Biofunctional Design and Scalable Preparation of Phytolith-Based Nanocomposites for Biomedical Engineering

Abstract

Natural inorganic nanomaterials, particularly silicon-based minerals, have gained attention as promising biofunctional carriers owing to their unique physicochemical properties and excellent biocompatibility. However, their practical application is often hindered by limited biofunctional integration and poor scalability. Herein, we developed a CeO₂–ZnO/phytolith nanocomposite via a strategy involving high-temperature pretreatment and controlled interfacial assembly. This nanocomposite synergistically combines hemostatic, antibacterial, and antioxidant functions: phytoliths rapidly absorb water to concentrate clotting factors; ZnO exhibits strong antibacterial activity, achieving >99.5% inhibition against both Escherichia coli and Staphylococcus aureus; and CeO₂ effectively scavenges H₂O₂ through reversible Ce³⁺/Ce⁴⁺ redox cycling. Moreover, a scalable synthesis route was established, enabling stable pilot-scale production of ∼370 g per batch with high reproducibility in the composition and performance. The resulting CeO₂–ZnO/phytolith-based hemostatic gauze demonstrates outstanding biocompatibility, ultrahigh fluid absorption capacity (1000%), and excellent in vivo hemostatic performance in a mouse liver injury model, comparable to commercial products. This work overcomes key challenges in biofunctional integration and process scalability of natural silicon-based minerals, offering an integrated development strategy from rational material design to biomedical application.

Introduction

Natural inorganic nanomaterials exhibit significant application prospects in the biomedical field due to their high specific surface area, excellent biocompatibility, and stable chemical properties. − Among these materials, silicon-based mineral inorganic nanomaterials, such as zeolites, , diatomite, , and clay minerals (e.g., kaolinite, montmorillonite, halloysite, etc.), − are abundant in reserves and cost-effective. They have been reported to be effective in various applications, including drug delivery, anti-inflammation, and biosensing, − demonstrating their potential as biofunctional carriers. However, current research predominantly focuses on exploiting their physicochemical properties (such as channel adsorption, interlayer loading, and surface modification). , In-depth studies concerning their intrinsic bioactivity or biofunctional integration remain scarce. A key factor hindering the advancement of these materials is the absence of directed design and synergistic enhancement strategies, particularly in the integration and mechanistic elucidation of multiple biological effects, including procoagulation, antibacterial, and antioxidant activities. − To overcome the limitations of traditional biofunctional carriers, it is crucial to develop silicon-based mineral biomaterials with controllable functions and clear structure–activity relationships, advancing natural inorganic nanomaterials from research to application.

In the development of silicon-based mineral biomaterials, considerable efforts have been dedicated to the functionalization of mineral carriers through methods such as surface modification, interfacial assembly, and tube loading. − These strategies have facilitated the creation of antibacterial and hemostatic materials by incorporating active metal species onto mineral substrates. − Our previous research has demonstrated the potential of these materials: kaolinite enhanced coagulation by enriching clotting factors and generated oxygen vacancies in ZnO to boost antibacterial activity through reactive oxygen species generation, and montmorillonite adjusted the d-band center of manganese oxide to improve cascade reactivity for the treatment of enteritis. Despite these advances, translational progress continues to be hindered by three engineering challenges: the difficulty in scaling up laboratory-dependent and condition-sensitive processes, the limited integration of synergistic biofunctions (such as hemostatic, antibacterial, and antioxidant properties) within a single carrier, and insufficient consistency and standardization between batches. , Collectively, these issues impede the transition to scalable, cost-effective, and quality-assured products. Therefore, the development of silicon-based biomaterial systems that allow for scalable production, multifunctional capabilities, and controlled quality is essential to bridging the gap between laboratory research and practical application.

Compared to other silicate minerals, phytoliths present several advantages as hemostatic materials: (1) Their simple mineral phase composition, devoid of toxic or harmful associated minerals, eliminates the necessity for complex sample pretreatment; (2) their high water absorption capacity enhances the concentration of blood components, thereby accelerating hemostasis; (3) the presence of surface hydroxyl groups offers binding sites for the loading of functional groups while simultaneously reducing particle agglomeration. Utilizing an abundant, layered phytolith as a carrier, a stable CeO₂–ZnO/phytolith (CeO₂–ZnO/Pl) composite was constructed through controlled interfacial assembly. Systematic characterization confirmed its microstructure, composition, and interfacial properties. Its multiple biological functions, including hemostasis, antibacterial, and antioxidant capabilities, were comprehensively evaluated. Furthermore, a reproducible pilot-scale preparation was achieved, yielding over 372.4 g per batch, while maintaining consistency with laboratory-scale samples. The composite was fabricated into hemostatic gauze that demonstrates excellent biocompatibility, high absorption capacity, and rapid clotting performance. Its superior in vivo hemostatic efficacy has been validated through a mouse liver injury model in comparison to commercial products. This study establishes a new research paradigm, from material design to scalable production, offering a viable route for a high-performance inorganic mineral-based multifunctional hemostatic agent.

