Single-atom Pt Doped Nanoceria for Enhanced Cell Phagocytosis and Nanozyme Activities in Keratitis Immune Regulation

Single-atom Pt Doped Nanoceria for Enhanced Cell Phagocytosis and Nanozyme Activities in Keratitis Immune Regulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Single-atom Pt Doped Nanoceria for Enhanced Cell Phagocytosis and Nanozyme Activities in Keratitis Immune Regulation Jianguo Zhao, Wanqing Lou, Yixin Wang, Lu Wang, Xiaoqian Jin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7588173/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract During keratitis treatment, oxidative stress and inflammation often result in corneal neovascularisation, scarring, and reduced light transmittance. In this study, single-atom Pt/CeO 2 is synthesised, exhibiting significantly enhanced catalase-like and superoxide dismutase-like activities for the elimination of superoxide anions (•O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (•OH). Doping single-atom Pt onto CeO 2 increases the Ce 3+ concentration in the Ce 3+ /Ce 4+ ratio from 39.12% to 58.66%, as confirmed by electron spin resonance, high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. In vitro studies demonstrate that single-atom Pt/CeO 2 effectively reduces intracellular ROS levels in H 2 O 2 -activated human corneal epithelial cells. Additionally, it exerts an anti-inflammatory effect on LPS-stimulated RAW264.7 macrophages, significantly decreasing the expression of interleukin-1β, interleukin-6, and tumour necrosis factor-α. In vivo , in an LPS-induced keratitis animal model, single-atom Pt/CeO 2 accelerates corneal ulcer healing and preserves corneal light transmittance, attributed to its anti-inflammatory properties, enzyme-like activities, and ability to promote cell migration. This study offers a novel approach for treating various inflammatory and autoimmune diseases. single-atom nanozymes CeO2 keratitis ROS elimination anti-inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Keratitis, a common ocular disease worldwide, is primarily classified into infectious and non-infectious categories and ranks as the fifth leading cause of global blindness. In recent years, the incidence of keratitis has been rising due to factors such as plant trauma, overuse of broad-spectrum antibiotics and hormones, improper contact lens use, ophthalmic surgeries, corneal transplants, and conditions like AIDS, diabetes, and immunodeficiency.[ 1 – 4 ] The main clinical manifestations of keratitis include redness, oedema, pain, reduced visual function, photophobia, and increased intraocular secretions. The onset of keratitis is typically acute, leading to severe inflammatory reactions, oxidative stress damage, corneal ulcers, and perforations. If left untreated, keratitis can progress to eyeball atrophy, blindness, and, in severe cases, life-threatening complications when inflammation spreads to the brain.[ 3 – 5 ] Clinically, the standard approach involves sterilisation followed by hormone treatment to suppress the inflammatory response. A significant challenge, however, is the inability to eliminate the inflammatory reaction and reactive oxygen species (ROS) generated by bacterial toxins in a timely manner, resulting in irreversible damage such as scarring, neovascularisation, perforation, and reduced corneal light transmittance.[ 5 , 6 ] Upon the onset of keratitis, the cornea experiences a severe inflammatory response, a complex process involving multiple cells, which triggers the release of various pro-inflammatory cytokines and the recruitment of immune cells to the site of inflammation. For example, lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa , is one of the key inducers of keratitis. LPS activates the NF-κB signalling pathway, generating transcription factors, stimulating ROS production, and inducing the secretion of various endogenous active factors, such as the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α).[ 7 – 9 ] In response, inflammatory cells produce large amounts of ROS to combat bacterial invasion, leading to oxidative stress in tissues. Oxidative stress refers to an imbalance between pro-oxidant and antioxidant substances, resulting in the infiltration of inflammatory cells, increased secretion of proteases, and the accumulation of oxidative by-products. These include pro-inflammatory substances such as leukotrienes and thromboxanes, which dilate blood vessels, promote leukocyte migration, and further exacerbate oxidative stress and inflammation.[ 10 , 11 ] Consequently, oxidative stress and inflammatory responses act synergistically, ultimately leading to irreversible corneal damage. Specifically, during keratitis, excessive ROS production induces oxidative damage to DNA, proteins, and lipids and leads to corneal cell apoptosis.[ 12 ] Elevated ROS levels are recognised as a critical factor in the progression of various ocular diseases, including ocular surface diseases (keratitis, xerophthalmia, alkali burns), cataracts, glaucoma, neurodegeneration, uveitis, and age-related macular degeneration.[ 13 ] Eliminating inflammation and ROS to regulate the microenvironment has proven to be an effective strategy for treating inflammatory diseases and promoting wound healing. In our previous study, nanozymes with catalase (CAT)-like activity were found to reduce secondary oxidative stress and inflammation following photodynamic therapy for choroidal neovascularisation, simultaneously alleviating hypoxia by catalytically degrading hydrogen peroxide (H 2 O 2 ) into oxygen. Nanozymes mimicking biological enzymes, such as superoxide dismutase (SOD), CAT, and peroxidase (POD), exhibit significant advantages in their synthesis, storage, transport, and structure-performance optimisation. For instance, CeO 2 with CAT-like activity demonstrated ROS scavenging abilities through the transition between Ce 3+ and Ce 4+ oxidation states, resulting in reduced inflammation in the treatment of bowel diseases, otitis media, ocular surface conditions, and intraocular diseases.[ 14 ] However, cell experiments have shown that CeO 2 becomes toxic at certain concentrations, which limits its application. Fortunately, recently developed single-atom catalysts (SACs), characterised by nearly 100% atomic dispersion, exhibit significantly enhanced catalytic performance compared to conventional nanozymes, thereby greatly reducing the use of precious metals.[ 15 , 16 ] For example, Yu et al. found that metal single atoms (Pt, Au, Cu, Ru) significantly improved the oxidase-like activity of PCN-222 by reducing the adsorption and activation energy of O 2 .[ 17 ] In this study, single-atom Pt-decorated CeO 2 was synthesised through a simple method involving the in situ reduction of the PtCl 4 2− precursor into single-atom Pt/CeO 2 by NaBH 4 , aiming to reduce the dosage of nanozymes (Scheme 1 ). In this process, key ROS components, including superoxide anions (•O 2 − ), H 2 O 2 , and hydroxyl radicals (•OH), were rapidly eliminated by single-atom Pt/CeO 2 , effectively reducing oxidative stress. Additionally, single-atom Pt/CeO 2 nanozymes demonstrated a strong scavenging effect on reactive nitrogen species (RNS), such as 1-Diphenyl-2-picrylhydrazyl (DPPH•) free radicals. The single-atom Pt/CeO 2 solution was administered as eye drops in a non-invasive, low-frequency manner (once daily). The ROS scavenging and anti-inflammatory properties of single-atom Pt/CeO 2 were further evaluated by measuring the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and assessing corneal epithelial healing. This study provides a valuable reference for the use of efficient single-atom catalytic nanozymes in the treatment of other inflammatory and autoimmune diseases. 2. Results and discussion 2.1 Synthesis and characterisation of CeO 2 and single-atom Pt/CeO 2 CeO 2 was first synthesised via a simple hydrothermal method.[ 18 , 19 ] Subsequently, negatively charged PtCl 4 2− was added and adsorbed onto the surface of positively charged CeO 2 nanocrystals, stabilised with 6-aminocaproic acid (AHA) through electrostatic interactions. The PtCl 4 2− was in situ reduced by NaBH 4 , yielding single-atom Pt/CeO 2 nanostructures.[ 20 ] [ 21 ]Transmission electron microscopy (TEM) images (Fig. 1 a) revealed that the synthesised CeO 2 exhibited a uniform spherical nanocrystal structure, with particle sizes ranging from 5 to 10 nm. High-resolution TEM (HRTEM) measurements indicated that the lattice spacing of CeO 2 nanozymes on the (111) plane was 0.3128 nm (Fig. 1 a and S1a, Supporting Information). The single-atom Pt appeared as clusters on the (111) surface, with a slightly broader lattice spacing of approximately 0.3272 nm (Fig. 1 b and S1b, Supporting Information), confirming the successful incorporation of Pt single atoms. The powder X-ray diffraction (XRD) patterns (Fig. 1 c) demonstrated characteristic diffraction peaks at 28.6°, 33.1°, 47.5°, 56.3°, 59.1°, 69.4°, 76.7°, 79.1°, and 88.4°, consistent with the standard card for CeO 2 (PDF#43-1002). These findings suggest that the incorporation of single-atom Pt did not alter the cubic fluorite structure of CeO 2 .[ 14 ] The doping of single-atom Pt resulted in an increased distance between crystal planes, causing a slight left shift in the XRD curve compared to CeO 2 . Additionally, no diffraction peaks related to Pt or PtO x (33°, 44°) were observed within the 10° to 90° range in single-atom Pt/CeO 2 , likely due to the extremely low concentration and excellent dispersion of Pt on the CeO 2 surface.[ 22 ] Figure 1 d presents the Raman spectra, showing that both CeO 2 and single-atom Pt/CeO 2 exhibited typical cubic fluorite structure characteristics. The Raman peaks of CeO 2 at 258 cm − 1 , 460 cm − 1 , and 606 cm − 1 corresponded to the second-order transverse acoustic vibration peak of CeO 2 (2TA), the symmetric stretching vibration characteristic peak of the cubic fluorite structure (F 2 g), and the defect induction mode (D), respectively. [ 23 ]New peaks at 550 cm − 1 and 650 cm − 1 were observed in the single-atom Pt/CeO 2 sample, corresponding to Pt-O-Ce and Pt-O bonds, respectively.[ 24 , 25 ] Further analysis using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) was conducted to examine the doping form of single-atom Pt on CeO 2 (Fig. 1 e). The electrons captured by the high-angle ring detector, scattered by heavy elements, created contrast related to the atomic number of the corresponding elements. The HAADF-STEM image revealed that the bright spot inside the red circle corresponded to the dispersed Pt single atoms on the CeO 2 surface. The chemical states of Ce and O elements in CeO 2 and single-atom Pt/CeO 2 were investigated by X-ray photoelectron spectroscopy (XPS). In comparison with CeO 2 , a peak around 75 eV appeared in the single-atom Pt/CeO 2 nanostructure, indicating successful Pt doping (Fig. 1 f and inset).[ 26 ] The Ce element in CeO 2 exists primarily in two valence states: Ce 3+ and Ce 4+ . The Ce 3+ centre scavenges •O 2 − and •OH through redox reactions or by mimicking SOD activity, while the Ce 4+ centre oxidises H 2 O 2 by mimicking CAT activity.[ 27 ] Since •O 2 − and •OH are directly associated with inflammatory responses and cell death, a high proportion of Ce 3+ in CeO 2 nanozymes is crucial for mitigating inflammation-related oxidative stress.[ 28 ] According to the literature, the absorption peaks at 901.9 eV, 898.6 eV, 883.9 eV, and 880.6 eV correspond to Ce 3+ .[ 29 ] As shown in Fig. 1 g and Figure S2 (Supporting Information), the doping of Pt single atoms increased the Ce 3+ content from 39.12% to 58.66%, as calculated from the absorption peak areas. The varying ratios of Ce 3+ and Ce 4+ in CeO 2 and single-atom Pt/CeO 2 clusters confirmed that Pt doping affected the redox state of Ce. The O 1s XPS spectrum could be deconvoluted into three binding energy peaks for CeO 2 and single-atom Pt/CeO 2 clusters. The characteristic peaks at 528.7–528.9 eV, 530.5–530.7 eV, and 533.5–533.7 eV corresponded to lattice oxygen O Lat (O − ), surface oxygen O Sur (O 2− , O − ), and other possible adsorbed oxygen species O Ads (CO 3 2− , OH − ), respectively (Fig. 1 h and Figure S3 , Supporting Information).[ 30 ]. Among these, the O 2 2− and O 2 − species in O Sur are ROS produced at oxygen vacancies. Thus, the concentration of O Sur is positively correlated with oxygen vacancy content.[ 31 ] From the integration of the peak areas, the O Sur concentration increased from 25.15% to 38.20% in CeO 2 and 1:10 Pt/CeO 2 , respectively. The single-atom Pt/CeO 2 sample with a doping ratio of 1:10 showed a higher oxygen vacancy concentration, consistent with the Ce3d XPS results mentioned earlier.[ 32 , 33 ] The Pt 4f XPS spectra of samples with varying Pt doping ratios are shown in Figure S4 (Supporting Information). The 4f 5/2 and 4f 7/2 peaks of Pt/CeO 2 were fitted into four Gaussian peaks, corresponding to PtO 2 and PtO, respectively.[ 34 ] This confirmed that PtO and PtO 2 enhance catalytic activity, acting as co-catalysts. As the Pt doping ratio increased, more PtO and PtO 2 species were present on the CeO 2 surface. 2.2 The enzyme-like properties of single-atom Pt/CeO 2 To achieve optimal ROS scavenging performance, single-atom Pt-doped CeO 2 was synthesised with varying Pt to CeO 2 feeding ratios of 1:40, 1:20, 1:10, and 1:7 (mass ratio of Pt to CeO 2 ) by adjusting the amount of Pt precursor. At the 1:7 doping ratio, the single-atom Pt/CeO 2 nanoparticles exhibited a lattice spacing of 0.227 nm on the (111) surface ( Figure S5 , Supporting Information), consistent with the previously reported lattice spacing of Pt monoliths.[ 35 ]. This indicated that the reduced Pt on CeO 2 existed as agglomerates, which substantially reduced the catalytic efficiency per Pt atom. [ 35 ]. H 2 O 2 decomposition and DPPH• removal tests were then performed to assess the catalytic performance of single-atom Pt/CeO 2 ( Figure S6 , Supporting Information). As the amount of single-atom Pt doping increased, the catalytic efficiency of the nanozymes also improved. Therefore, the 1:10 Pt doping ratio was chosen as the optimal ratio for obtaining monoatomically dispersed Pt with the best ROS scavenging effect. Single-atom Pt/CeO 2 with a 1:10 doping ratio was selected for further investigation in subsequent studies. Various ROS produced by oxidative stress during immune responses include hydroxyl free radicals (•OH), H 2 O 2 , superoxide radicals (•O 2 − ), superoxide anion free radicals, lipoxygen free radicals, and ozone.