X03 phage-cerium oxide nanoszyme composite therapeutic platform based on chemical cross-linking of polyethylene glycol

Abstract

The application belongs to the field of nanomaterials and biomedicine, and provides a X03 phage-cerium oxide nanoscale enzyme composite treatment platform based on chemical cross-linking of polyethylene glycol, a synergistic treatment platform integrating bactericidal and detoxification functions, namely X03@CeO2, is constructed by covalently coupling phage X03 targeting pseudomonas aeruginosa and CeO2 nanoscale enzyme with phosphatase activity, which solves the problem that antibiotic and phage therapy exacerbate systemic inflammatory storm due to massive release of lipopolysaccharide caused by bacterial lysis when killing multiple drug-resistant gram-negative bacteria. In vitro and in vivo experiments show that the complex can efficiently remove bacteria and biofilm, simultaneously degrade released LPS, significantly inhibit the production of inflammatory factors, active oxygen burst and TLR4 pathway activation, greatly improve the survival rate in a lethal sepsis mouse model and reverse multiple organ damage. The application is suitable for treating sepsis caused by drug-resistant gram-negative bacteria.

Description

Technical Field

[0001] This invention belongs to the fields of nanomaterials and biomedicine, and relates to a complex for treating inflammatory diseases, specifically to an X03 phage-cerium oxide nanoenzyme composite therapeutic platform based on polyethylene glycol chemical cross-linking. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by an abnormal host response to infection. Gram-negative bacterial infection is one of the main causes of sepsis, with the core pathogenic factor being the lipopolysaccharide (LPS, or endotoxin) released by bacterial lysis. LPS activates the Toll-like receptor 4 (TLR4) signaling pathway, triggering an excessive inflammatory cascade and oxidative stress, ultimately leading to multiple organ dysfunction syndrome (MODS), a key pathological mechanism contributing to the high mortality rate of sepsis.

[0003] Currently, standard treatment for sepsis relies on antibiotics. However, antibiotic therapy has two inherent drawbacks: first, the rapid evolution of bacterial resistance severely weakens the efficacy of antibiotics; second, while antibiotics kill bacteria, they also cause bacterial lysis, releasing large amounts of LPS and triggering treatment-associated toxin release (TAS). This antibacterial process itself can become a "second blow," exacerbating the systemic inflammatory response, thereby limiting treatment effectiveness and even accelerating disease progression. Therefore, developing a novel treatment strategy that can simultaneously achieve highly effective antibacterial action and immediate endotoxin detoxification has become an urgent need to overcome the current bottlenecks in sepsis treatment.

[0004] Phage therapy, due to its high strain specificity, self-replication ability, and low likelihood of inducing classical drug resistance, is considered a promising alternative to combating drug-resistant bacterial infections. However, its systemic application, similar to antibiotics, involves the release of LPS (endotoxins) along with the lysis of bacteria, posing a risk of exacerbating inflammation. Secondly, phage preparations themselves struggle to completely remove endotoxins. Existing endotoxin removal processes (such as ultrafiltration, affinity chromatography, and density gradient centrifugation) are typically complex, time-consuming, costly, and prone to phage titer loss. Consequently, the endotoxin levels of most phage preparations far exceed the safe threshold for intravenous injection (5 EU / mL), leading regulatory agencies to strictly limit systemic administration and severely hindering the clinical application of phage therapy in treating systemic infections such as sepsis.

[0005] Cerium oxide (CeO2) nanozymes exhibit unique advantages in addressing the need for LPS detoxification. Possessing phosphatase-like activity, they can specifically hydrolyze the phosphate groups of the lipid A moiety, the core of LPS toxicity, thereby directly neutralizing endotoxin toxicity. Unlike traditional adsorption-based removal methods, CeO2 nanozymes achieve complete LPS detoxification through catalytic degradation and are stable, making them suitable for functioning in complex in vivo environments.

[0006] In summary, the current treatment of sepsis urgently requires novel antibacterial methods that can overcome drug resistance and avoid the "toxin release" effect; however, promising phage therapy is hampered by its own endotoxin safety and insufficient in vivo detoxification capacity. The simple approach of "killing bacteria first, then detoxifying" or relying on complex in vitro purification methods is no longer sufficient to meet treatment needs. Therefore, developing an innovative platform that organically integrates precise antibacterial and immediate in-situ detoxification functions, achieving temporal and spatial synchronization and synergy of "killing bacteria and detoxifying," is key to fundamentally improving the efficacy of sepsis treatment and promoting the clinical translation of systemic phage therapy. Summary of the Invention

[0007] The purpose of this invention is to provide a polyethylene glycol-based XO3 phage-cerium oxide nanozyme composite therapeutic platform. By covalently coupling XO3 phage with specific bactericidal function with CeO2 nanozyme with efficient endotoxin degradation function, a synergistic therapeutic system integrating "targeted antibacterial" and "in-situ detoxification" functions is constructed. This addresses the problems in existing sepsis treatment where bacterial lysis leads to the release of large amounts of endotoxins, exacerbating systemic inflammation, and the high endotoxin content of existing phage preparations, making them unsafe for systemic treatment.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: an X03 phage-cerium oxide nanozyme complex based on polyethylene glycol chemical cross-linking, wherein the complex is X03@CeO2.