Results and Discussion

Synthesis and Structural Characterizations

The raw phytoliths contain a small amount of organic carbon impurities. To reduce impurities, a high-temperature calcination process is employed to pretreat the raw phytoliths. As shown in Figure S1, the phytoliths exhibit a layered structure and are primarily composed of three elements: C, O, and Si. As depicted in Figure a, the phytoliths display a distinct endothermic peak at 612.5 °C, accompanied by heat absorption and a sharp mass loss, indicating the decomposition of organic carbon within this temperature range. The reaction is essentially completed at 653.1 °C, with the remaining mass being 90.2% of the initial mass, and the total mass loss during the entire heating process is 10.22%. Based on these results, the calcination temperature was determined to be 700 °C to ensure the effective removal of organic carbon. After calcination at 700 °C, the organic carbon content in the phytoliths decreased, with the mass fraction of carbon dropping from 40.3% to 7.8%. The relative contents of elements such as Si, O, and Fe increased correspondingly (Figure b). Phase analysis results indicate that the primary phase of the phytoliths remains SiO₂ both before and after calcination. The diffraction peaks align well with the standard card PDF #86-2237 (Figure S2), demonstrating that the high-temperature pretreatment did not alter the main crystalline phase structure. Scanning electron microscopy (SEM) images reveal that phytoliths are composed of stacked lamellae, with no obvious changes in their microstructure observed before and after calcination (Figure S3). The calcination process results in a decrease in the specific surface area of phytoliths from 7.39 to 5.61 m²/g (Figure S4), which may be attributed to partial collapse of the pore structure.

1.

(a) TG-DSC curves of phytoliths. (b) Elemental composition of the phytoliths before and after calcination at 700 °C. (c) XRD patterns of the samples. (d) SEM and (e) TEM with elemental mapping of CeO₂–ZnO/Pl. (f) High-resolution XPS spectra of Zn 2p and Ce 3d for CeO₂–ZnO and CeO₂–ZnO/Pl. (g) ²⁹Si NMR spectra of Pl and CeO₂–ZnO/Pl.

Using phytoliths treated with high-temperature calcination as a carrier, a multifunctional composite system was constructed by employing an interface assembly strategy to load CeO₂ and ZnO nanoparticles, which possess antioxidant and antibacterial functions. This composite design fully leverages the inherent hemostatic capability of phytoliths, the valence state transition characteristics of CeO₂ (Ce³⁺/Ce⁴⁺), and the sustained Zn²⁺ release capability of ZnO, , enabling a synergistic enhancement of hemostasis, antioxidant, and antibacterial functions. The CeO₂–ZnO/Pl composite was prepared by using a one-step ball milling method. The characteristic diffraction peaks of SiO₂, CeO₂, and ZnO are clearly observed in the X-ray diffraction (XRD) patterns (Figure c), indicating their coexistence within the composite. SEM observations further confirm the uniform distribution of nanoparticles on the surface of phytoliths (Figure d), while energy-dispersive X-ray spectroscopy (EDS) reveals that the composite predominantly contains the O, Zn, Si, and Ce elements (Figure e). In contrast, the CeO₂–ZnO sample prepared without the phytoliths displays a combination of nanoparticles and lamellar structures, primarily consisting of the elements O, Zn, and Ce (Figure S5).

The valence states of surface elements in the material were analyzed by using X-ray photoelectron spectroscopy (XPS). In the Zn 2p spectrum (Figure f), the peaks at binding energies of 1044.9 and 1021.9 eV correspond to Zn 2p_1/2 and Zn 2p_3/2, respectively, indicating that the Zn element exists in the +2 valence state. In contrast to CeO₂–ZnO, the Zn 2p peaks in CeO₂–ZnO/Pl shift toward lower binding energies, suggesting the presence of interfacial interactions and charge transfer between the phytolith support and CeO₂–ZnO. In the Ce 3d spectrum, eight characteristic peaks were obtained through fitting, with six attributed to Ce⁴⁺ (yellow region) and two to Ce³⁺ (green region). The calculated proportions of Ce³⁺ in CeO₂–ZnO and CeO₂–ZnO/Pl are 13.4% and 15.7%, respectively, while those of Ce⁴⁺ are 86.6% and 84.3%, respectively. This indicates that phytoliths, acting as a carrier, enhance the content of Ce³⁺ in the surface oxides through interfacial interactions.

