CeO 2 @PAA@RGD had a time-dependent manner. The above results effectively illustrated the preferential uptake of CeO 2 @PAA@RGD by HUVEC cells via RGD-mediated endocytosis, which was consistent with the previous reports [35,51].

ROS-Scavenging Effects
The ROS-scavenging effects of CeO 2 @PAA@RGD on Raw264.7 cell were evaluated under a cellular oxidative microenvironment induced by 100 µm of H 2 O 2 . Intracellular ROS content was indicated via the ROS probe DCFH-DA (green color). As depicted in Figure 3b, H 2 O 2 (100 µM) triggered a remarkably higher ROS in Raw264.7 than untreated control group (p < 0.001). Negligible fluorescence (p < 0.001) was observed in Raw264.7 cells treated with CeO 2 @PAA@RGD (100 µg/mL), which was comparable to the negative control (p > 0.05), suggesting the potent ROS-scavenging effect of CeO 2 @PAA@RGD. A similar significant decrease in fluorescence intensity was observed in the CeO 2 @PAA group compared to the H 2 O 2 group (p < 0.001), but no significant difference was found compared to the CeO 2 @PAA@RGD group (p > 0.05). It was speculated that the comparable ROSscavenging property of two tested cerium oxide nanoparticles resulted from the redox properties of Ce 3+ and Ce 4+ on the surface; the surface modifications had no significant effect on its redox properties. The effect of ROS scavenging was further confirmed by flow cytometry analysis (Figure 3c,d); CeO 2 @PAA and CeO 2 @PAA@RGD consistently showed significant ROS-scavenging levels in Raw264.7 cells, showing that the average fluorescence intensity of CeO 2 @PAA and CeO 2 @PAA@RGD treated cells was 2.87-and 3.89-fold lower than that of H 2 O 2 (100 µM) (p < 0.001). The above results demonstrate that CeO 2 @PAA@RGD nanoparticles could effectively scavenge the intracellular ROS level and exhibit the antioxidant potential.

Anti-Inflammatory Effects
The potential anti-inflammatory abilities of CeO 2 @PAA@RGD were evaluated by macrophage phenotype transition and subsequent inflammatory cytokine detection. Firstly, CD86 (a common phenotypic marker of M1 macrophages) and CD206 (a common phenotypic marker of M2 macrophages) were detected for cells after different treatments, and the levels of inflammatory cytokines were assessed by flow cytometry after different treatments. The CD86 and CD206 expression levels are shown in Figure 4a. Raw264.7 cells treated with LPS (2 µg/mL) for 24 h produced 96.50% of CD86 + marker without notable CD206 + marker expression (Figure 4a). However, compared to the LPS group, the expression of CD86 + were significantly reduced in the cells treated with CeO 2 @PAA (2.57%) or CeO 2 @PAA@RGD (2.24%) groups. Moreover, a higher increment of CD206 + expression was observed in the cells treated with CeO 2 @PAA (45.60%) or CeO 2 @PAA@RGD (60.10%). Among them, the expression of CD206 + was 14.5% higher in the CeO 2 @PAA@RGD group than that in the CeO 2 @PAA group, suggesting that CeO 2 @PAA@RGD has a more potent ability to switch macrophage phenotype from M1 to M2 than CeO 2 @PAA. The inflammation-related cytokine expression was also tested by qRT-PCR to further show the anti-inflammatory effect of CeO 2 @PAA@RGD nanoparticles. As shown in Figure 4b, the expression of proinflammatory cytokines including CD86, TNF-α, and IL-6 were downregulated in either CeO 2 @PAA or CeO 2 @PAA@RGD groups compared to the LPS group. We also noted that the expression of anti-inflammatory cytokines such as CD206, VEGF, and IL-10 was increased accordingly compared to the LPS group (Figure 4c). These results demonstrate that CeO 2 @PAA@RGD has a promising anti-inflammatory effect.