[ 26 ] In the present study, classic and significant biological enzyme-like activities related to free radical scavenging, including SOD-like and CAT-like activities, were used to explore the enzyme-like properties of single-atom Pt/CeO 2 (Fig. 2 a). In physiological conditions, the SOD enzyme plays a pivotal role in maintaining intracellular redox balance by catalysing the disproportionation of •O 2 − into H 2 O 2 . To test the SOD-like activity of single-atom Pt/CeO 2 , the generated •O 2 − in the xanthine reaction, catalysed by xanthine oxidase, was scavenged using tetrazolium salt- 2 - [2 – methoxy – 4 - nitrophenyl] − 3 - [4 - nitrophenyl] − 5 - [2, 4 - disulfophenyl] − 2H - tetrazole (WST-8), which reacted with •O 2 − to form formazan. The quantity of formazan was measured spectrophotometrically at 450 nm. As shown in Fig. 2 b, the scavenging rates of CeO 2 were less than 30% within the concentration range of 20–100 µg/mL, much lower than those of single-atom Pt/CeO 2 . The •O 2 − scavenging rate of single-atom Pt/CeO 2 reached 87.22% even at a concentration of 20 µg/mL. Furthermore, electron spin resonance (ESR) analysis revealed that CeO 2 could not completely eliminate •OH free radicals, even at a concentration of 100 µg/mL for over 2 hours. In contrast, the ESR curve for single-atom Pt/CeO 2 showed a flat profile, indicating complete removal of •OH free radicals (Fig. 2 c). To assess the CAT-like catalytic activity, harmful H 2 O 2 was used as the substrate to be degraded into harmless H 2 O and O 2 . The process of H 2 O 2 degradation was quantitatively measured by tracking changes in H 2 O 2 concentration, monitored by absorbance at 240 nm (Fig. 2 d). The results showed that more than 50% of H 2 O 2 was rapidly decomposed within 300 seconds by single-atom Pt/CeO 2 (20 µg/mL). At the end of the reaction, the H 2 O 2 scavenging ratio by single-atom Pt/CeO 2 was 12 times higher than that of CeO 2 clusters. Additionally, the production of O 2 bubbles was observed immediately upon contact between the H 2 O 2 solution and single-atom Pt/CeO 2 ( Figure S7 , Supporting Information). The O 2 production rate was found to be highly dependent on the concentration of single-atom Pt/CeO 2 (Fig. 2 e). In contrast, CeO 2 exhibited negligible CAT-like activity in catalysing H 2 O 2 degradation and O 2 production. The repeatability of the CAT-like catalytic activity and the stability of single-atom Pt/CeO 2 were further evaluated by measuring the H 2 O 2 concentration change over four cycles (Fig. 2 f). The linear slope of H 2 O 2 decomposition did not significantly change over time, indicating that the catalytic reaction did not affect the binding and catalytic properties of single-atom Pt on the CeO 2 surface. To further verify the scavenging ability of single-atom Pt/CeO 2 towards •OH, ESR and UV-Vis spectroscopy were used to quantify •OH. ESR analysis revealed that CeO 2 showed a weak ability to scavenge •OH, whereas single-atom Pt/CeO 2 was able to completely scavenge all free radicals after just 2 minutes of incubation (Fig. 2 g). Furthermore, methylene blue (MB), a common trap for •OH produced in the Fenton reaction between FeSO 4 and H 2 O 2 , reacts with •OH to form hydroxylated methylene blue (MB-OH), causing a colour change from blue to colourless. Therefore, the scavenging ability of single-atom Pt/CeO 2 towards •OH was indirectly evaluated by the change in MB concentration, measured by absorbance at 664 nm. As shown in Fig. 2 h, the presence of single-atom Pt/CeO 2 led to strong absorbance at 664 nm, confirming that MB was not oxidised by •OH, which demonstrated the strong scavenging ability of single-atom Pt/CeO 2 . Moreover, the single-atom Pt/CeO 2 showed a significant concentration dependence in its scavenging activity towards •OH (Fig. 2 i). DPPH•, a stable nitrogen-centred chromogenic radical dissolved in ethanol, exhibits a purplish-red colour with a characteristic absorption peak at 517 nm. In the single-atom Pt/CeO 2 -treated group, the DPPH• solution rapidly faded to a colourless and transparent solution, with no distinct absorption peak at 517 nm (Fig. 2 j). In contrast, no notable colour change was observed in the CeO 2 -treated group, with an absorption curve similar to that of the control group. This demonstrated that single-atom Pt/CeO 2 exhibited excellent DPPH• scavenging ability, while CeO 2 had negligible scavenging activity. Further statistical analysis revealed that the scavenging activity of CeO 2 towards DPPH• increased by 48.9 times after doping with single-atom Pt. Additionally, a clear concentration dependence of the simulated enzyme activity was observed for concentrations below 25 µg/mL (Fig. 2 i and Figure S8 , Supporting Information). In summary, the engineered single-atom Pt/CeO 2 nanozymes displayed significantly higher scavenging abilities for ROS and RNS compared to CeO 2 . 2.3 In vitro ROS-scavenging and anti-inflammatory activities of single-atom Pt/CeO 2 . Upon the onset of keratitis, corneal epithelial cells and inflammatory cells, under severe oxidative stress, generate an excessive amount of oxidative free radicals, exacerbating the inflammatory response. Corneal epithelial cells, as the outermost layer of the cornea, act as the first line of defence against external pathogens, engaging in innate immunity, detecting pathogens, and signalling the activation of the corneal defence system. [ 36 ] Macrophages, essential phagocytes, are activated into the M1 phenotype by interferon-γ and LPS, secreting pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, and IL-6 to induce immune responses, phagocytose, and eliminate bacteria. However, prolonged M1 macrophage activation can exacerbate metalloproteinase secretion, leading to corneal perforation, delayed healing, and scar formation.[ 37 ] Therefore, timely removal of oxidative stress and modulation of the inflammatory response are essential for mitigating corneal tissue damage.[ 38 , 39 ] To assess the protective effect of single-atom Pt/CeO 2 on human corneal epithelial cells (HCECs) against oxidative stress-induced injury, the uptake of Pt/CeO 2 by HCECs was evaluated using fluorescein isothiocyanate (FITC) as a fluorescent marker. FITC-labelled single-atom Pt/CeO 2 was incubated with HCECs, followed by fluorescence detection using a confocal laser scanning microscope (CLSM). As shown in Figure S9 (Supporting Information), HCECs treated with single-atom Pt/CeO 2 exhibited stronger fluorescence intensity than those treated with CeO 2 after 0.5 hours of incubation, indicating a higher uptake of single-atom Pt-doped CeO 2 by HCECs. Furthermore, the fluorescence of FITC-labelled nanoparticles primarily indicated their distribution within the cytoplasm ( Figure S10 , Supporting Information). After 12 hours, nearly no green fluorescence was observed in the CeO 2 -treated group, whereas HCECs in the single-atom Pt/CeO 2 group maintained strong green fluorescence, indicating that single-atom Pt/CeO 2 exhibited faster cellular uptake and longer intracellular retention compared to CeO 2 . This enhanced uptake may be attributed to the increased antioxidant activity of single-atom Pt/CeO 2 , with 58.66% Ce 3+ in the Ce 3+ /Ce 4+ ratio, compared to 39.12% Ce 3+ in CeO 2 (Fig. 1 i). The cytotoxicity and cytoprotective effects of CeO 2 and single-atom Pt/CeO 2 nanozymes on HCECs were assessed using the cell counting kit-8 (CCK-8) assay. As shown in Fig. 3 a, both CeO 2 and single-atom Pt/CeO 2 demonstrated low cytotoxicity, even at concentrations up to 120 µg/mL. The protective effect of CeO 2 and single-atom Pt/CeO 2 on HCECs against H 2 O 2 -induced oxidative stress was then evaluated. HCECs were incubated with various concentrations of H 2 O 2 for 24 hours, and the cell survival rate was approximately 50% at a H 2 O 2 concentration of 1 mmol/L ( Figure S11 , Supporting Information). When HCECs were treated with H 2 O 2 and different concentrations of CeO 2 and single-atom Pt/CeO 2 , CeO 2 showed no significant cytoprotective effect due to its lack of CAT-like activity (Fig. 3 b). In contrast, single-atom Pt/CeO 2 demonstrated significant protection in a concentration-dependent manner (Fig. 3 c). A cell live/dead staining assay was performed on different treatment groups to assess the protective effect of the nanozymes under oxidative stress. CLSM images confirmed that at a concentration of 40 µg/mL, single-atom Pt/CeO 2 restored HCEC viability to nearly normal levels (Fig. 3 d and 3 e). Intracellular ROS levels were quantified using DCFH-DA as an indicator, with green fluorescence intensity measured to assess oxidative stress (Fig. 3 f). In the negative control group, where cells also produced ROS during normal physiological activities, fluorescence was sparse. In the positive control group, H 2 O 2 stimulation significantly increased intracellular fluorescence, indicating severe oxidative stress. CeO 2 showed minimal ROS scavenging activity, with fluorescence intensity similar to that of the positive control group. However, in the single-atom Pt/CeO 2 -treated group, green fluorescence was markedly reduced, indicating effective inhibition of intracellular oxidative stress. Further statistical analysis confirmed that intracellular oxidative stress levels returned to normal after single-atom Pt/CeO 2 treatment (Fig. 3 g). These results demonstrate that single-atom Pt/CeO 2 effectively protects cells from oxidative stress-induced cytotoxicity. In keratitis, both corneal epithelial cells and macrophages produce oxidative stress in response to LPS or toxins, playing a key role in generating inflammatory cytokines. Therefore, LPS was used to stimulate RAW264.7 cells to investigate the role of single-atom Pt/CeO 2 in modulating the inflammatory response. Quantitative real-time PCR (qPCR) was performed to analyse the expression levels of inflammatory factors by measuring RNA content. As shown in Fig. 3 h-j, after LPS stimulation, the RNA expressions of TNF-α, IL-1β, and IL-6 in RAW264.7 cells increased approximately 7-fold, 300-fold, and 1,300-fold, respectively. In the CeO 2 -treated group, the expressions of these inflammatory factors showed some reduction. However, in the single-atom Pt/CeO 2 -treated group, the expression of all three inflammatory factors was significantly decreased, indicating a potent anti-inflammatory effect. 2.4 In vivo biosafety of single-atom Pt/CeO 2 In vivo biocompatibility of single-atom Pt/CeO 2 was assessed using slit-lamp examination, fluorescein sodium staining, and tissue section analysis to evaluate corneal permeability, wound size, and inflammatory cell infiltration. Specific Pathogen-Free (SPF) grade C57BL/6 male mice, aged 6 to 8 weeks with healthy anterior segments, were treated with PBS, CeO 2 , and single-atom Pt/CeO 2 . Solutions of PBS, CeO 2 , and single-atom Pt/CeO 2 (100 µg/mL, 10 µL) were administered once daily for one week. As shown in Figure S12 (Supporting Information), the corneal morphology and structure remained intact, with no significant reduction in light transmittance or inflammatory response. Fluorescein sodium staining revealed no green fluorescence in any group, indicating the absence of corneal epithelial defects. Hematoxylin and eosin (H&E) staining of tissue sections on day 7 showed no significant oedema or inflammatory cell infiltration, confirming that the corneal structure was intact. These results demonstrated the good biological safety and in vivo biocompatibility of the nanomaterials. To assess the retention time of single-atom Pt/CeO 2 , the nanomaterial was labelled with FITC and applied to the ocular surface of healthy mice. Fluorescence was observed at various time points to monitor retention on the corneal surface. As shown in Figure S13 (Supporting Information), after 12 hours of incubation, substantial fluorescence remained in the corneal layer, indicating that single-atom Pt/CeO 2 nanozymes had a prolonged retention time on the ocular surface. 2.5 In vivo anti-inflammatory activity of single-atom Pt/CeO 2 To evaluate the therapeutic effect of single-atom Pt/CeO 2 in treating keratitis, a non-infectious keratitis animal model was established by scraping part of the corneal epithelial cells with an insulin needle and applying LPS to the cornea. Mice were randomly assigned to three groups: PBS, CeO 2 , and single-atom Pt/CeO 2 , with eye drop treatments administered once daily for 7 days (Fig. 4 a). Chronic inflammation and oxidative stress in keratitis can lead to corneal ulcers and non-healing wounds, ultimately resulting in corneal neovascularisation, scarring, and reduced light transmittance.[ 40 ] Therefore, promoting corneal epithelial migration and reducing inflammation are critical factors in facilitating corneal ulcer healing. A cell scratch assay was performed to assess the effect of single-atom Pt/CeO 2 on HCEC migration. As presented in Fig. 4 b, HCEC migration was minimal in the PBS and CeO 2 -treated groups, with significant gaps remaining between the cells. In contrast, the scratch area in the single-atom Pt/CeO 2 -treated group was almost completely covered by HCECs, leading to the disappearance of the wound. Statistical analysis in Fig. 4 c further confirmed that single-atom Pt/CeO 2 significantly enhanced HCEC migration during wound healing. The scratch assay showed that oxidative stress and inflammation delayed HCEC migration in the control groups.. However, single-atom Pt/CeO 2 promoted HCEC migration by alleviating oxidative stress and inflammation in the cornea. In the in vivo keratitis experiment, slit-lamp observation revealed a large area of corneal opacity from day 1 to day 7 in the PBS-treated group, indicating severe inflammation and reduced corneal transparency (Fig. 4 d and 4 e). Sodium fluorescein staining showed that corneal epithelial defects gradually decreased in the PBS-treated group but did not fully heal after 7 days. In the CeO 2 -treated group, corneal defects were healed by day 3, as indicated by the disappearance of green fluorescence. However, a significant area of corneal opacity persisted on day 7, suggesting continued inflammation and collagen accumulation. In the single-atom Pt/CeO 2 -treated group, corneal transparency recovered to near-normal levels by day 7, and slit-lamp observations confirmed that light transmittance was restored to pre-treatment levels. Furthermore, the wound area decreased dramatically by day 3 in the single-atom Pt/CeO 2 -treated group, demonstrating that reducing inflammation through ROS elimination effectively promoted cell migration and preserved corneal transparency. The change in corneal epithelial defect area during keratitis treatment was quantitatively measured and depicted in Fig. 4 e. On day 0, prior to treatment, there were no significant differences in the epithelial defect area among the three groups. After one day of treatment, the corneal epithelium in the single-atom Pt/CeO 2 -treated group showed rapid healing, with the epithelial defect area significantly smaller compared to the PBS and CeO 2 -treated groups. Clinical scores of keratitis, measured on day 3, showed no notable differences among the three groups (Fig. 4 f), indicating that the immune system remained in a state of oxidative stress and inflammation. However, by days 5 and 7, the clinical scores of keratitis in the single-atom Pt/CeO 2 -treated group were significantly lower than those in the PBS and CeO 2 -treated groups. No significant differences were observed between the CeO 2 and PBS-treated groups, likely due to the limited ROS scavenging ability of CeO 2 . The doping of single-atom Pt greatly enhanced