Further investigation into the changes in the coordination environment of silicon atoms was conducted by using ²⁹Si solid-state nuclear magnetic resonance (NMR) spectroscopy (Figure g). Qⁿ denotes the structural state in which each [SiO₄]^4– tetrahedral unit is connected to n other silicon atoms via bridging oxygens. In the pure phytolith sample, the chemical shifts of Q³ and Q⁴ were measured at −95.0 and −107.4 ppm, with proportions of 44.0% and 56.0%, respectively. In the CeO₂–ZnO/Pl composite, the proportion of Q³ increased to 59.6%, suggesting the formation of chemical bonding between CeO₂–ZnO and the changes in the coordination number of certain silicon atoms. This finding corroborates the interfacial interactions observed in XPS. In summary, the characterization results confirm the successful preparation of the CeO₂–ZnO/Pl composite. By integrating the findings from XPS and NMR, we infer that new chemical bonds may have formed at the interface between the two materials.

Biological Functional Evaluation of the CeO₂–ZnO/Pl Composite

A systematic evaluation of the biological functions of CeO₂–ZnO/Pl composites was conducted, focusing on hemostatic, antibacterial, and antioxidant performances. By variation of the precursor ratio to phytoliths, a series of composites with different CeO₂–ZnO loadings were successfully synthesized to investigate the impact of loading on their biological effects. The in vitro hemostatic performance and blood compatibility of the materials were assessed using the blood clotting index (BCI) and hemolysis rate tests. Changes in the absorbance value of the reaction system at 540 nm were monitored to quantitatively evaluate the relative release of hemoglobin, thereby reflecting the coagulation ability of different composites. As illustrated in Figure a, the CeO₂–ZnO/Pl composite accelerates the coagulation process by rapidly absorbing water molecules from whole blood, concentrating coagulation factors and platelets, and promoting the conversion of fibrinogen to fibrin. Quantitative results demonstrate that both phytoliths and CeO₂–ZnO exhibit procoagulation effects compared to the negative control group (Figure b). Notably, the CeO₂–ZnO/Pl composite displays the most remarkable coagulation performance with a coagulation index obviously lower than that of the individual components. This finding indicates a synergistic enhancement effect between the carrier and the active components.

2.

(a) Schematic illustration of the coagulation process on CeO₂–ZnO/Pl. (b) BCI and (c) hemolysis rates of the materials. (d) Schematic of the proposed antibacterial mechanism. (e) Antibacterial rates against E. coli and S. aureus. (f) Schematic of the ROS scavenging antioxidant process. (g) Catalytic O₂ generation profiles and (h) total production of total O₂ from the reaction with H₂O₂.

The hemolysis rate was assessed by directly incubating the composite with a diluted suspension of rabbit red blood cells to evaluate the blood compatibility of the materials. As illustrated in Figure c, under the specified test conditions, the hemolysis rates of all materials remained below the safety threshold of 5%, thereby indicating that these materials satisfy the fundamental requirements for biomedical applications. Notably, the hemolysis rate demonstrated a dependence on the component composition: at an equivalent concentration, a higher loading of CeO₂–ZnO was correlated with an increased hemolysis rate of the material. This phenomenon may be attributed to the physicochemical properties of the material and the intensity of its interaction with the red blood cell, suggesting that in practical applications, an optimal balance between hemostatic performance and blood compatibility must be achieved. These findings affirm the potential of the CeO₂–ZnO/Pl composite as effective hemostatic agents.

The antibacterial properties of various materials against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were evaluated by using the plate counting method. The composites separately loaded with ZnO and CeO₂ were prepared for comparison, confirming the successful synthesis of the ZnO/Pl and CeO₂/Pl composites through XRD patterns (Figure S6). The photographs of colonies after coincubation with ZnO/Pl and CeO₂/Pl composites are presented in Figure S7, demonstrating that the ZnO component exerts a key antibacterial effect. The ZnO component in the CeO₂–ZnO/Pl composite exhibits antibacterial functions, primarily through mechanisms such as bacterial capture via contact and the release of zinc ions that disrupt cell structures, ultimately leading to bacterial death (as illustrated in Figure d). The colony plate photographs of the treated materials in each group are presented in Figure S8. The antibacterial experimental results for E. coli indicated that the phytolith carrier alone did not display obvious antibacterial activity against either bacterium (Figure e); however, CeO₂–ZnO demonstrated a notable bactericidal effect on E. coli, achieving an antibacterial rate of 66.9%. In contrast, the CeO₂–ZnO/Pl composite exhibited obviously enhanced antibacterial performance, with the antibacterial rate against E. coli increasing to 97.2% when the loading of CeO₂–ZnO reached 20%. When the loading is further increased to 40%, the antibacterial rate reaches as high as 99.9%, indicating that the material almost completely inhibits the growth of E. coli. In contrast, S. aureus did not exhibit obvious antibacterial effects with phytoliths, while both the CeO₂–ZnO and CeO₂–ZnO/Pl composites demonstrated excellent antibacterial activity. At a loading amount of 20%, the antibacterial rate of the composite against S. aureus reached 97.1%; when the loading amount increased to 40%, the antibacterial rate reached 98.7%, thereby proving its potent inhibitory effect against Gram-positive bacteria as well. More importantly, testing conducted by a China national accreditation service for conformity assessment (CNAS)-accredited laboratory, in accordance with GB/T 21510-2024 Appendix A (Antimicrobial property testing and evaluation methods for nanoinorganic materials), revealed that the composite exhibits an antibacterial rate of 99.6% against E. coli and 99.9% against S. aureus. The test report can be found in the Supporting Information. These data confirm that the composite has a potent inhibitory effect on the aforementioned common pathogenic bacteria.