Anti-Inflammatory Effects
The potential anti-inflammatory abilities of CeO2@PAA@RGD were evaluated by macrophage phenotype transition and subsequent inflammatory cytokine detection. Firstly, CD86 (a common phenotypic marker of M1 macrophages) and CD206 (a common phenotypic marker of M2 macrophages) were detected for cells after different treatments, and the levels of inflammatory cytokines were assessed by flow cytometry after different treatments. The CD86 and CD206 expression levels are shown in Figure  4a. Raw264.7 cells treated with LPS (2 µg/mL) for 24 h produced 96.50% of CD86 + marker without notable CD206 + marker expression (Figure 4a). However, compared to the LPS group, the expression of CD86 + were significantly reduced in the cells treated with CeO2@PAA (2.57%) or CeO2@PAA@RGD (2.24%) groups. Moreover, a higher increment of CD206 + expression was observed in the cells treated with CeO2@PAA (45.60%) or CeO2@PAA@RGD (60.10%). Among them, the expression of CD206 + was 14.5% higher in the CeO2@PAA@RGD group than that in the CeO2@PAA group, suggesting that CeO2@PAA@RGD has a more potent ability to switch macrophage phenotype from M1 to M2 than CeO2@PAA. The inflammation-related cytokine expression was also tested by qRT-PCR to further show the anti-inflammatory effect of CeO2@PAA@RGD nanoparticles. As shown in Figure 4b, the expression of proinflammatory cytokines including (c) histogram of ROS levels in Raw264.7 cells treated with CeO 2 @PAA@RGD nanoparticles for 24 h by flow cytometry analysis; (d) mean fluorescence intensity of intracellular ROS levels was calculated. Error bars = mean ± standard deviation (SD) (n = 3); ns > 0.05, ** p < 0.01, *** p < 0.001, using a one-way ANOVA (and nonparametric or mixed).

Promotion of HUVEC Angiogenesis by CeO 2 @PAA@RGD
Tube formation assays of HUVEC were used to assess the CeO 2 @PAA@RGD effect on angiogenesis in vitro. The diagram shows the process of tube network formation (Figure 5a). The results showed that only a few tube networks were observed after incubating PBS for 8 h. Additionally, compared to the CeO 2 @PAA group, the CeO 2 @PAA@RGD induced more tube networks. With increasing time, the effect of promoting tube formation became more remarkable, and the number of node points and total tube length per field at 8 h were 2.14-fold and 1.50-fold higher than CeO 2 @PAA (Figure 5b,c), respectively. The CeO 2 @PAA@RGD showed stronger angiogenesis compared to the CeO 2 @PAA, which was due to the targeting property of RGD leading to greater cellular uptake of CeO 2 @PAA@RGD [52]. Collectively, these results suggested that the CeO 2 @PAA@RGD had a promoted angiogenesis of endothelial cells.

Promotion of HUVEC Angiogenesis by CeO2@PAA@RGD
Tube formation assays of HUVEC were used to assess the CeO2@PAA@RGD effect on angiogenesis in vitro. The diagram shows the process of tube network formation (Figure 5a). The results showed that only a few tube networks were observed after incubating PBS for 8 h. Additionally, compared to the CeO2@PAA group, the CeO2@PAA@RGD induced more tube networks. With increasing time, the effect of promoting tube formation became more remarkable, and the number of node points and total tube length per field at 8 h were 2.14-fold and 1.50-fold higher than CeO2@PAA (Figure 5b,c), respectively. The CeO2@PAA@RGD showed stronger angiogenesis compared to the CeO2@PAA, which was due to the targeting property of RGD leading to greater @PAA or CeO 2 @PAA@RGD treated; (b) qRT-PCR was performed to detect the expression of proinflammatory factors CD86, TNF-α, and IL-6; (c) qRT-PCR was used to examine the expression of anti-inflammatory factors CD206, VEGF, and IL-10. Error bars = mean ± standard deviation (SD) (n = 3); ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, using a one-way ANOVA (and nonparametric or mixed).