the ROS scavenging effect, eliminating the inflammatory response and modulating the immune system to promote corneal epithelial migration and maintain corneal transparency. After 7 days of treatment, immunohistochemical analysis was conducted to assess oxidative stress and inflammatory reactions by detecting the expression of F4/80, lymphocyte antigen 6 complex, locus G (Ly-6G), IL-6, IL-1β, and TNF-α. The intensity of red fluorescence indicated the expression levels of inflammatory factors in the corneal tissue layers (Fig. 5 a). Both F4/80 and Ly-6G, markers for macrophages and neutrophils, respectively, were widely distributed throughout the corneal tissue in the PBS-treated group, with red fluorescent spots indicating severe infiltration of these inflammatory cells. Similarly, high expression of these cells was also observed in the CeO 2 -treated group. In contrast, the single-atom Pt/CeO 2 -treated group showed minimal infiltration of both inflammatory cell types, suggesting that keratitis was effectively controlled after 7 days of treatment. Similar results were observed for the three inflammatory factors (IL-6, IL-1β, and TNF-α), with the PBS and CeO 2 -treated groups showing a significant inflammatory response, including an unhealed corneal epithelial layer at the wound site. However, expression of these inflammatory factors was significantly reduced in the single-atom Pt/CeO 2 -treated group. Statistical analysis of the fluorescence intensity of these markers further confirmed that the doping of single-atom Pt substantially enhanced the anti-inflammatory effect in keratitis treatment (Fig. 5 b-f). High levels of oxidative stress during keratitis are a key factor contributing to the exacerbation of inflammation. The animal model experiments thus validated the effectiveness of single-atom Pt/CeO 2 in alleviating inflammation by scavenging ROS in the treatment of keratitis. Histological evaluation was performed by H&E staining after the mice were sacrificed (Fig. 5 j). Observation of the corneal morphology revealed that the arrangement of corneal epithelial cells and stroma in the single-atom Pt/CeO 2 -treated group was more organised compared to the PBS-treated group. The PBS-treated group exhibited extensive infiltration of inflammatory cells (red arrow), indicating severe inflammation even on day 7. While inflammatory cell infiltration was significantly reduced in the CeO 2 -treated group, corneal neovascularisation (black arrow) was evident. In the single-atom Pt/CeO 2 -treated group, only a few inflammatory cells were present, and the corneal layers appeared clear with no signs of oedema or neovascularisation. Persistent inflammation and oxidative stress damage can lead to corneal neovascularisation, structural damage, and significant visual impairment.[ 42 ] Therefore, timely removal of oxidative stress and inflammation is an effective approach to preserving corneal transparency. 3. Conclusion Keratitis, a significant cause of irreversible corneal damage, is characterised primarily by inflammation and oxidative stress within the microenvironment. In this study, single-atom Pt was doped onto the surface of CeO 2 using a simple in-situ reduction method. It was found that the catalytic activity, including CAT-like and SOD-like activities, was greatly enhanced by adjusting the doping ratio. The cytotoxicity and in vitro cellular oxidative stress models demonstrated the good biocompatibility and protective function of single-atom Pt/CeO 2 in shielding HCECs from oxidative stress-induced damage. In the LPS-induced keratitis animal model, single-atom Pt/CeO 2 significantly promoted corneal epithelial cell migration and wound healing. Further histological analysis confirmed that single-atom Pt/CeO 2 treatment notably reduced neutrophil and macrophage infiltration in the corneal tissue. The expression levels of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, were significantly lower in the single-atom Pt/CeO 2 -treated group compared to the CeO 2 and PBS-treated groups, particularly in the corneal epithelial layer. Consequently, oxidative stress and inflammation in the cornea were effectively controlled following single-atom Pt/CeO 2 treatment, which preserved both the structural integrity and light transmittance of the cornea. Thus, the elimination of oxidative stress in the keratitis microenvironment is an effective strategy to reduce inflammation and promote corneal epithelial cell migration during treatment. 4. Experimental Section 4.1. Materials Cerium (III) nitrate hexahydrate (Ce(NO 3 ) 3 ·6H 2 O), 6-aminohexanoic acid (AHA), potassium tetrachloroplatinate (II), methylene blue, 1,1-diphenyl-2-picrylhydrazyl (DPPH•), and lipopolysaccharide (LPS) were purchased from Macklin (Shanghai, China). Hydrochloric acid (37%) was sourced from Zhejiang Zhongxing Chemical Co., Ltd. (Zhejiang, China). Polyvinylpyrrolidone (PVP, MW = 58,000) and ammonium hydroxide were acquired from Aladdin (Shanghai, China). Sodium borohydride was purchased from Adamas. H 2 O 2 was obtained from Sinopharm Chemical Reagent Co., Ltd. Fluorescein isothiocyanate (FITC), 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO), and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (USA). Cell Counting Kit-8 (CCK-8) and Live/Dead Cell Staining Kit (L7012) were acquired from Beyotime Biotechnology (Shanghai, China). All immunofluorescence reagents were sourced from Abcam (USA). Ultrapure water was obtained from a Milli-Q system (Millipore, USA). All other chemical reagents, unless specified, were purchased from Aladdin (China). 4.2. Characterisations TEM and HRTEM images were captured using a transmission electron microscope (FEI Company, USA). The crystal structure of single-atom Pt/CeO 2 nanozymes was analysed by XRD (Bruker Corporation, Germany). Chemical bonding information was obtained using Raman spectroscopy. Atomic-resolution HAADF-STEM imaging with spherical aberration correction was performed to examine single-atom Pt/CeO 2 . XPS provided full-spectrum analysis of both CeO 2 and single-atom Pt/CeO 2 . Cellular fluorescence images were captured using a Leica DMi8 microscope (Leica Microsystems, Germany). Corneal H&E sections underwent fluorescence imaging and immunofluorescence staining using a Leica DM4B microscope (Leica Microsystems, Germany). 4.3. Synthesis of single-atom Pt/CeO 2 4.3.1. Synthesis of CeO 2 nanomaterials The synthesis of CeO 2 followed a method previously reported in the literature.[ 43 ] A solution of 131.2 mg AHA and 5 µL hydrochloric acid was dissolved in 6 mL deionised water. Separately, 108.6 mg cerium (III) nitrate hexahydrate (Ce(NO 3 ) 3 ·6H 2 O) was dissolved in 5 mL deionised water. Ammonia (2 mL) was then added to the solution and magnetically stirred for 1 minute at room temperature to obtain an aqueous cerium nitrate solution. The AHA solution was quickly mixed with the cerium nitrate solution and stirred at 95°C for 6 hours. Finally, CeO 2 was obtained through centrifugation and repeated washing with deionised water. 4.3.2. Doping of single-atom Pt onto CeO 2 nanomaterials The preparation of single-atom Pt/CeO 2 followed a method reported previously. [ 21 ] To 7 mL deionised water, 1 mL of the CeO 2 solution obtained above was added. Then, 16 mg PVP (MW ≈ 58,000) and an additional 1 mL aqueous K 2 PtCl 4 solution with varying Pt mass doping ratios (1:7, 1:10, 1:20, and 1:40) were added to the mixture, which was stirred at 95°C for 2 hours. Afterwards, 3.52 mg sodium borohydride (NaBH 4 ) was dissolved in 1 mL deionised water and added dropwise to the solution, followed by stirring for 30 minutes. The single-atom Pt/CeO 2 aqueous solution was then obtained by centrifugation and washed three times with deionised water. 4.4. Enzyme-like activity tests 4.4.1. SOD activity determination The scavenging ability of nanomaterials against superoxide anion radicals (•O 2 − ) was assessed using a total SOD activity assay kit based on the reaction of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), following the manufacturer's instructions. Briefly, WST-8 reacts with •O 2 − , catalysed by xanthine oxidase, to produce a water-soluble formazan dye, which can be inhibited by SOD. The enzymatic activity of SOD was determined by colourimetric analysis of the WST-8 product. The enzyme marker was preheated at 37°C and set to a single wavelength time scan mode at 450 nm. The experimental procedure was performed according to the instruction manual, starting with the addition of the reaction starter, with measurements taken every 5 minutes over a 30-minute period. Five replicates were tested for each group, with samples added in a concentration gradient. The •O 2 − scavenging ability of the nanomaterials was also evaluated by ESR spectroscopy at room temperature. After reacting CeO 2 and single-atom Pt/CeO 2 with •O 2 − for 2 minutes, 100 µL of the solution was taken, and 50 mM DMPO (a spin trap agent) was added for ESR spectrum recording. 4.4.2. CAT activity determination The consumption of H 2 O 2 (10 mM) by CeO 2 and single-atom Pt/CeO 2 (1 mg/mL) was monitored directly at 240 nm using a UV-Vis spectrophotometer (n = 3). Single-atom Pt/CeO 2 catalysed the conversion of H 2 O 2 to H 2 O and O 2 , which was measured by detecting the amount of dissolved O 2 in the solution using a dissolved oxygen meter. 4.4.3. •OH scavenging activity The •OH scavenging efficiency of single-atom Pt/CeO 2 was determined by ESR spectroscopy at room temperature. The generation of •OH was initiated by reacting ferrous sulfate with aqueous H 2 O 2 in a Fenton-like reaction, and the •OH was captured by DMPO to form the spin adduct DMPO/•OH. After 2 minutes of incubation with single-atom Pt/CeO 2 , the remaining •OH was captured with DMPO for another 2 minutes. ESR spectra were recorded, and the content of •OH was calculated by comparing the peak position and intensity with the negative control group. The •OH scavenging ability was further evaluated using MB as an indicator, with a UV-Vis spectrophotometer. Ferrous sulfate and H 2 O 2 were reacted for 2 minutes, and MB along with CeO 2 or single-atom Pt/CeO 2 was added to the solution. After 5 minutes, the remaining MB content was measured at the characteristic absorption peak at 662 nm. 4.4.4. DPPH• scavenging activity DPPH• was dissolved in ethanol to create a homogeneous purplish-red solution (1 mM) with a characteristic absorption peak at 517 nm. In a 48-well plate, 900 µL of DPPH• (100 µM) and 100 µL of CeO 2 or single-atom Pt/CeO 2 (100 µg/mL) were added to each well. The spectra were measured between 200 and 800 nm intervals using an enzyme marker after 3 minutes at 2 nm intervals. Additionally, DPPH• was incubated with various concentrations of single-atom Pt/CeO 2 to evaluate the scavenging activity against DPPH• radicals. 4.5. Biocompatibility and in vitro biological function evaluation 4.5.1. Cell cultures HCECs were purchased from Procell and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum. The cells were digested with 0.25% Trypsin-EDTA (Procell Life Science & Technology Co., Ltd.) and incubated in a 37°C, 5% CO 2 incubator. 4.5.2. Cytotoxicity assay The cytotoxicity of CeO 2 and single-atom Pt/CeO 2 nanozymes was evaluated using the CCK-8 assay. Pre-inoculated HCECs in a 96-well plate were incubated with varying concentrations of CeO 2 and single-atom Pt/CeO 2 for 24 hours, and cell viability was measured using the CCK-8 kit. 4.5.3. Cellular uptake assay To prepare FITC-labelled CeO 2 and single-atom Pt/CeO 2 , 2 mg of FITC and 1 mg of nanomaterials were added to 3 mL of deionised water and stirred for 24 hours under light protection. The solutions were then centrifuged and washed three times. Pre-inoculated HCECs in a 24-well plate were incubated with 1 mL of FITC-labelled nanomaterials (10 µg/mL) in the dark for 0.5 and 12 hours, respectively. The plate was removed, and cells were stained with DAPI for 10 minutes at room temperature. After gentle washing with PBS, images were captured using CLSM. 4.5.4. Cell protection from oxidative stress damage For the ROS and cell viability assay, 100 µL of HCECs was counted and inoculated in a 96-well plate at a concentration of 1 × 10 4 cells/well with five replicate samples. To prevent evaporation, 100 µL of PBS was added around the plate’s perimeter. After 24 hours, the original medium was replaced with complete medium containing 1 mM H 2 O 2 , along with various concentrations of CeO 2 and single-atom Pt/CeO 2 (20, 40, 60, 80, and 100 µg/mL). After 24 hours, cell viability and ROS levels were assessed using cell death staining and ROS content evaluation assays. 4.5.5. Cell migration assay For the cell migration assay, HCECs in the logarithmic growth phase were inoculated into a 24-well plate and incubated for 24 hours to form a monolayer. A vertical line was drawn across the monolayer using a 200 µL tip, and cells at the edge of the scratch were removed by gentle washing with sterile PBS three times. The scratched cell layer was then incubated with serum-free medium containing CeO 2 and single-atom Pt/CeO 2 (4 µg/mL). The migration areas were photographed at the beginning and after 12 hours using a microscope camera system (DMi8), and the migration was quantified using ImageJ software. 4.6. In vivo animal experimental model study 4.6.1. In vivo biocompatibility analysis All animal experiments were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University (Ethics Approval No.: YSG25050602). To evaluate the in vivo biocompatibility of nanomaterials, PBS, CeO 2 , and single-atom Pt/CeO 2 nanozymes were applied to the corneas of normal mice. The concentration and frequency of the drops were consistent with those used in clinical keratitis treatment. Corneal transparency was observed using slit-lamp white light. Corneal epithelial damage was examined by applying cobalt blue light after dropping 0.1% fluorescein sodium solution onto the eyes and capturing images. H&E-stained tissue sections were observed and photographed under a microscope. 4.6.2. Aseptic keratitis mouse model establishment To establish an aseptic keratitis animal model, 8-week-old male C57BL/6J mice were housed in the Experimental Animal Center of Wenzhou Medical University (Zhejiang, China). Mice were anaesthetised via intraperitoneal injection of 10% chloral hydrate and local anaesthesia with procaine eye drops. After routine topical disinfection, a ring drill with a 2.5 mm diameter was gently rotated at the centre of the cornea to create a visible circle under a microscope. A corneal epithelial defect model was then created by scraping the corneal epithelium within the marked area using an insulin needle. Following this, 3 µL of LPS solution (10 mg/mL) was applied to the corneal wound. On the following day, a slit-lamp examination was performed to confirm the successful establishment of the keratitis model. On day 2 post-modelling, mice were treated with 10 µL of normal saline, CeO 2 (100 µg/mL), or single-atom Pt/CeO 2 (100 µg/mL) eye drops once daily for seven consecutive days. The therapeutic effect was evaluated and recorded on days 0, 1, 3, 5, and 7 after the different treatments. 