It is hypothesized that the antibacterial efficacy of the material is closely linked to the sustained release behavior of zinc ions. The concentration of zinc ions released in the material extract was quantitatively assessed by using inductively coupled plasma spectroscopy (ICP) (Figure S9). The results indicated that the zinc ion concentrations released from CeO₂–ZnO and CeO₂–ZnO/Pl were 216.5 and 784.0 μg/L, respectively. These data align closely with the antibacterial experimental results obtained through the plate counting method, suggesting that a higher concentration of zinc ion release correlates with enhanced antibacterial performance.

Oxidative stress is a trigger for various pathological processes, primarily due to the excessive accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). These highly reactive molecules can oxidize critical cellular components, including proteins and lipids, and induce DNA damage, ultimately resulting in cellular dysfunction or apoptosis. In recent years, cerium (Ce)-containing nanomaterials have attracted considerable attention for their unique antioxidant properties. These materials can efficiently catalyze the decomposition of ROS through the reversible valence transition between Ce³⁺ and Ce⁴⁺, thereby effectively alleviating oxidative stress. To systematically evaluate the antioxidant performance of the CeO₂–ZnO/Pl composite, this study quantitatively characterized its catalytic ability to decompose H₂O₂ by monitoring the O₂ generation (Figure f). The experimental results demonstrated that all sample groups exhibited a gradual increase in O₂ release over time following their reaction with H₂O₂ (Figure g). Notably, the catalytic performance of the composite was enhanced upon the incorporation of CeO₂–ZnO onto phytoliths, with the rate of the generation of the O₂ showing a positive correlation to the loading amount of CeO₂–ZnO (Figure h). This observation confirms that the CeO₂–ZnO/Pl composite possesses an antioxidant capacity. Though visual observation of bubble formation demonstrates that the CeO₂ component plays a key role in oxidation resistance (Figure S10), it was primarily attributed to the valence state cycling of cerium ions within the CeO₂ nanoparticles. Furthermore, the phytolith carrier provides a support for the active component, which collectively facilitates the efficient decomposition of H₂O₂. Furthermore, we evaluated the antioxidant capacity of the CeO₂–ZnO/Pl composite through in vitro antioxidant activity assays. The results indicate that the CeO₂–ZnO/Pl composite efficiently eliminates reactive oxygen species, such as H₂O₂, thereby protecting cells from oxidative damage (Figure S11).

Scalable Production and Engineering Applications of the CeO₂–ZnO/Pl Composite

The scalable and controllable preparation of functional materials is a crucial prerequisite and key step for their engineering applications. To this end, based on the preliminary small-scale process, this study successfully achieved the pilot-scale batch production of the CeO₂–ZnO/Pl composite. Each batch consistently yielded 372.4 g of the composite, with the overall process demonstrating stability and operational control, as illustrated in Figure a. A total of three batches were completed, resulting in 1091.3 g of the CeO₂–ZnO/Pl composite (Figure S12), thereby providing a sufficient material foundation for subsequent performance evaluation and the development of hemostatic gauze products.

3.

(a) Pilot-scale preparation process of CeO₂–ZnO/Pl. (b) XRD patterns and (c) elemental composition of the samples from lab and pilot batches. (d) SEM images of the CeO₂–ZnO/Pl gauze. (f) Absorption capacity of the gauzes in water and whole blood. (g) In vitro BCI at different time points (right: digital image after 120 s). (e) Viability of BJ cells after 24 h of exposure to the extracts of the gauzes.

To verify the stability of the batch preparation process and the consistency of the products, a systematic comparative analysis of the phase structure and chemical composition was conducted between the pilot-scale products and small-scale samples. The XRD patterns (Figure b) revealed that the phase composition of the pilot-scale products was highly consistent with that of the small-scale samples, with no diffraction peaks from impurities observed. This finding indicates that no impurities were introduced during the batch preparation process. Furthermore, the results of the chemical composition analysis demonstrated that the contents of key elements, such as Zn, Ce, and Si, in the pilot-scale products were in good agreement with those in the small-scale samples (Figure c), suggesting that the process exhibited good reproducibility and stability during scale-up.