[0009] Preferably, the linker for CeO2 and XO3 in the composite is one of HS-PEG-NHS, NHS-PEG-COOH, and EDC-NHS, and the ratio of CeO2, linker, and XO3 is CeO2:linker:XO3 = 2 mg: 1 mg: 2 × 10⁻⁶ mg. 11 PFU.

[0010] Preferably, the binder is NHS-PEG-COOH.

[0011] The present invention further provides a method for preparing the above-mentioned complex.

[0012] Preferably, the steps include:

[0013] S1. Preparation of CeO2 nanozymes via hydrothermal method;

[0014] Coupling of S2.CeO2@PEG: NHS-PEG-COOH and CeO2 were mixed at a volume ratio of 1:1 for 30 min to form CeO2@PEG;

[0015] Preparation of S3.XO3@CeO2: In an ice-water bath, CeO2@PEG was mixed with 1×10 10 PFU / mL screened X03 was mixed for 2 hours to form X03@CeO2; then X03@CeO2 was salted out with 25% PEG-6k to remove unbound CeO2@PEG, and finally resuspended in 100 μL PBS.

[0016] Preferably, the CeO2 nanozyme prepared in step S1 has an average particle size of 2.80 ± 0.86 nm, and the CeO2 nanozyme contains Ce 3+ and Ce 4+ They accounted for 38.26% and 61.74% of the total Ce, respectively, with an oxygen vacancy concentration of 47.89%.

[0017] The present invention further provides applications of the above-described complex.

[0018] Preferably, the complex is used to prepare a product for treating sepsis caused by multidrug-resistant Gram-negative bacteria, and a limited component of the product includes XO3@CeO2.

[0019] Preferably, CeO2 in the complex is a detoxification unit and XO3 is a bactericidal unit; when the bactericidal unit lyses bacteria, its covalently linked detoxification unit can simultaneously degrade the lipopolysaccharide released by the lysis, i.e., LPS, thereby blocking the activation of the TLR4-NF-κB inflammatory signaling pathway mediated by LPS at the source.

[0020] Preferably, the product achieves a 99.99% sterilization rate against Pseudomonas aeruginosa within two hours, wherein the concentration of Pseudomonas aeruginosa is 1×10⁻⁶. 9 CFU / mL.

[0021] Preferably, the product can increase the survival rate of mouse models infected with a lethal dose of Pseudomonas aeruginosa to over 85% when treating sepsis.

[0022] Preferably, after application, the product can reduce the plasma LPS residue rate in sepsis model animals to below 3%.

[0023] The beneficial effects of this invention are:

[0024] 1. Spatiotemporal synchronization of sterilization and detoxification: By covalently coupling CeO2 nanozymes to X03 bacteriophages, an integrated "sterilization-detoxification" platform is constructed, which achieves in-situ degradation at the moment when the bacteriophage lyses the bacteria and releases endotoxin (LPS), solving the problem of spatiotemporal mismatch between antibacterial and anti-endotoxin effects in traditional treatments.

[0025] 2. Highly efficient endotoxin clearance: The CeO2 nanozyme in the complex can specifically hydrolyze the OP bond of the lipid A moiety of LPS. In in vitro experiments, the plasma LPS clearance rate reached 97.4%, which is significantly better than the physical mixing group (only 46.2%), thus blocking the endotoxin toxicity at the source.

[0026] 3. Excellent antibacterial properties: Effective against high concentrations of Pseudomonas aeruginosa (1×10⁻⁶). 9 The sterilization rate of CFU / mL reached 99.99% within 2 hours, which is comparable to that of free bacteriophages, proving that the chemical coupling process did not impair the natural infection and lysis capabilities of the bacteriophages.

[0027] 4. Synergistic biofilm removal effect: 24-hour treatment can achieve a biofilm removal rate of 63.76%, which is significantly higher than that of the single component (38.24% for the phage group and 12.57% for the CeO2 group), which is attributed to the synergistic effect of the efficient lysis of bacteria by phage and the degradation of LPS by nanozymes. Attached Figure Description

[0028] Figure 1 This invention describes the characterization, catalytic performance, and coupling verification of X03@CeO2 (A is a TEM image of cerium oxide, B is a TEM image of X03 bacteriophage, C is a TEM image of X03@CeO2, D is the diameter distribution of CeO2, E is the XPS image of CeO2, F is the XRD image of CeO2, G is the LPS scavenging efficiency of CeO2, CeO2@PEG, and X03@CeO2, H is the degradation of LPS by different concentrations of CeO2 at different incubation time points, and I is the potential effect of NHS-PEG-COOH on X03).

[0029] Figure 2 This invention describes the characterization, catalytic performance, and coupling verification of XO3@CeO2 (A is the XPS spectrum of cerium, B is the XPS spectrum of oxygen, C is the Zeta potential of CeO2, D is the effect of CeO2 on the potential of XO3, E is the effect of different linking molecules on LPS scavenging activity, F is the effect of different contents of NHS-PEG-COOH on CeO2 enzyme activity, and G is the effect of different ratios of CeO2 to NHS-PEG-COOH on the grafting rate).