4.6.3. Immunofluorescence staining On the 7th day post-treatment, mice were euthanised by cervical dislocation. The whole eyeball was quickly excised by clamping the optic nerve with curved forceps after reaching the orbit from the temporal side of the mouse. The eyeball was then placed on ice, and the cornea was carefully excised while other tissues outside the corneal rim and iris were gently removed with forceps. After washing with PBS, the corneal samples were fixed, dehydrated, embedded, and sectioned to produce frozen sections. Immunofluorescence staining was performed following the manufacturer's instructions to detect the presence of neutrophils, macrophages, and inflammatory factors (IL-6, IL-1β, and TNF-α) in the corneal samples. Immunofluorescence images were captured using a Leica DM4B fluorescence microscope and analysed semi-quantitatively using ImageJ software. Statistical Analysis Statistical analysis was performed using GraphPad Prism 8.4.3 software. All data are presented as mean ± standard deviation unless otherwise stated. Unpaired two-tailed Student's t-test was used to compare means between two groups, while one-way ANOVA was applied for comparisons involving multiple groups. Statistical significance was set at *P < 0.05, **P < 0.001, and ****P<0.0001. All experiments were repeated at least three times. Declarations Supporting Information Supplementary data to this article can be found online at . Conflict of Interest The authors declare no conflict of interest. Author Contribution J. 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Schematic illustration of single-atom Pt/CeO 2 fabrication and application in treating LPS-induced keratitis. Cite Share Download PDF Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 01 Oct, 2025 Reviews received at journal 28 Sep, 2025 Reviews received at journal 28 Sep, 2025 Reviews received at journal 25 Sep, 2025 Reviewers agreed at journal 24 Sep, 2025 Reviewers agreed at journal 24 Sep, 2025 Reviewers agreed at journal 24 Sep, 2025 Reviewers invited by journal 24 Sep, 2025 Editor assigned by journal 12 Sep, 2025 Submission checks completed at journal 12 Sep, 2025 First submitted to journal 11 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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bar = 10\u0026nbsp;nm, 2 nm in enlarged image). (b) TEM and HRTEM images of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (d111 = 0.3272A) (Scale bar = 10\u0026nbsp;nm, 2 nm in enlarged image). (c) XRD patterns of CeO\u003csub\u003e2\u003c/sub\u003e clusters and the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e compared with the standard card of CeO\u003csub\u003e2\u003c/sub\u003e. (d) The Raman spectra of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e. (e) Aberration-corrected HAADF-STEM images of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e. Isolated Pt single atoms were circled in red. (Scale bar = 2\u0026nbsp;nm) (f) XPS spectra, (g) Ce3d and (h) O1s of CeO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/a0738f25cb8ffdd7c0907d0a.png"},{"id":92890304,"identity":"9922e942-106e-4bf0-a601-b97aa0ebb9b7","added_by":"auto","created_at":"2025-10-06 17:47:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":329479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe enzyme-like properties of CeO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eand single-atom Pt/CeO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003enanozymes in ROS/RNS scavenging\u003c/strong\u003e. (a) Schematic illustration of ROS-scavenging activities of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes. (b) SOD-like activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes at different concentrations. (c) The •O\u003csup\u003e2− \u003c/sup\u003escavenging activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes demonstrated by ESR spectroscopy. (d) CAT-like activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e through measuring H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition (initial 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). e) CAT-like activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e at different concentration by tracking DO with initial 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.（f) Repetitive CAT-like activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e determined by adding H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM) every 5 min for 4 times. (g) The •OH scavenging activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e by ESR spectroscopy. (h) The •OH scavenging efficiency of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e tested by UV-Vis absorption spectra with MB as indicator. (1) MB, (2) MB+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, (3) MB+FeSO\u003csub\u003e4\u003c/sub\u003e, (4) MB+Pt/CeO\u003csub\u003e2\u003c/sub\u003e, (5) MB+FeSO\u003csub\u003e4\u003c/sub\u003e+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and (6) MB+FeSO\u003csub\u003e4\u003c/sub\u003e+H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e+Pt/CeO\u003csub\u003e2\u003c/sub\u003e, in which •OH were generated in FeSO\u003csub\u003e4\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reaction. (i) •OH scavenging efficiency of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e at different concentrations. (j) The DPPH• scavenging efficiency of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e tested by UV-Vis absorption spectra and (k) corresponding statistical analysis of absorbance value at 517 nm. (l) UV-Vis absorption spectra of DPPH• after reaction with different concentrations of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/cc091dd21dea6ea3f8dd0fd7.png"},{"id":92890305,"identity":"55d745c6-a090-480a-b954-eff94fa563cd","added_by":"auto","created_at":"2025-10-06 17:47:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":386907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntracellular ROS scavenging and cell protection effect of CeO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and single-atom Pt/CeO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. \u003c/strong\u003e(a) Cell viability of HCECs after incubation with CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes for 24 h. Cell viability of HCECs after incubation with 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and different concentrations of (b) CeO\u003csub\u003e2\u003c/sub\u003e and (c) single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes. (d) CLSM images and (e) corresponding quantitative analysis of HCECs stained with Calcein-AM/PI after different treatments. (Scale bar = 200\u0026nbsp;µm) (f) CLSM images and (g) corresponding quantitative analysis of HCECs cells stained with DCFH-DA after different treatments. (Scale bar = 200\u0026nbsp;µm) Intracellular levels of (h) TNF-α, (i) IL-1β and (j) IL-6 induced by LPS using qPCR measurement after different treatments. Data are presented as the mean ± SD (n ≥ 3). significances are determined by one-way ANOVA with Tukey's correction. *p \u0026lt; 0.05, **p\u0026lt;0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001; ns, no significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/0bb476871fd1cdcbf44bbc9d.png"},{"id":92890307,"identity":"9783802b-5234-4925-91a6-09f5e12f443e","added_by":"auto","created_at":"2025-10-06 17:47:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":438847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etherapeutic effect of CeO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and single-atom Pt/CeO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e eye drops in LPS induced aseptic keratitis treatment.\u003c/strong\u003e (a) Scheme of\u003cem\u003e in vivo \u003c/em\u003ekeratitis modeling and treatments. (b) The bright field microscopic images of HCECs after being treated by serum-free medium supplemented with PBS (Control), CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e for 12 h. (Scale bar = 200 μm) (c) Statistical analysis of scratch area to analyze the migration of HCECs. (d) Representative slit lamp images and fluorescein staining of corneas after different treatments on days 0, 1, 3, 5, and 7 to observe inflammation site (white arrow) and corneal epithelial defect (red arrow). (e) Corneal epithelial defect shapes at different time points. (f) Clinical scores of LPS induced aseptic keratitis in Control, CeO\u003csub\u003e2\u003c/sub\u003e, and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e treated groups at days 0, 1, 3, 5 and 7. (g) Quantitative analysis of epithelial defect area through fluorescence labeling. Data are presented as the mean ± SD (n ≥ 3). significances are determined by one-way ANOVA with Tukey's correction. *p \u0026lt; 0.05, **p\u0026lt;0.01, and ****p \u0026lt; 0.0001; ns, no significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/26cb0652bc79bbad65675fa2.png"},{"id":92891003,"identity":"ee5b7fe2-c88f-46ae-9185-f3fc1922bc09","added_by":"auto","created_at":"2025-10-06 17:55:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":493459,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Immunofluorescence images of F4/80, Ly6g, IL-6, IL-1β and TNF-α expressions in corneas of mice after different treatments. (Scale bar = 100 μm) (b-f) Quantitative results of F4/80, Ly6g, IL-6, IL-1β and TNF-α, respectively, in corneas after different treatments. Data are presented as the mean ± SD (n = 3). significances are determined by one-way ANOVA with Tukey's correction. **p\u0026lt;0.01, ***p\u0026lt;0.001, ns, no significant. (j) H\u0026amp;E stained corneas obtained from mice treated by PBS, CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e for 7 days. (Red arrows: inflammatory infiltration, black arrows: neovascular area, scale bar = 100 μm)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/48badee871d3b08c832d7809.png"},{"id":100617619,"identity":"63446565-2cec-430d-9a0d-e9dd3dda398a","added_by":"auto","created_at":"2026-01-19 17:54:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3405684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/97607cc4-ce03-487b-9448-bc486b838bdc.pdf"},{"id":92890328,"identity":"2d734784-1092-4133-8abf-36d9d3b37821","added_by":"auto","created_at":"2025-10-06 17:47:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":48525212,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedSIinblack.docx","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/5c5406bd42b39c68b36ce9b1.docx"},{"id":92891316,"identity":"ef6a0ba1-77d8-4676-ad94-0fea99367708","added_by":"auto","created_at":"2025-10-06 18:03:35","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":650493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. Schematic illustration of single-atom Pt/CeO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e fabrication and application in treating LPS-induced keratitis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7588173/v1/e29859ed8bd234d753f5914c.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single-atom Pt Doped Nanoceria for Enhanced Cell Phagocytosis and Nanozyme Activities in Keratitis Immune Regulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eKeratitis, a common ocular disease worldwide, is primarily classified into infectious and non-infectious categories and ranks as the fifth leading cause of global blindness. In recent years, the incidence of keratitis has been rising due to factors such as plant trauma, overuse of broad-spectrum antibiotics and hormones, improper contact lens use, ophthalmic surgeries, corneal transplants, and conditions like AIDS, diabetes, and immunodeficiency.[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] The main clinical manifestations of keratitis include redness, oedema, pain, reduced visual function, photophobia, and increased intraocular secretions. The onset of keratitis is typically acute, leading to severe inflammatory reactions, oxidative stress damage, corneal ulcers, and perforations. If left untreated, keratitis can progress to eyeball atrophy, blindness, and, in severe cases, life-threatening complications when inflammation spreads to the brain.[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Clinically, the standard approach involves sterilisation followed by hormone treatment to suppress the inflammatory response. A significant challenge, however, is the inability to eliminate the inflammatory reaction and reactive oxygen species (ROS) generated by bacterial toxins in a timely manner, resulting in irreversible damage such as scarring, neovascularisation, perforation, and reduced corneal light transmittance.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eUpon the onset of keratitis, the cornea experiences a severe inflammatory response, a complex process involving multiple cells, which triggers the release of various pro-inflammatory cytokines and the recruitment of immune cells to the site of inflammation. For example, lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria like \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, is one of the key inducers of keratitis. LPS activates the NF-κB signalling pathway, generating transcription factors, stimulating ROS production, and inducing the secretion of various endogenous active factors, such as the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α).[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] In response, inflammatory cells produce large amounts of ROS to combat bacterial invasion, leading to oxidative stress in tissues. Oxidative stress refers to an imbalance between pro-oxidant and antioxidant substances, resulting in the infiltration of inflammatory cells, increased secretion of proteases, and the accumulation of oxidative by-products. These include pro-inflammatory substances such as leukotrienes and thromboxanes, which dilate blood vessels, promote leukocyte migration, and further exacerbate oxidative stress and inflammation.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Consequently, oxidative stress and inflammatory responses act synergistically, ultimately leading to irreversible corneal damage.\u003c/p\u003e\u003cp\u003eSpecifically, during keratitis, excessive ROS production induces oxidative damage to DNA, proteins, and lipids and leads to corneal cell apoptosis.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Elevated ROS levels are recognised as a critical factor in the progression of various ocular diseases, including ocular surface diseases (keratitis, xerophthalmia, alkali burns), cataracts, glaucoma, neurodegeneration, uveitis, and age-related macular degeneration.