In terms of product transformation, phytoliths and the CeO₂–ZnO/Pl composite were further applied to medical gauze to create hemostatic gauzes. SEM and EDS results demonstrated that the phytoliths and CeO₂–ZnO/Pl composite were effectively loaded onto the surfaces of the fibers (Figure d and Figures S13 and S14). Thermogravimetric analysis (TGA) revealed that the loading amount of the CeO₂–ZnO/Pl composite on the gauze was approximately 2.1% (Figure S15). To verify the biosafety of the hemostatic gauze, BJ cells were cocultured with different concentrations of gauze extract for 24 h to assess cytotoxicity. The results demonstrated that within the concentration range of 0.2–1.0 mg/mL for the extracts, the cell survival rate remained above 80% (Figure e), surpassing the basic requirement of a cell survival rate greater than 75%. This indicates that both phytolith gauze and CeO₂–ZnO/Pl gauze possess good biocompatibility. These hemostatic gauzes enhance the coagulation reaction by absorbing water molecules from the blood, thereby concentrating platelets, red blood cells, and coagulation factors. In terms of liquid absorption performance, both phytolith gauze and CeO₂–ZnO/Pl gauze demonstrated exceptional liquid absorption capabilities (Figure f). After immersion in ultrapure water for 5 min, the water absorption rates of both materials surpassed 600%, indicating that the mass of the absorbed liquid exceeded six times their own weight. In anticoagulated whole blood, the blood absorption rate of phytolith gauze was 800%, whereas that of CeO₂–ZnO/Pl gauze reached 1000%. This finding suggests that the composite exhibits an obviously enhanced liquid absorption capability in a blood, which is advantageous for promoting coagulation. Further in vitro coagulation assays were conducted to evaluate the hemostatic properties of the gauze (Figure g). As the incubation time increased, the coagulation indices for both gauze groups consistently decreased. At 120 s, the coagulation indices for phytolith gauze and CeO₂–ZnO/Pl gauze dropped to 37% and 25%, respectively, indicating that both types effectively accelerated blood coagulation and exhibited superior overall hemostatic performance.

Assessment of Hemostatic Performance of the CeO₂–ZnO/Pl Gauze

To evaluate the hemostatic performance of phytolith gauze and CeO₂–ZnO/Pl gauze in a realistic wound environment, a mouse liver injury bleeding model was established using commercial zeolite hemostatic gauze as the positive control. Hemostasis time and blood loss were set as the core evaluation indicators, allowing for a comprehensive comparison of the in vivo hemostatic effects of various materials. Figure a illustrates the establishment process of the mouse liver injury bleeding model and the complete procedural schematic for the gauze hemostasis operation. The images depicting the typical hemostasis process reveal that all gauze groups effectively controlled bleeding compared with the untreated blank group (Figure b), demonstrating obvious hemostatic effects. In terms of quantitative metrics, CeO₂–ZnO/Pl gauze exhibited optimal hemostatic efficiency, achieving a hemostasis time of 37.07 ± 3.18 s, which was shorter than that of phytolith gauze (51.93 ± 5.07 s, p < 0.001) and comparable to that of the commercial zeolite hemostatic gauze (36.81 ± 4.26 s, Figure c). Regarding blood loss (Figure d), the CeO₂–ZnO/Pl gauze group recorded a blood loss of only 86.50 ± 7.24 mg, which is lower than the 101.41 ± 7.27 mg recorded by the phytolith gauze group (p < 0.05), indicating a superior blood retention capability. The in vivo experimental results further demonstrate that the CeO₂–ZnO/Pl composite effectively accelerates the coagulation process and reduces blood loss in real biological environments. Its overall hemostatic performance is superior to that of pure phytolith gauze and is comparable to existing commercial products.

4.

(a) Schematic illustration of the experimental setup for liver hemorrhage. (b) Representative photographs capturing the hemostatic process. Quantitative analysis of the (c) hemostatic time and (d) blood loss. Data are presented as the mean ± SD (n = 6). Statistical significance was determined by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; NS, not significant.

Conclusions

This study presents significant advancements in the functionalization and scalable production of natural inorganic nanomaterials for biomedical and engineering applications. By innovatively employing natural phytoliths as structural carriers, a multifunctional CeO₂–ZnO/Pl composite was constructed through interfacial assembly strategies. The composite synergistically integrates hemostatic, antibacterial, and antioxidant activities: it accelerates coagulation by rapidly concentrating clotting components through water absorption; exhibits potent antibacterial action with 99.6% and 99.9% inhibition against E. coli and S. aureus, respectively, as confirmed by CNAS certification; and scavenges H₂O₂ via reversible Ce³⁺/Ce⁴⁺ valence cycling. A scalable manufacturing process was established, yielding 372.4 g per batch with high reproducibility in phase and chemical composition. The composite-based gauze demonstrates excellent biocompatibility, a blood absorption capacity of 1000%, and obviously enhanced hemostatic performance. In a murine liver injury model, the composite exhibited efficacy comparable to similar commercial hemostats, highlighting its strong potential for clinical application.