Promotion of Cell Migration
HUVEC cell migration is regarded as one of the critical steps in the angiogenesis process. We conducted a scratch wound healing assay to further evaluate the migrationpromoting effect of CeO 2 @PAA@RGD on HUVEC. The images of scratch experiments for each group at different time points (0, 12, 24, 36 h) are displayed in Figure 6a, and it was observed that both the PBS group and CeO 2 @PAA-treated HUVEC cells did not fill the scratch area within 36 h. In contrast, the CeO 2 @PAA@RGD group presented the maximum wound recovery rate (97.95 ± 0.41%) after 36 h of incubation, which revealed almost a 2.72-fold and 1.40-fold increment compared to PBS (35.99 ± 2.81%) and CeO 2 @PAA (69.77 ± 0.78%), respectively (Figure 6b). These results suggested that the CeO 2 @PAA@RGD significantly promoted cell migration, consistent with the previous report [53].

Promotion of Cell Migration
HUVEC cell migration is regarded as one of the critical steps in the angiogenesis process. We conducted a scratch wound healing assay to further evaluate the migrationpromoting effect of CeO2@PAA@RGD on HUVEC. The images of scratch experiments for each group at different time points (0, 12, 24, 36 h) are displayed in Figure 6a, and it was observed that both the PBS group and CeO2@PAA-treated HUVEC cells did not fill the scratch area within 36 h. In contrast, the CeO2@PAA@RGD group presented the maximum wound recovery rate (97.95 ± 0.41%) after 36 h of incubation, which revealed almost a 2.72-fold and 1.40-fold increment compared to PBS (35.99 ± 2.81%) and CeO2@PAA (69.77 ± 0.78%), respectively (Figure 6b). These results suggested that the CeO2@PAA@RGD significantly promoted cell migration, consistent with the previous report [53].
RGD peptides are well known to bind preferentially to integrins αvβ3 [33], which are highly expressed in endothelial cells and play an important role in angiogenesis. Therefore, RGD peptide-modified nanoparticles are a promising approach to modulate the interaction between nanoparticles and endothelial cells. In this study, we found that CeO2@PAA@RGD significantly enhanced angiogenesis and cell migration in HUVEC cells. This may be attributed to the increased uptake of cerium oxide nanoparticles by endothelial cells after RGD modification of CeO2@PAA, thus further enhancing the interaction between nanoparticles and cells. It has been reported that low levels of intracel- Figure 5. Angiogenic effect of HUVEC cells observed by microscopy. (a) Microscopic photomicrographs were used to observe the effect of angiogenesis in HUVEC cells after treatment with PBS, CeO 2 @PAA, and CeO 2 @PAA@RGD for 2 h, 4 h, and 8 h (scale bar = 250 µm); quantitative measurement of the branch length (b) and node points (c) (n = 3) after treatment of HUVEC cells with PBS, CeO 2 @PAA, and CeO 2 @PAA@RGD nanoparticles at 2 h, 4 h, and 8 h. Error bars = mean ± standard deviation (SD) (n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001, using a one-way ANOVA (and nonparametric or mixed). RGD peptides are well known to bind preferentially to integrins αvβ3 [33], which are highly expressed in endothelial cells and play an important role in angiogenesis. Therefore, RGD peptide-modified nanoparticles are a promising approach to modulate the interaction between nanoparticles and endothelial cells. In this study, we found that CeO 2 @PAA@RGD significantly enhanced angiogenesis and cell migration in HUVEC cells. This may be attributed to the increased uptake of cerium oxide nanoparticles by endothelial cells after RGD modification of CeO 2 @PAA, thus further enhancing the interaction between nanoparticles and cells. It has been reported that low levels of intracellular ROS [54] and appropriate oxygen concentration [55] in the HUVEC cells are essential for anagenesis. The results of this paper demonstrate that CeO 2 @PAA@RGD has excellent CAT and SOD enzymelike activities. It can effectively reduce the intracellular ROS level. CeO 2 @PAA@RGD has a greater potential in regulating intracellular oxygen concentration, which is mainly attributed to the fact that CeO 2 @PAA@RGD can catalyze the decomposition of H 2 O 2 to produce oxygen. Based on the above hypothesis, it can effectively promote endothelial cell migration and angiogenesis. tial for anagenesis. The results of this paper demonstrate that CeO2@PAA@RGD has excellent CAT and SOD enzyme-like activities. It can effectively reduce the intracellular ROS level. CeO2@PAA@RGD has a greater potential in regulating intracellular oxygen concentration, which is mainly attributed to the fact that CeO2@PAA@RGD can catalyze the decomposition of H2O2 to produce oxygen. Based on the above hypothesis, it can effectively promote endothelial cell migration and angiogenesis.