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Eliminating inflammation and ROS to regulate the microenvironment has proven to be an effective strategy for treating inflammatory diseases and promoting wound healing. In our previous study, nanozymes with catalase (CAT)-like activity were found to reduce secondary oxidative stress and inflammation following photodynamic therapy for choroidal neovascularisation, simultaneously alleviating hypoxia by catalytically degrading hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) into oxygen. Nanozymes mimicking biological enzymes, such as superoxide dismutase (SOD), CAT, and peroxidase (POD), exhibit significant advantages in their synthesis, storage, transport, and structure-performance optimisation. For instance, CeO\u003csub\u003e2\u003c/sub\u003e with CAT-like activity demonstrated ROS scavenging abilities through the transition between Ce\u003csup\u003e3+\u003c/sup\u003e and Ce\u003csup\u003e4+\u003c/sup\u003e oxidation states, resulting in reduced inflammation in the treatment of bowel diseases, otitis media, ocular surface conditions, and intraocular diseases.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] However, cell experiments have shown that CeO\u003csub\u003e2\u003c/sub\u003e becomes toxic at certain concentrations, which limits its application. Fortunately, recently developed single-atom catalysts (SACs), characterised by nearly 100% atomic dispersion, exhibit significantly enhanced catalytic performance compared to conventional nanozymes, thereby greatly reducing the use of precious metals.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] For example, Yu \u003cem\u003eet al.\u003c/em\u003e found that metal single atoms (Pt, Au, Cu, Ru) significantly improved the oxidase-like activity of PCN-222 by reducing the adsorption and activation energy of O\u003csub\u003e2\u003c/sub\u003e.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn this study, single-atom Pt-decorated CeO\u003csub\u003e2\u003c/sub\u003e was synthesised through a simple method involving the in situ reduction of the PtCl\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e precursor into single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e by NaBH\u003csub\u003e4\u003c/sub\u003e, aiming to reduce the dosage of nanozymes (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In this process, key ROS components, including superoxide anions (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and hydroxyl radicals (\u0026bull;OH), were rapidly eliminated by single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, effectively reducing oxidative stress. Additionally, single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes demonstrated a strong scavenging effect on reactive nitrogen species (RNS), such as 1-Diphenyl-2-picrylhydrazyl (DPPH\u0026bull;) free radicals. The single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e solution was administered as eye drops in a non-invasive, low-frequency manner (once daily). The ROS scavenging and anti-inflammatory properties of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e were further evaluated by measuring the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and assessing corneal epithelial healing. This study provides a valuable reference for the use of efficient single-atom catalytic nanozymes in the treatment of other inflammatory and autoimmune diseases.\u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Synthesis and characterisation of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eCeO\u003csub\u003e2\u003c/sub\u003e was first synthesised \u003cem\u003evia\u003c/em\u003e a simple hydrothermal method.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Subsequently, negatively charged PtCl\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e was added and adsorbed onto the surface of positively charged CeO\u003csub\u003e2\u003c/sub\u003e nanocrystals, stabilised with 6-aminocaproic acid (AHA) through electrostatic interactions. The PtCl\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e was in situ reduced by NaBH\u003csub\u003e4\u003c/sub\u003e, yielding single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanostructures.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]Transmission electron microscopy (TEM) images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) revealed that the synthesised CeO\u003csub\u003e2\u003c/sub\u003e exhibited a uniform spherical nanocrystal structure, with particle sizes ranging from 5 to 10 nm. High-resolution TEM (HRTEM) measurements indicated that the lattice spacing of CeO\u003csub\u003e2\u003c/sub\u003e nanozymes on the (111) plane was 0.3128 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and S1a, Supporting Information). The single-atom Pt appeared as clusters on the (111) surface, with a slightly broader lattice spacing of approximately 0.3272 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and S1b, Supporting Information), confirming the successful incorporation of Pt single atoms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe powder X-ray diffraction (XRD) patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) demonstrated characteristic diffraction peaks at 28.6\u0026deg;, 33.1\u0026deg;, 47.5\u0026deg;, 56.3\u0026deg;, 59.1\u0026deg;, 69.4\u0026deg;, 76.7\u0026deg;, 79.1\u0026deg;, and 88.4\u0026deg;, consistent with the standard card for CeO\u003csub\u003e2\u003c/sub\u003e (PDF#43-1002). These findings suggest that the incorporation of single-atom Pt did not alter the cubic fluorite structure of CeO\u003csub\u003e2\u003c/sub\u003e.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] The doping of single-atom Pt resulted in an increased distance between crystal planes, causing a slight left shift in the XRD curve compared to CeO\u003csub\u003e2\u003c/sub\u003e. Additionally, no diffraction peaks related to Pt or PtO\u003csub\u003ex\u003c/sub\u003e (33\u0026deg;, 44\u0026deg;) were observed within the 10\u0026deg; to 90\u0026deg; range in single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, likely due to the extremely low concentration and excellent dispersion of Pt on the CeO\u003csub\u003e2\u003c/sub\u003e surface.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed presents the Raman spectra, showing that both CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e exhibited typical cubic fluorite structure characteristics. The Raman peaks of CeO\u003csub\u003e2\u003c/sub\u003e at 258 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 460 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 606 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponded to the second-order transverse acoustic vibration peak of CeO\u003csub\u003e2\u003c/sub\u003e (2TA), the symmetric stretching vibration characteristic peak of the cubic fluorite structure (F\u003csub\u003e2\u003c/sub\u003eg), and the defect induction mode (D), respectively. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]New peaks at 550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were observed in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e sample, corresponding to Pt-O-Ce and Pt-O bonds, respectively.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Further analysis using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) was conducted to examine the doping form of single-atom Pt on CeO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The electrons captured by the high-angle ring detector, scattered by heavy elements, created contrast related to the atomic number of the corresponding elements. The HAADF-STEM image revealed that the bright spot inside the red circle corresponded to the dispersed Pt single atoms on the CeO\u003csub\u003e2\u003c/sub\u003e surface.\u003c/p\u003e\u003cp\u003eThe chemical states of Ce and O elements in CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e were investigated by X-ray photoelectron spectroscopy (XPS). In comparison with CeO\u003csub\u003e2\u003c/sub\u003e, a peak around 75 eV appeared in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanostructure, indicating successful Pt doping (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef and inset).[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] The Ce element in CeO\u003csub\u003e2\u003c/sub\u003e exists primarily in two valence states: Ce\u003csup\u003e3+\u003c/sup\u003e and Ce\u003csup\u003e4+\u003c/sup\u003e. The Ce\u003csup\u003e3+\u003c/sup\u003e centre scavenges \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and \u0026bull;OH through redox reactions or by mimicking SOD activity, while the Ce\u003csup\u003e4+\u003c/sup\u003e centre oxidises H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by mimicking CAT activity.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Since \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and \u0026bull;OH are directly associated with inflammatory responses and cell death, a high proportion of Ce\u003csup\u003e3+\u003c/sup\u003e in CeO\u003csub\u003e2\u003c/sub\u003e nanozymes is crucial for mitigating inflammation-related oxidative stress.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] According to the literature, the absorption peaks at 901.9 eV, 898.6 eV, 883.9 eV, and 880.6 eV correspond to Ce\u003csup\u003e3+\u003c/sup\u003e.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and \u003cb\u003eFigure S2\u003c/b\u003e (Supporting Information), the doping of Pt single atoms increased the Ce\u003csup\u003e3+\u003c/sup\u003e content from 39.12% to 58.66%, as calculated from the absorption peak areas. The varying ratios of Ce\u003csup\u003e3+\u003c/sup\u003e and Ce\u003csup\u003e4+\u003c/sup\u003e in CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e clusters confirmed that Pt doping affected the redox state of Ce. The O 1s XPS spectrum could be deconvoluted into three binding energy peaks for CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e clusters. The characteristic peaks at 528.7\u0026ndash;528.9 eV, 530.5\u0026ndash;530.7 eV, and 533.5\u0026ndash;533.7 eV corresponded to lattice oxygen O\u003csub\u003eLat\u003c/sub\u003e (O\u003csup\u003e\u0026minus;\u003c/sup\u003e), surface oxygen O\u003csub\u003eSur\u003c/sub\u003e (O\u003csup\u003e2\u0026minus;\u003c/sup\u003e, O\u003csup\u003e\u0026minus;\u003c/sup\u003e), and other possible adsorbed oxygen species O\u003csub\u003eAds\u003c/sub\u003e (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, OH\u003csup\u003e\u0026minus;\u003c/sup\u003e), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and \u003cb\u003eFigure S3\u003c/b\u003e, Supporting Information).[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong these, the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e species in O\u003csub\u003eSur\u003c/sub\u003e are ROS produced at oxygen vacancies. Thus, the concentration of O\u003csub\u003eSur\u003c/sub\u003e is positively correlated with oxygen vacancy content.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] From the integration of the peak areas, the O\u003csub\u003eSur\u003c/sub\u003e concentration increased from 25.15% to 38.20% in CeO\u003csub\u003e2\u003c/sub\u003e and 1:10 Pt/CeO\u003csub\u003e2\u003c/sub\u003e, respectively. The single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e sample with a doping ratio of 1:10 showed a higher oxygen vacancy concentration, consistent with the Ce3d XPS results mentioned earlier.[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] The Pt 4f XPS spectra of samples with varying Pt doping ratios are shown in \u003cb\u003eFigure S4\u003c/b\u003e (Supporting Information). The 4f\u003csub\u003e5/2\u003c/sub\u003e and 4f\u003csub\u003e7/2\u003c/sub\u003e peaks of Pt/CeO\u003csub\u003e2\u003c/sub\u003e were fitted into four Gaussian peaks, corresponding to PtO\u003csub\u003e2\u003c/sub\u003e and PtO, respectively.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] This confirmed that PtO and PtO\u003csub\u003e2\u003c/sub\u003e enhance catalytic activity, acting as co-catalysts. As the Pt doping ratio increased, more PtO and PtO\u003csub\u003e2\u003c/sub\u003e species were present on the CeO\u003csub\u003e2\u003c/sub\u003e surface.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 The enzyme-like properties of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eTo achieve optimal ROS scavenging performance, single-atom Pt-doped CeO\u003csub\u003e2\u003c/sub\u003e was synthesised with varying Pt to CeO\u003csub\u003e2\u003c/sub\u003e feeding ratios of 1:40, 1:20, 1:10, and 1:7 (mass ratio of Pt to CeO\u003csub\u003e2\u003c/sub\u003e) by adjusting the amount of Pt precursor. At the 1:7 doping ratio, the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanoparticles exhibited a lattice spacing of 0.227 nm on the (111) surface (\u003cb\u003eFigure S5\u003c/b\u003e, Supporting Information), consistent with the previously reported lattice spacing of Pt monoliths.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This indicated that the reduced Pt on CeO\u003csub\u003e2\u003c/sub\u003e existed as agglomerates, which substantially reduced the catalytic efficiency per Pt atom. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition and DPPH\u0026bull; removal tests were then performed to assess the catalytic performance of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (\u003cb\u003eFigure S6\u003c/b\u003e, Supporting Information). As the amount of single-atom Pt doping increased, the catalytic efficiency of the nanozymes also improved. Therefore, the 1:10 Pt doping ratio was chosen as the optimal ratio for obtaining monoatomically dispersed Pt with the best ROS scavenging effect. Single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e with a 1:10 doping ratio was selected for further investigation in subsequent studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eVarious ROS produced by oxidative stress during immune responses include hydroxyl free radicals (\u0026bull;OH), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, superoxide radicals (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), superoxide anion free radicals, lipoxygen free radicals, and ozone.