Experimental Section

Chemicals and Instrumentations

Zinc chloride (ZnCl₂), anhydrous cerium chloride (CeCl₃), sodium carbonate (Na₂CO₃), sodium chloride (NaCl), and calcium chloride (CaCl₂) were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Polyvinylpyrrolidone (PVP) and hydrogen peroxide (H₂O₂) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). The phytoliths were collected from the Shilukeng area of Fengcheng City, Jiangxi Province, and provided by the Jiangxi Province Natural Resources Interests and Reserve Security Center. All purchased reagents were utilized directly without further purification.

The microstructure and elemental distribution of the prepared materials were examined by using a Czech TESCAN MIRA LMS scanning electron microscope and a Japanese JEOL JEM-2100F transmission electron microscope. X-ray photoelectron spectroscopy (XPS) data were collected with an American Thermo Scientific ESCALAB Xi+, utilizing the C 1s peak at a binding energy of 284.80 eV as the reference. X-ray diffraction (XRD) patterns were obtained using a Bruker D8 Advance 25 with Cu Kα radiation (λ = 0.15 nm) at a scanning rate of 5°/min over a 2θ range of 10–80°. Thermal and mass change information during heating was acquired by using a Netzsch STA449F5 synchronous thermal analyzer (DSC/TG). X-ray fluorescence (XRF) signals were obtained using a SHIMADZU XRF-1800 instrument to analyze changes in elemental content. The dissolved ion concentration of the material was analyzed with an inductively coupled plasma (ICP) spectrometer, model Agilent 5110 (OES) from the USA. N₂ adsorption–desorption isotherms were obtained using a Micromeritics Tristar3030 physical adsorption instrument to analyze the specific surface area and pore structure characteristics of the material. The bonding forms of Si atoms in the material were analyzed by using a German Bruker 400M nuclear magnetic resonance (NMR) spectrometer. The amount of oxygen produced during the experiment was measured with an AZ8403 dissolved oxygen meter. The absorbance values of the test liquid were detected by using a microplate reader (Tecan Spark, Switzerland). The cell morphology and fluorescence intensity were observed using a Nikon C2Si laser confocal microscope.

Preparation of the CeO₂–ZnO/Pl Composite

The CeO₂–ZnO/Pl composite was synthesized by using a chemical ball milling method. The phytolith was calcined at 700 °C to remove a portion of the organic carbon, with the samples before and after calcination designated as Pl-1 and Pl-2, respectively. The synthesis procedure for CeO₂–ZnO/Pl with varying loading amounts was as follows: ZnCl₂ and anhydrous CeCl₃ were predried in an oven at 120 °C for 12 h. Subsequently, 2.33 g of ZnCl₂, 0.47 g of anhydrous CeCl₃, 2.50 g of Na₂CO₃, 2.24 g of NaCl, and varying amounts of calcined phytoliths were combined and ball-milled for 30 min. The resulting product was calcined at 200 °C for 1 h at a heating rate of 5 °C/min. Following calcination, the product was centrifuged and washed with deionized water five times and then dried at 60 °C and ground into a powder for subsequent use. The calcined phytolith was added in masses of 12.51, 5.56, 2.09, and 0.93 g, with the corresponding composite materials labeled as (2), (3), (4), and (5), respectively. For comparison, Pl (only phytolith added, labeled as (1)) and CeO₂–ZnO (no phytolith added, labeled as (6)) were prepared using the same method described above.

Preparation of CeO₂–ZnO/Pl Gauze

The impregnation solution was prepared by combining the loading material with polyvinylpyrrolidone (PVP) at a mass ratio of 2:1, utilizing ultrapure water as the solvent, resulting in a material concentration of 6.00 g/L. This solution was subjected to ultrasonic dispersion for 30 min. Subsequently, the impregnation solution was transferred into a water tank, where the nonwoven fabric was impregnated using rollers under airflow agitation. Finally, the material-loaded gauze was laid flat and allowed to air-dry naturally. The gauzes prepared using PVP and CeO₂–ZnO/PVP were designated as PVP gauze and CeO₂–ZnO/PVP gauze, respectively.

In Vitro Coagulation Assay

For powdered materials, a 10.00 mg amount of various samples was accurately weighed into 2.00 mL centrifuge tubes. We added 100.00 μL of anticoagulated whole blood, followed immediately by the addition of 10.00 μL of 0.10 mol/L CaCl₂ solution to initiate coagulation. We incubated the mixture in a constant-temperature water bath at 37 °C for 5 min and then slowly added 1 mL of deionized water. We centrifuged the incubated mixture at 3000 rpm for 5 min, collected the supernatant, and measured the absorbance at 540 nm by using a microplate reader. For gauze samples, we placed 1 cm × 1 cm pieces of gauze of equal size into the wells of a microplate. We added 50.00 μL of anticoagulated whole blood onto the gauze surface, followed by 5.00 μL of 0.10 mol/L CaCl₂ solution to initiate coagulation. As a blank control group, 50.00 μL of whole blood was added directly into empty wells. We incubated the plate at 37 °C for 30, 60, 90, and 120 s and then slowly added 3 mL of deionized water. We continued incubation for 1 min to release hemoglobin from uncoagulated red blood cells. We aspirated 100.00 μL of the supernatant and measured the absorbance at 540 nm using a microplate reader. Each group should include three replicates. The blood clotting index (BCI) was calculated using the following formula:

$$\mathrm{BCI}(\%)=\frac{{\mathrm{OD}}_{\mathrm{sample}}}{{\mathrm{OD}}_{\mathrm{control}}}\times 100\%$$ 1