Conclusions
In summary, CeO2@PAA@RGD nanoparticles maintained the excellent CAT-like and SOD-like enzymatic activities after the surface modifications. Due to the RGD modification, CeO2@PAA@RGD nanoparticles increased the cellular uptake by integrinmediated endocytosis, thus enhancing the ROS-scavenging capability and exerting more potent antioxidant effects. In addition, the anti-inflammatory functions of CeO2@PAA@RGD were further confirmed by modulating the conversion of macrophages from M1 to M2 type, indicated by the upregulation of anti-inflammatory cytokines and downregulation of proinflammatory cytokines expression. Finally, the RGD modification promoted the uptake of CeO2@PAA@RGD by HUVEC cells, thus promoting the effects of angiogenesis and cell migration. Collectively, our study shed light on the development and application of CeO2@PAA@RGD nanoparticles for some inflammatory diseases, vascular-compromised diseases, diabetes, and tissue repair. The representative pictures of cell migration after treatment with PBS, CeO 2 @PAA, and CeO 2 @PAA@RGD nanoparticles for 0 h, 12 h, 24 h, and 36 h (red areas indicate scratch areas; scale bar = 500 µm); (b) quantification of scratch healing rate of HUVEC cells treated with PBS, CeO 2 @PAA, and CeO 2 @PAA@RGD nanoparticles. Error bars = mean ± standard deviation (SD) (n = 3); ns > 0.05, ** p < 0.01, *** p < 0.001, using a one-way ANOVA (and nonparametric or mixed).

Conclusions
In summary, CeO 2 @PAA@RGD nanoparticles maintained the excellent CAT-like and SOD-like enzymatic activities after the surface modifications. Due to the RGD modification, CeO 2 @PAA@RGD nanoparticles increased the cellular uptake by integrin-mediated endocytosis, thus enhancing the ROS-scavenging capability and exerting more potent antioxidant effects. In addition, the anti-inflammatory functions of CeO 2 @PAA@RGD were further confirmed by modulating the conversion of macrophages from M1 to M2 type, indicated by the upregulation of anti-inflammatory cytokines and downregulation of proinflammatory cytokines expression. Finally, the RGD modification promoted the uptake of CeO 2 @PAA@RGD by HUVEC cells, thus promoting the effects of angiogenesis and cell migration. Collectively, our study shed light on the development and application of CeO 2 @PAA@RGD nanoparticles for some inflammatory diseases, vascular-compromised diseases, diabetes, and tissue repair.
Supplementary Materials: The following supporting materials can be obtained by website download: https://www.mdpi.com/article/10.3390/biom12091277/s1, Figure S1: Schematic diagram of the preparation of CeO 2 @PAA; Figure S2: UV-Vis absorption spectra of CeO 2 @PAA and CeO 2 @PAA@RGD for (a) O 2 − , (b) H 2 O 2 , and (c) ·OH; Figure S3: In vitro oxygen bubbles production of nanoparticles was observed; Table S1: Reaction system for detecting SOD-like enzyme activity; Table S2: Reaction system for detecting ·OH; Table S3: Synthesis by reverse transcription of cDNA reaction system; Table S4: Primers were used to detect mRNA expression of pro-and anti-inflammatory-related genes.