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] In the present study, classic and significant biological enzyme-like activities related to free radical scavenging, including SOD-like and CAT-like activities, were used to explore the enzyme-like properties of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In physiological conditions, the SOD enzyme plays a pivotal role in maintaining intracellular redox balance by catalysing the disproportionation of \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. To test the SOD-like activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, the generated \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the xanthine reaction, catalysed by xanthine oxidase, was scavenged using tetrazolium salt- 2 - [2 \u0026ndash; methoxy \u0026ndash; 4 - nitrophenyl] \u0026minus;\u0026thinsp;3 - [4 - nitrophenyl] \u0026minus;\u0026thinsp;5 - [2, 4 - disulfophenyl] \u0026minus;\u0026thinsp;2H - tetrazole (WST-8), which reacted with \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to form formazan. The quantity of formazan was measured spectrophotometrically at 450 nm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, the scavenging rates of CeO\u003csub\u003e2\u003c/sub\u003e were less than 30% within the concentration range of 20\u0026ndash;100 \u0026micro;g/mL, much lower than those of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e. The \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e scavenging rate of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e reached 87.22% even at a concentration of 20 \u0026micro;g/mL. Furthermore, electron spin resonance (ESR) analysis revealed that CeO\u003csub\u003e2\u003c/sub\u003e could not completely eliminate \u0026bull;OH free radicals, even at a concentration of 100 \u0026micro;g/mL for over 2 hours. In contrast, the ESR curve for single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e showed a flat profile, indicating complete removal of \u0026bull;OH free radicals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eTo assess the CAT-like catalytic activity, harmful H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used as the substrate to be degraded into harmless H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e. The process of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e degradation was quantitatively measured by tracking changes in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration, monitored by absorbance at 240 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The results showed that more than 50% of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was rapidly decomposed within 300 seconds by single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (20 \u0026micro;g/mL). At the end of the reaction, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging ratio by single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e was 12 times higher than that of CeO\u003csub\u003e2\u003c/sub\u003e clusters. Additionally, the production of O\u003csub\u003e2\u003c/sub\u003e bubbles was observed immediately upon contact between the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (\u003cb\u003eFigure S7\u003c/b\u003e, Supporting Information). The O\u003csub\u003e2\u003c/sub\u003e production rate was found to be highly dependent on the concentration of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). In contrast, CeO\u003csub\u003e2\u003c/sub\u003e exhibited negligible CAT-like activity in catalysing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e degradation and O\u003csub\u003e2\u003c/sub\u003e production. The repeatability of the CAT-like catalytic activity and the stability of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e were further evaluated by measuring the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration change over four cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The linear slope of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition did not significantly change over time, indicating that the catalytic reaction did not affect the binding and catalytic properties of single-atom Pt on the CeO\u003csub\u003e2\u003c/sub\u003e surface.\u003c/p\u003e\u003cp\u003eTo further verify the scavenging ability of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e towards \u0026bull;OH, ESR and UV-Vis spectroscopy were used to quantify \u0026bull;OH. ESR analysis revealed that CeO\u003csub\u003e2\u003c/sub\u003e showed a weak ability to scavenge \u0026bull;OH, whereas single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e was able to completely scavenge all free radicals after just 2 minutes of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Furthermore, methylene blue (MB), a common trap for \u0026bull;OH produced in the Fenton reaction between FeSO\u003csub\u003e4\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, reacts with \u0026bull;OH to form hydroxylated methylene blue (MB-OH), causing a colour change from blue to colourless. Therefore, the scavenging ability of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e towards \u0026bull;OH was indirectly evaluated by the change in MB concentration, measured by absorbance at 664 nm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, the presence of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e led to strong absorbance at 664 nm, confirming that MB was not oxidised by \u0026bull;OH, which demonstrated the strong scavenging ability of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e. Moreover, the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e showed a significant concentration dependence in its scavenging activity towards \u0026bull;OH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003eDPPH\u0026bull;, a stable nitrogen-centred chromogenic radical dissolved in ethanol, exhibits a purplish-red colour with a characteristic absorption peak at 517 nm. In the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group, the DPPH\u0026bull; solution rapidly faded to a colourless and transparent solution, with no distinct absorption peak at 517 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). In contrast, no notable colour change was observed in the CeO\u003csub\u003e2\u003c/sub\u003e-treated group, with an absorption curve similar to that of the control group. This demonstrated that single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e exhibited excellent DPPH\u0026bull; scavenging ability, while CeO\u003csub\u003e2\u003c/sub\u003e had negligible scavenging activity. Further statistical analysis revealed that the scavenging activity of CeO\u003csub\u003e2\u003c/sub\u003e towards DPPH\u0026bull; increased by 48.9 times after doping with single-atom Pt. Additionally, a clear concentration dependence of the simulated enzyme activity was observed for concentrations below 25 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei and \u003cb\u003eFigure S8\u003c/b\u003e, Supporting Information). In summary, the engineered single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes displayed significantly higher scavenging abilities for ROS and RNS compared to CeO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 In vitro ROS-scavenging and anti-inflammatory activities of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e.\u003c/h2\u003e\u003cp\u003eUpon the onset of keratitis, corneal epithelial cells and inflammatory cells, under severe oxidative stress, generate an excessive amount of oxidative free radicals, exacerbating the inflammatory response. Corneal epithelial cells, as the outermost layer of the cornea, act as the first line of defence against external pathogens, engaging in innate immunity, detecting pathogens, and signalling the activation of the corneal defence system. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] Macrophages, essential phagocytes, are activated into the M1 phenotype by interferon-γ and LPS, secreting pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, and IL-6 to induce immune responses, phagocytose, and eliminate bacteria. However, prolonged M1 macrophage activation can exacerbate metalloproteinase secretion, leading to corneal perforation, delayed healing, and scar formation.[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] Therefore, timely removal of oxidative stress and modulation of the inflammatory response are essential for mitigating corneal tissue damage.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eTo assess the protective effect of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e on human corneal epithelial cells (HCECs) against oxidative stress-induced injury, the uptake of Pt/CeO\u003csub\u003e2\u003c/sub\u003e by HCECs was evaluated using fluorescein isothiocyanate (FITC) as a fluorescent marker. FITC-labelled single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e was incubated with HCECs, followed by fluorescence detection using a confocal laser scanning microscope (CLSM). As shown in \u003cb\u003eFigure S9\u003c/b\u003e (Supporting Information), HCECs treated with single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e exhibited stronger fluorescence intensity than those treated with CeO\u003csub\u003e2\u003c/sub\u003e after 0.5 hours of incubation, indicating a higher uptake of single-atom Pt-doped CeO\u003csub\u003e2\u003c/sub\u003e by HCECs. Furthermore, the fluorescence of FITC-labelled nanoparticles primarily indicated their distribution within the cytoplasm (\u003cb\u003eFigure S10\u003c/b\u003e, Supporting Information). After 12 hours, nearly no green fluorescence was observed in the CeO\u003csub\u003e2\u003c/sub\u003e-treated group, whereas HCECs in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e group maintained strong green fluorescence, indicating that single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e exhibited faster cellular uptake and longer intracellular retention compared to CeO\u003csub\u003e2\u003c/sub\u003e. This enhanced uptake may be attributed to the increased antioxidant activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, with 58.66% Ce\u003csup\u003e3+\u003c/sup\u003e in the Ce\u003csup\u003e3+\u003c/sup\u003e/Ce\u003csup\u003e4+\u003c/sup\u003e ratio, compared to 39.12% Ce\u003csup\u003e3+\u003c/sup\u003e in CeO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe cytotoxicity and cytoprotective effects of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes on HCECs were assessed using the cell counting kit-8 (CCK-8) assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, both CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e demonstrated low cytotoxicity, even at concentrations up to 120 \u0026micro;g/mL. The protective effect of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e on HCECs against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress was then evaluated. HCECs were incubated with various concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours, and the cell survival rate was approximately 50% at a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration of 1 mmol/L (\u003cb\u003eFigure S11\u003c/b\u003e, Supporting Information). When HCECs were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and different concentrations of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, CeO\u003csub\u003e2\u003c/sub\u003e showed no significant cytoprotective effect due to its lack of CAT-like activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In contrast, single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e demonstrated significant protection in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). A cell live/dead staining assay was performed on different treatment groups to assess the protective effect of the nanozymes under oxidative stress. CLSM images confirmed that at a concentration of 40 \u0026micro;g/mL, single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e restored HCEC viability to nearly normal levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003eIntracellular ROS levels were quantified using DCFH-DA as an indicator, with green fluorescence intensity measured to assess oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). In the negative control group, where cells also produced ROS during normal physiological activities, fluorescence was sparse. In the positive control group, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation significantly increased intracellular fluorescence, indicating severe oxidative stress. CeO\u003csub\u003e2\u003c/sub\u003e showed minimal ROS scavenging activity, with fluorescence intensity similar to that of the positive control group. However, in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group, green fluorescence was markedly reduced, indicating effective inhibition of intracellular oxidative stress. Further statistical analysis confirmed that intracellular oxidative stress levels returned to normal after single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). These results demonstrate that single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e effectively protects cells from oxidative stress-induced cytotoxicity.\u003c/p\u003e\u003cp\u003eIn keratitis, both corneal epithelial cells and macrophages produce oxidative stress in response to LPS or toxins, playing a key role in generating inflammatory cytokines. Therefore, LPS was used to stimulate RAW264.7 cells to investigate the role of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e in modulating the inflammatory response. Quantitative real-time PCR (qPCR) was performed to analyse the expression levels of inflammatory factors by measuring RNA content. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-j, after LPS stimulation, the RNA expressions of TNF-α, IL-1β, and IL-6 in RAW264.7 cells increased approximately 7-fold, 300-fold, and 1,300-fold, respectively. In the CeO\u003csub\u003e2\u003c/sub\u003e-treated group, the expressions of these inflammatory factors showed some reduction. However, in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group, the expression of all three inflammatory factors was significantly decreased, indicating a potent anti-inflammatory effect.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 In vivo biosafety of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e biocompatibility of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e was assessed using slit-lamp examination, fluorescein sodium staining, and tissue section analysis to evaluate corneal permeability, wound size, and inflammatory cell infiltration. Specific Pathogen-Free (SPF) grade C57BL/6 male mice, aged 6 to 8 weeks with healthy anterior segments, were treated with PBS, CeO\u003csub\u003e2\u003c/sub\u003e, and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e. Solutions of PBS, CeO\u003csub\u003e2\u003c/sub\u003e, and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;g/mL, 10 \u0026micro;L) were administered once daily for one week. As shown in \u003cb\u003eFigure S12\u003c/b\u003e (Supporting Information), the corneal morphology and structure remained intact, with no significant reduction in light transmittance or inflammatory response. Fluorescein sodium staining revealed no green fluorescence in any group, indicating the absence of corneal epithelial defects. Hematoxylin and eosin (H\u0026amp;E) staining of tissue sections on day 7 showed no significant oedema or inflammatory cell infiltration, confirming that the corneal structure was intact. These results demonstrated the good biological safety and \u003cem\u003ein vivo\u003c/em\u003e biocompatibility of the nanomaterials. To assess the retention time of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, the nanomaterial was labelled with FITC and applied to the ocular surface of healthy mice. Fluorescence was observed at various time points to monitor retention on the corneal surface. As shown in Figure S13 (Supporting Information), after 12 hours of incubation, substantial fluorescence remained in the corneal layer, indicating that single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes had a prolonged retention time on the ocular surface.