Hemolysis Test

Anticoagulated whole blood was subjected to centrifugation to remove serum, followed by washing three times with phosphate-buffered saline (PBS) to isolate pure red blood cells. The isolated red blood cells were then diluted with PBS to create a 4% (v/v) red blood cell suspension. Different powdered materials (20.00 mg each) were weighed and mixed with 2.00 mL of the prepared 4% red blood cell suspension and 1.00 mL of PBS, and the mixtures were subsequently incubated in a 37 °C water bath for 3 h. Both negative and positive control groups were established. In the negative control group (no hemolysis), 1.00 mL of PBS was combined with 2.00 mL of the prepared 4% red blood cell suspension. Conversely, in the positive control group (hemolysis occurred), 1 mL of 1% Triton-X-100 was mixed with 2.00 mL of the prepared 4% red blood cell suspension. Three parallel experiments were conducted for each sample. Following the incubation, the mixed system was centrifuged at 3000 rpm for 5 min, after which the supernatant was collected, and the absorbance was measured at a wavelength of 540 nm using a microplate reader. The hemolysis rate was calculated as follows:

$$\mathrm{hemolysis}\mathrm{rate}(100\%)=\frac{{\mathrm{OD}}_{\mathrm{sample}}-{\mathrm{OD}}_{\mathrm{negative}}}{{\mathrm{OD}}_{\mathrm{positive}}-{\mathrm{OD}}_{\mathrm{negative}}}\times 100\%$$ 2

Cytotoxicity Assay

The complete medium for culturing BJ cells was prepared using RPMI 1640 basal medium supplemented with 1% double antibiotics and 10% fetal bovine serum. Cryopreserved BJ cells were thawed by shaking at 37 °C, and 1.00 mL of BJ cell cryopreservation solution was transferred into 10.00 mL of the complete medium. After thorough mixing, the solution was centrifuged at 1300 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in a complete medium. The cell suspension was then transferred to a culture dish and incubated for 12–24 h to facilitate cell adhesion. Subsequently, trypsin was used to digest the cells, detaching them from the adherent state for resuspension. The density of the cell suspension was adjusted, and the cells were seeded into a 96-well plate at a density of 1 × 10⁴ cells per well. The plate was incubated at 37 °C with 5% CO₂ in a cell culture incubator for 4 h until optimal adherent growth was achieved. The original culture medium was discarded, and 100.00 μL of fresh complete medium was added to the control wells. For powdered materials, 100.00 μL of sample culture medium at varying concentrations was added to the test wells. For gauze, 100.00 μL of extract from the gauze, filtered through a 0.45 μm syringe filter at different concentrations, was added to the experimental wells. After continuing cultivation for 24 h postmaterial addition, cytotoxicity was measured as follows: 10% CCK-8 reagent-containing complete medium was added to each well, followed by a 1 h incubation. The absorbance of the culture medium at 450 nm was measured using a microplate reader with wells without material addition serving as blank controls. The cell survival rate was calculated based on absorbance measurements using the following formula:

$$\mathrm{cell}\mathrm{viability}(100\%)=\frac{{\mathrm{OD}}_{\mathrm{sample}}-{\mathrm{OD}}_{\mathrm{blank}}}{{\mathrm{OD}}_{\mathrm{control}}-{\mathrm{OD}}_{\mathrm{blank}}}\times 100\%$$ 3

H₂O₂ Decomposition Experiments

We prepared different sample solutions and ultrasonically dispersed them for 10 min. We added 5.00 mL of H₂O₂ solution (final concentration of 60.00 mol/L) and 1.00 mL of various sample suspensions (final concentration of 1.00 mg/mL) into a six-well plate. The calibrated dissolved oxygen meter was immersed below the liquid surface. After the reaction was allowed to stabilize for 4 min, counting was begun and measurements were recorded every 2 min for a total of 8 recordings. We weighed 10 mg of different materials into 1.5 mL of H₂O and ultrasonically dispersed them for 10 min. Then, we added 0.5 mL of 30% H₂O₂ solution, let the reaction stand for 10 min, observed the bubble generation, and photographed it for documentation.