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 In vivo anti-inflammatory activity of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eTo evaluate the therapeutic effect of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e in treating keratitis, a non-infectious keratitis animal model was established by scraping part of the corneal epithelial cells with an insulin needle and applying LPS to the cornea. Mice were randomly assigned to three groups: PBS, CeO\u003csub\u003e2\u003c/sub\u003e, and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, with eye drop treatments administered once daily for 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Chronic inflammation and oxidative stress in keratitis can lead to corneal ulcers and non-healing wounds, ultimately resulting in corneal neovascularisation, scarring, and reduced light transmittance.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] Therefore, promoting corneal epithelial migration and reducing inflammation are critical factors in facilitating corneal ulcer healing. A cell scratch assay was performed to assess the effect of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e on HCEC migration. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, HCEC migration was minimal in the PBS and CeO\u003csub\u003e2\u003c/sub\u003e-treated groups, with significant gaps remaining between the cells. In contrast, the scratch area in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group was almost completely covered by HCECs, leading to the disappearance of the wound. Statistical analysis in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec further confirmed that single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e significantly enhanced HCEC migration during wound healing. The scratch assay showed that oxidative stress and inflammation delayed HCEC migration in the control groups.. However, single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e promoted HCEC migration by alleviating oxidative stress and inflammation in the cornea.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the \u003cem\u003ein vivo\u003c/em\u003e keratitis experiment, slit-lamp observation revealed a large area of corneal opacity from day 1 to day 7 in the PBS-treated group, indicating severe inflammation and reduced corneal transparency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Sodium fluorescein staining showed that corneal epithelial defects gradually decreased in the PBS-treated group but did not fully heal after 7 days. In the CeO\u003csub\u003e2\u003c/sub\u003e-treated group, corneal defects were healed by day 3, as indicated by the disappearance of green fluorescence. However, a significant area of corneal opacity persisted on day 7, suggesting continued inflammation and collagen accumulation. In the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group, corneal transparency recovered to near-normal levels by day 7, and slit-lamp observations confirmed that light transmittance was restored to pre-treatment levels. Furthermore, the wound area decreased dramatically by day 3 in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group, demonstrating that reducing inflammation through ROS elimination effectively promoted cell migration and preserved corneal transparency.\u003c/p\u003e\u003cp\u003eThe change in corneal epithelial defect area during keratitis treatment was quantitatively measured and depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee. On day 0, prior to treatment, there were no significant differences in the epithelial defect area among the three groups. After one day of treatment, the corneal epithelium in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group showed rapid healing, with the epithelial defect area significantly smaller compared to the PBS and CeO\u003csub\u003e2\u003c/sub\u003e-treated groups. Clinical scores of keratitis, measured on day 3, showed no notable differences among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), indicating that the immune system remained in a state of oxidative stress and inflammation. However, by days 5 and 7, the clinical scores of keratitis in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group were significantly lower than those in the PBS and CeO\u003csub\u003e2\u003c/sub\u003e-treated groups. No significant differences were observed between the CeO\u003csub\u003e2\u003c/sub\u003e and PBS-treated groups, likely due to the limited ROS scavenging ability of CeO\u003csub\u003e2\u003c/sub\u003e. The doping of single-atom Pt greatly enhanced the ROS scavenging effect, eliminating the inflammatory response and modulating the immune system to promote corneal epithelial migration and maintain corneal transparency.\u003c/p\u003e\u003cp\u003eAfter 7 days of treatment, immunohistochemical analysis was conducted to assess oxidative stress and inflammatory reactions by detecting the expression of F4/80, lymphocyte antigen 6 complex, locus G (Ly-6G), IL-6, IL-1β, and TNF-α. The intensity of red fluorescence indicated the expression levels of inflammatory factors in the corneal tissue layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Both F4/80 and Ly-6G, markers for macrophages and neutrophils, respectively, were widely distributed throughout the corneal tissue in the PBS-treated group, with red fluorescent spots indicating severe infiltration of these inflammatory cells. Similarly, high expression of these cells was also observed in the CeO\u003csub\u003e2\u003c/sub\u003e-treated group. In contrast, the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group showed minimal infiltration of both inflammatory cell types, suggesting that keratitis was effectively controlled after 7 days of treatment. Similar results were observed for the three inflammatory factors (IL-6, IL-1β, and TNF-α), with the PBS and CeO\u003csub\u003e2\u003c/sub\u003e-treated groups showing a significant inflammatory response, including an unhealed corneal epithelial layer at the wound site. However, expression of these inflammatory factors was significantly reduced in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group. Statistical analysis of the fluorescence intensity of these markers further confirmed that the doping of single-atom Pt substantially enhanced the anti-inflammatory effect in keratitis treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-f). High levels of oxidative stress during keratitis are a key factor contributing to the exacerbation of inflammation. The animal model experiments thus validated the effectiveness of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e in alleviating inflammation by scavenging ROS in the treatment of keratitis.\u003c/p\u003e\u003cp\u003eHistological evaluation was performed by H\u0026amp;E staining after the mice were sacrificed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej). Observation of the corneal morphology revealed that the arrangement of corneal epithelial cells and stroma in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group was more organised compared to the PBS-treated group. The PBS-treated group exhibited extensive infiltration of inflammatory cells (red arrow), indicating severe inflammation even on day 7. While inflammatory cell infiltration was significantly reduced in the CeO\u003csub\u003e2\u003c/sub\u003e-treated group, corneal neovascularisation (black arrow) was evident. In the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group, only a few inflammatory cells were present, and the corneal layers appeared clear with no signs of oedema or neovascularisation. Persistent inflammation and oxidative stress damage can lead to corneal neovascularisation, structural damage, and significant visual impairment.[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] Therefore, timely removal of oxidative stress and inflammation is an effective approach to preserving corneal transparency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eKeratitis, a significant cause of irreversible corneal damage, is characterised primarily by inflammation and oxidative stress within the microenvironment. In this study, single-atom Pt was doped onto the surface of CeO\u003csub\u003e2\u003c/sub\u003e using a simple in-situ reduction method. It was found that the catalytic activity, including CAT-like and SOD-like activities, was greatly enhanced by adjusting the doping ratio. The cytotoxicity and \u003cem\u003ein vitro\u003c/em\u003e cellular oxidative stress models demonstrated the good biocompatibility and protective function of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e in shielding HCECs from oxidative stress-induced damage. In the LPS-induced keratitis animal model, single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e significantly promoted corneal epithelial cell migration and wound healing. Further histological analysis confirmed that single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e treatment notably reduced neutrophil and macrophage infiltration in the corneal tissue. The expression levels of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, were significantly lower in the single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e-treated group compared to the CeO\u003csub\u003e2\u003c/sub\u003e and PBS-treated groups, particularly in the corneal epithelial layer. Consequently, oxidative stress and inflammation in the cornea were effectively controlled following single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e treatment, which preserved both the structural integrity and light transmittance of the cornea. Thus, the elimination of oxidative stress in the keratitis microenvironment is an effective strategy to reduce inflammation and promote corneal epithelial cell migration during treatment.\u003c/p\u003e"},{"header":"4. Experimental Section","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Materials\u003c/h2\u003e\u003cp\u003eCerium (III) nitrate hexahydrate (Ce(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO), 6-aminohexanoic acid (AHA), potassium tetrachloroplatinate (II), methylene blue, 1,1-diphenyl-2-picrylhydrazyl (DPPH\u0026bull;), and lipopolysaccharide (LPS) were purchased from Macklin (Shanghai, China). Hydrochloric acid (37%) was sourced from Zhejiang Zhongxing Chemical Co., Ltd. (Zhejiang, China). Polyvinylpyrrolidone (PVP, MW\u0026thinsp;=\u0026thinsp;58,000) and ammonium hydroxide were acquired from Aladdin (Shanghai, China). Sodium borohydride was purchased from Adamas. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was obtained from Sinopharm Chemical Reagent Co., Ltd. Fluorescein isothiocyanate (FITC), 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO), and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich. Dulbecco\u0026rsquo;s modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (USA). Cell Counting Kit-8 (CCK-8) and Live/Dead Cell Staining Kit (L7012) were acquired from Beyotime Biotechnology (Shanghai, China). All immunofluorescence reagents were sourced from Abcam (USA). Ultrapure water was obtained from a Milli-Q system (Millipore, USA). All other chemical reagents, unless specified, were purchased from Aladdin (China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Characterisations\u003c/h2\u003e\u003cp\u003eTEM and HRTEM images were captured using a transmission electron microscope (FEI Company, USA). The crystal structure of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes was analysed by XRD (Bruker Corporation, Germany). Chemical bonding information was obtained using Raman spectroscopy. Atomic-resolution HAADF-STEM imaging with spherical aberration correction was performed to examine single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e. XPS provided full-spectrum analysis of both CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e. Cellular fluorescence images were captured using a Leica DMi8 microscope (Leica Microsystems, Germany). Corneal H\u0026amp;E sections underwent fluorescence imaging and immunofluorescence staining using a Leica DM4B microscope (Leica Microsystems, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Synthesis of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e4.3.1. Synthesis of CeO\u003csub\u003e2\u003c/sub\u003e nanomaterials\u003c/h2\u003e\u003cp\u003eThe synthesis of CeO\u003csub\u003e2\u003c/sub\u003e followed a method previously reported in the literature.[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] A solution of 131.2 mg AHA and 5 \u0026micro;L hydrochloric acid was dissolved in 6 mL deionised water. Separately, 108.6 mg cerium (III) nitrate hexahydrate (Ce(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO) was dissolved in 5 mL deionised water. Ammonia (2 mL) was then added to the solution and magnetically stirred for 1 minute at room temperature to obtain an aqueous cerium nitrate solution. The AHA solution was quickly mixed with the cerium nitrate solution and stirred at 95\u0026deg;C for 6 hours. Finally, CeO\u003csub\u003e2\u003c/sub\u003e was obtained through centrifugation and repeated washing with deionised water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e4.3.2. Doping of single-atom Pt onto CeO\u003csub\u003e2\u003c/sub\u003e nanomaterials\u003c/h2\u003e\u003cp\u003eThe preparation of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e followed a method reported previously. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] To 7 mL deionised water, 1 mL of the CeO\u003csub\u003e2\u003c/sub\u003e solution obtained above was added. Then, 16 mg PVP (MW\u0026thinsp;\u0026asymp;\u0026thinsp;58,000) and an additional 1 mL aqueous K\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e4\u003c/sub\u003e solution with varying Pt mass doping ratios (1:7, 1:10, 1:20, and 1:40) were added to the mixture, which was stirred at 95\u0026deg;C for 2 hours. Afterwards, 3.52 mg sodium borohydride (NaBH\u003csub\u003e4\u003c/sub\u003e) was dissolved in 1 mL deionised water and added dropwise to the solution, followed by stirring for 30 minutes. The single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e aqueous solution was then obtained by centrifugation and washed three times with deionised water.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Enzyme-like activity tests\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e4.4.1. SOD activity determination\u003c/h2\u003e\u003cp\u003eThe scavenging ability of nanomaterials against superoxide anion radicals (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) was assessed using a total SOD activity assay kit based on the reaction of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), following the manufacturer's instructions. Briefly, WST-8 reacts with \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, catalysed by xanthine oxidase, to produce a water-soluble formazan dye, which can be inhibited by SOD. The enzymatic activity of SOD was determined by colourimetric analysis of the WST-8 product. The enzyme marker was preheated at 37\u0026deg;C and set to a single wavelength time scan mode at 450 nm. The experimental procedure was performed according to the instruction manual, starting with the addition of the reaction starter, with measurements taken every 5 minutes over a 30-minute period. Five replicates were tested for each group, with samples added in a concentration gradient.\u003c/p\u003e\u003cp\u003eThe \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e scavenging ability of the nanomaterials was also evaluated by ESR spectroscopy at room temperature. After reacting CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e with \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e for 2 minutes, 100 \u0026micro;L of the solution was taken, and 50 mM DMPO (a spin trap agent) was added for ESR spectrum recording.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e4.4.2. CAT activity determination\u003c/h2\u003e\u003cp\u003eThe consumption of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM) by CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (1 mg/mL) was monitored directly at 240 nm using a UV-Vis spectrophotometer (n\u0026thinsp;=\u0026thinsp;3). Single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e catalysed the conversion of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e, which was measured by detecting the amount of dissolved O\u003csub\u003e2\u003c/sub\u003e in the solution using a dissolved oxygen meter.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e4.4.3. \u0026bull;OH scavenging activity\u003c/h2\u003e\u003cp\u003eThe \u0026bull;OH scavenging efficiency of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e was determined by ESR spectroscopy at room temperature. The generation of \u0026bull;OH was initiated by reacting ferrous sulfate with aqueous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in a Fenton-like reaction, and the \u0026bull;OH was captured by DMPO to form the spin adduct DMPO/\u0026bull;OH. After 2 minutes of incubation with single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, the remaining \u0026bull;OH was captured with DMPO for another 2 minutes. ESR spectra were recorded, and the content of \u0026bull;OH was calculated by comparing the peak position and intensity with the negative control group. The \u0026bull;OH scavenging ability was further evaluated using MB as an indicator, with a UV-Vis spectrophotometer. Ferrous sulfate and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were reacted for 2 minutes, and MB along with CeO\u003csub\u003e2\u003c/sub\u003e or single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e was added to the solution. After 5 minutes, the remaining MB content was measured at the characteristic absorption peak at 662 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e4.4.4. DPPH\u0026bull; scavenging activity\u003c/h2\u003e\u003cp\u003eDPPH\u0026bull; was dissolved in ethanol to create a homogeneous purplish-red solution (1 mM) with a characteristic absorption peak at 517 nm. In a 48-well plate, 900 \u0026micro;L of DPPH\u0026bull; (100 \u0026micro;M) and 100 \u0026micro;L of CeO\u003csub\u003e2\u003c/sub\u003e or single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;g/mL) were added to each well. The spectra were measured between 200 and 800 nm intervals using an enzyme marker after 3 minutes at 2 nm intervals. Additionally, DPPH\u0026bull; was incubated with various concentrations of single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e to evaluate the scavenging activity against DPPH\u0026bull; radicals.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.5. Biocompatibility and in vitro biological function evaluation\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e4.5.1. Cell cultures\u003c/h2\u003e\u003cp\u003eHCECs were purchased from Procell and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum. The cells were digested with 0.25% Trypsin-EDTA (Procell Life Science \u0026amp; Technology Co., Ltd.) and incubated in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e4.5.2. Cytotoxicity assay\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes was evaluated using the CCK-8 assay. Pre-inoculated HCECs in a 96-well plate were incubated with varying concentrations of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e for 24 hours, and cell viability was measured using the CCK-8 kit.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e4.5.3. Cellular uptake assay\u003c/h2\u003e\u003cp\u003eTo prepare FITC-labelled CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e, 2 mg of FITC and 1 mg of nanomaterials were added to 3 mL of deionised water and stirred for 24 hours under light protection. The solutions were then centrifuged and washed three times. Pre-inoculated HCECs in a 24-well plate were incubated with 1 mL of FITC-labelled nanomaterials (10 \u0026micro;g/mL) in the dark for 0.5 and 12 hours, respectively. The plate was removed, and cells were stained with DAPI for 10 minutes at room temperature. After gentle washing with PBS, images were captured using CLSM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e4.5.4. Cell protection from oxidative stress damage\u003c/h2\u003e\u003cp\u003eFor the ROS and cell viability assay, 100 \u0026micro;L of HCECs was counted and inoculated in a 96-well plate at a concentration of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well with five replicate samples. To prevent evaporation, 100 \u0026micro;L of PBS was added around the plate\u0026rsquo;s perimeter. After 24 hours, the original medium was replaced with complete medium containing 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, along with various concentrations of CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (20, 40, 60, 80, and 100 \u0026micro;g/mL). After 24 hours, cell viability and ROS levels were assessed using cell death staining and ROS content evaluation assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e4.5.5. Cell migration assay\u003c/h2\u003e\u003cp\u003eFor the cell migration assay, HCECs in the logarithmic growth phase were inoculated into a 24-well plate and incubated for 24 hours to form a monolayer. A vertical line was drawn across the monolayer using a 200 \u0026micro;L tip, and cells at the edge of the scratch were removed by gentle washing with sterile PBS three times. The scratched cell layer was then incubated with serum-free medium containing CeO\u003csub\u003e2\u003c/sub\u003e and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (4 \u0026micro;g/mL). The migration areas were photographed at the beginning and after 12 hours using a microscope camera system (DMi8), and the migration was quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.6. In vivo animal experimental model study\u003c/h2\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e4.6.1. In vivo biocompatibility analysis\u003c/h2\u003e\u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University (Ethics Approval No.: YSG25050602). To evaluate the \u003cem\u003ein vivo\u003c/em\u003e biocompatibility of nanomaterials, PBS, CeO\u003csub\u003e2\u003c/sub\u003e, and single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e nanozymes were applied to the corneas of normal mice. The concentration and frequency of the drops were consistent with those used in clinical keratitis treatment. Corneal transparency was observed using slit-lamp white light. Corneal epithelial damage was examined by applying cobalt blue light after dropping 0.1% fluorescein sodium solution onto the eyes and capturing images. H\u0026amp;E-stained tissue sections were observed and photographed under a microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e4.6.2. Aseptic keratitis mouse model establishment\u003c/h2\u003e\u003cp\u003eTo establish an aseptic keratitis animal model, 8-week-old male C57BL/6J mice were housed in the Experimental Animal Center of Wenzhou Medical University (Zhejiang, China). Mice were anaesthetised \u003cem\u003evia\u003c/em\u003e intraperitoneal injection of 10% chloral hydrate and local anaesthesia with procaine eye drops. After routine topical disinfection, a ring drill with a 2.5 mm diameter was gently rotated at the centre of the cornea to create a visible circle under a microscope. A corneal epithelial defect model was then created by scraping the corneal epithelium within the marked area using an insulin needle. Following this, 3 \u0026micro;L of LPS solution (10 mg/mL) was applied to the corneal wound. On the following day, a slit-lamp examination was performed to confirm the successful establishment of the keratitis model. On day 2 post-modelling, mice were treated with 10 \u0026micro;L of normal saline, CeO\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;g/mL), or single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;g/mL) eye drops once daily for seven consecutive days. The therapeutic effect was evaluated and recorded on days 0, 1, 3, 5, and 7 after the different treatments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e4.6.3. Immunofluorescence staining\u003c/h2\u003e\u003cp\u003eOn the 7th day post-treatment, mice were euthanised by cervical dislocation. The whole eyeball was quickly excised by clamping the optic nerve with curved forceps after reaching the orbit from the temporal side of the mouse. The eyeball was then placed on ice, and the cornea was carefully excised while other tissues outside the corneal rim and iris were gently removed with forceps. After washing with PBS, the corneal samples were fixed, dehydrated, embedded, and sectioned to produce frozen sections. Immunofluorescence staining was performed following the manufacturer's instructions to detect the presence of neutrophils, macrophages, and inflammatory factors (IL-6, IL-1β, and TNF-α) in the corneal samples. Immunofluorescence images were captured using a Leica DM4B fluorescence microscope and analysed semi-quantitatively using ImageJ software.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.4.3 software. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation unless otherwise stated. Unpaired two-tailed Student's t-test was used to compare means between two groups, while one-way ANOVA was applied for comparisons involving multiple groups. Statistical significance was set at *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****P\u0026lt;0.0001. All experiments were repeated at least three times.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eSupporting Information\u003c/h2\u003e\u003cp\u003eSupplementary data to this article can be found online at .\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ. Zhao, W, Lou, Y. Wang and L. Wang prepared figure 1-4. X. Jin, C. Wang, J. Zhang, W. Zhuang, J. Wei, D. Lin, and S. Guo prepared figure 5. J. Zhao, W. Lou and Y Wang wrote the main manuscript. Y. Shao, Z. Jiang and B. Wang designed the work and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by the Zhejiang Provincial Natural Science Foundation of China (LBY21H120001, LR21H180001), the Wenzhou Key Program of Scientific and Technological Innovation (ZY2019017), and the Zhejiang Provincial Traditional Chinese Medicine Science and Technology Project (2021ZA091).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCheng XJ, Chen HY, Yang F, Hong JX, Cheng YY, Hu JJ. All-small-molecule supramolecular hydrogels assembled from guanosine 5\u0026prime;-monophosphate disodium salt and tobramycin for the treatment of bacterial keratitis. 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Angew Chem-Int Edit. 2010;49(26):4484\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"single-atom nanozymes, CeO2, keratitis, ROS elimination, anti-inflammation","lastPublishedDoi":"10.21203/rs.3.rs-7588173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7588173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring keratitis treatment, oxidative stress and inflammation often result in corneal neovascularisation, scarring, and reduced light transmittance. In this study, single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e is synthesised, exhibiting significantly enhanced catalase-like and superoxide dismutase-like activities for the elimination of superoxide anions (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and hydroxyl radicals (\u0026bull;OH). Doping single-atom Pt onto CeO\u003csub\u003e2\u003c/sub\u003e increases the Ce\u003csup\u003e3+\u003c/sup\u003e concentration in the Ce\u003csup\u003e3+\u003c/sup\u003e/Ce\u003csup\u003e4+\u003c/sup\u003e ratio from 39.12% to 58.66%, as confirmed by electron spin resonance, high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. \u003cem\u003eIn vitro\u003c/em\u003e studies demonstrate that single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e effectively reduces intracellular ROS levels in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-activated human corneal epithelial cells. Additionally, it exerts an anti-inflammatory effect on LPS-stimulated RAW264.7 macrophages, significantly decreasing the expression of interleukin-1β, interleukin-6, and tumour necrosis factor-α. \u003cem\u003eIn vivo\u003c/em\u003e, in an LPS-induced keratitis animal model, single-atom Pt/CeO\u003csub\u003e2\u003c/sub\u003e accelerates corneal ulcer healing and preserves corneal light transmittance, attributed to its anti-inflammatory properties, enzyme-like activities, and ability to promote cell migration. This study offers a novel approach for treating various inflammatory and autoimmune diseases.\u003c/p\u003e","manuscriptTitle":"Single-atom Pt Doped Nanoceria for Enhanced Cell Phagocytosis and Nanozyme Activities in Keratitis Immune Regulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 17:47:30","doi":"10.21203/rs.3.rs-7588173/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-01T13:55:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T17:07:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T14:23:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T06:33:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169386880129292147874839274857510752804","date":"2025-09-24T12:41:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286088640032360673181624316033448548262","date":"2025-09-24T12:28:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151382425478661439849605684778835816277","date":"2025-09-24T11:24:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-24T11:08:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-12T04:37:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T04:37:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-09-11T05:54:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a4bde2e-2672-4857-ba55-84f391d53d8a","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T17:22:39+00:00","versionOfRecord":{"articleIdentity":"rs-7588173","link":"https://doi.org/10.1186/s12951-025-03869-y","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-01-14 16:28:38","publishedOnDateReadable":"January 14th, 2026"},"versionCreatedAt":"2025-10-06 17:47:30","video":"","vorDoi":"10.1186/s12951-025-03869-y","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03869-y","workflowStages":[]},"version":"v1","identity":"rs-7588173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7588173","identity":"rs-7588173","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}