ROS Scavenging in RAW 264.7 Macrophages

The reactive oxygen species (ROS) content was measured using the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to analyze the ROS scavenging capacity of different materials. The procedure is as follows: RAW 264.7 macrophages were seeded at a density of 1 × 10⁵ cells per well in a 6-well plate and cultured for 24 h for attachment. Cells were treated with 1 mL of 20 mg/mL CeO₂–ZnO/Pl suspension and 1 mL of 200 μM H₂O₂ solution (diluted with RPMI 1640 medium), followed by continued culture for 12 h. After removing the spent medium, we added 1 mL of DCFH-DA (at a dilution ratio of 1:2000) and incubated it for an additional 20 min. Following incubation, we washed away excess DCFH-DA with PBS and observed cell images and fluorescence intensity under a laser confocal microscope.

Antibacterial Experiments

Using Escherichia coli (ATCC-25922) and Staphylococcus aureus (ATCC-25923) as test strains, with the no-sample group serving as the control, a single colony from the plate streak was inoculated into 10.00 mL of LB liquid medium and cultured at 37 °C in a constant-temperature shaker for 13 h. The bacterial suspension was then diluted sequentially three times (1000-fold), and 100.00 μL of this suspension was added to 10.00 mL of liquid medium. Subsequently, 10 mL of different samples was introduced for coincubation for 4 h, followed by another three dilution steps. Finally, 50.00 μL of the mixture was spread evenly on the surface of UV-sterilized LB solid medium in three directions for 2 min each, with three plates prepared per sample. The plates were then incubated at 37 °C for 12 h, after which image acquisition and counting were performed by using a fully automated gel imaging analysis system to calculate the antibacterial rate.

Liquid Absorption Performance Test

The specific gravity method was employed to determine the liquid absorption rate of the gauze. The liquid absorption capacity of the gauze was tested using ultrapure water and anticoagulant whole blood. The gauze was cut into 1 cm × 1 cm pieces and weighed, denoted as W ₀. The gauze pieces were then immersed separately in ultrapure water and whole blood. After 5 min, the gauze was removed, and any surface liquid was absorbed using filter paper before being weighed again, recorded as W ₁. Three parallel groups were established for each gauze sample. The liquid absorption rate (A) was calculated using the following formula:

$$A(\%)=\left(\frac{W_1-W_0}{W_0}\right)\times 100\%$$ 4

In Vivo Hemostatic Performance

A mouse liver injury bleeding model was established to evaluate the hemostatic efficacy of the gauze with commercially available zeolite hemostatic gauze serving as a control. Hemostasis time and blood loss mass were utilized as evaluation indicators. The mice used in the experiment were provided by Wuhan Myhalic Biotechnology Co., Ltd. (Laboratory Animal Production License No. SCXK­(E)­2023-0033 and Laboratory Animal Use License No. SYXK­(E)­2023-0104). The animal subjects consisted of female BALB/c mice, aged 8–9 weeks, with a weight range of 20–25 g. The specific experimental procedure was as follows: First, the mice were anesthetized via intramuscular injection of 50.00 mg/mL of chloral hydrate. Each mouse was positioned supine with its limbs secured to a fixation board using rubber bands. The abdominal skin was disinfected sequentially with iodophor and 75% ethanol. An incision was made in the abdominal epithelial tissue to expose the liver, and filter paper was placed beneath the liver to absorb excess tissue fluid. A surgical scalpel was employed to create a linear incision wound approximately 1 cm in length and 0.5 cm in depth on the right upper lobe of the liver. Experimental gauze was subsequently applied to cover the liver wounds, and bleeding was monitored. Timing was halted when the bleeding ceased. The gauze was weighed before and after hemostasis to document the hemostatic time and blood loss. This animal experiment received approval from the Animal Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd., with Animal Welfare and Ethical Review Approval Number HLK-20250716–001 (see the Supporting Information).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12255.

• TEM image of Pl and its corresponding elemental mapping; XRD patterns, SEM images, and N₂ adsorption–desorption isotherms of Pl-1 and Pl-2; SEM and TEM images of CeO₂–ZnO and its corresponding elemental mapping; XRD patterns and photographs of flat-bed coating of CeO₂/Pl and ZnO/Pl; photographs of flat-bed coating in different groups; release concentration of Zn²⁺ in suspensions of CeO₂–ZnO/Pl and CeO₂–ZnO; pictures of bubble formation in the reaction with H₂O₂; ROS staining of RAW 264.7 macrophages using DCFH-DA under different treatments; three-batch preparation of product photograph; photographs of Pl gauze samples and corresponding SEM images; SEM images of Pl gauze and CeO₂–ZnO/Pl gauze and their corresponding elemental mapping diagrams; TG curve of CeO₂–ZnO/Pl gauze (PDF)

Linyun Lu synthesized and characterized the materials and wrote the initial drafts of the paper. Xi Liu and Qing Zhang analyzed the data and carried out the formal analysis. Tengyun Zuo and Feng Liu provided mineral samples and conducted analyses on them. Hao Wang designed the experiments and wrote the final paper. Huaming Yang designed and conceived the project. All authors discussed the results and commented on the manuscript.

The authors declare no competing financial interest.