[0030] Figure 3 This is an explanation of the cerium oxide bond breaking mechanism in this invention (A is a schematic diagram before LPS degradation, and B is a schematic diagram after LPS degradation).

[0031] Figure 4This invention evaluates the in vitro inhibitory effect of X03@CeO2 on LPS-induced inflammatory response (A is the ELISA result of TNF-α production after 24 hours of stimulation with different concentrations of LPS (n=2), B is the ELISA result of IL-6 production after 24 hours of stimulation with different concentrations of LPS (n=3), C is the effect of 24 hours of stimulation with different concentrations of LPS on THP-1 cell viability (n=4), and D is the average fluorescence intensity of reactive oxygen species (ROS)).

[0032] Figure 5 This invention presents the in vitro antibacterial activity of X03@CeO2 (A represents the bactericidal rate of TOB, CeO2@PEG, X03, X03+CeO2 and X03@CeO2 against Pseudomonas aeruginosa after 2 hours of culture (n=3); B represents the quantitative analysis of biofilm biomass at 595 nm UV absorption using the crystal violet method (n=3); C represents the effect of different treatments on the scavenging efficiency of lipopolysaccharide released by Pseudomonas aeruginosa (n=3); D represents representative images of bacterial viable staining by different components; and E represents scanning electron microscopy images of Pseudomonas aeruginosa biofilms after 12 hours of different treatments).

[0033] Figure 6 This invention evaluates the antioxidant and anti-inflammatory effects of X03@CeO2 on LPS-induced THP-1 cells (A: protective effect of different treatments on THP-1 cells exposed to 800 ng / mL (n=3); B: therapeutic effect of X03@CeO2 on THP-1 cells exposed to 800 ng / mL LPS (n=5); C: TNF-α expression detected by ELISA 24 hours after different treatments; D: IL-6 expression detected by ELISA 24 hours after different treatments; E: representative results of ROS level images; F: representative immunofluorescence images of TLR4 expression in THP-1 cells).

[0034] Figure 7 This invention describes the biosafety of X03@CeO2 (A represents hemolysis caused by different concentrations of X03@CeO2, B represents the biosafety of different concentrations of X03@CeO2 on THP-1 cells, CJ represents routine blood tests and biochemical indicators on the seventh day after administration: where C represents creatinine, D represents aspartate aminotransferase / alanine, E represents blood urea nitrogen, F represents white blood cells, G represents lymphocytes, H represents neutrophils, I represents platelets, J represents red blood cells, and K represents H&E staining of the heart, liver, spleen, lungs, and kidneys on the seventh day after administration).

[0035] Figure 8This invention relates to the application of X03@CeO2 in the treatment of severe sepsis in mice (A is a schematic diagram of the animal experimental design for testing the therapeutic effect of X03@CeO2 in a sepsis mouse model; B is the 24-hour mouse survival rate (n=3); C is the clearance effect of LPS in mice (n=3); D is the clearance effect of bacteria in mice (n=3); E is the change of TNF-α in mice after different treatments (n=3); F is the change of IL-6 in mice after different treatments (n=3); G is H&E staining of heart, liver, spleen, and lung tissues).

[0036] Figure 9 These are the blood routine and blood biochemical index test results of mice in different treatment groups in this invention (A is the white blood cell count in different treatment groups, B is the neutrophil count in different treatment groups, C is the lymphocyte count in different treatment groups, D is the platelet count in different treatment groups, E is the percentage of neutrophils in different treatment groups, F is the percentage of lymphocytes in different treatment groups, G is the concentration of aspartate aminotransferase in different treatment groups, and H is the concentration of alanine aminotransferase in different treatment groups).

[0037] Figure 10 These are the physiological indicators and blood biochemical test results of mice in different treatment groups in this invention (A is the change in body weight of mice in different treatment groups within 24 hours, B is the change in body temperature of mice in different treatment groups within 24 hours, C is the creatinine concentration under different treatment methods, and D is the blood urea nitrogen concentration under different treatment methods). Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] Example 1: Preparation and performance testing of CeO2 nanozymes

[0040] 1. Preparation

[0041] A CeO2 nanozyme with high lipopolysaccharide degradation efficiency was synthesized via a hydrothermal method. The specific steps were as follows: 0.86 M Ce(NH4)2(NO3)6 was dissolved in 4 mL of deionized water and placed in a 50 mL beaker. Then, 16 mL of 1,2-propanediamine was added, and the mixture was stirred for 10 minutes to form a homogeneous suspension. The suspension was then transferred to a three-necked flask and heated in an oil bath at 180 °C for 12 hours under aeration. The resulting sample was then washed three times with deionized water, centrifuged at 10,000 rpm for 5 minutes, and then dispersed in water. Subsequently, the sample was dialyzed overnight using a 3500 DA dialysis bag to remove other impurities that did not form ions. The liquid cerium oxide was then freeze-dried for 48 hours to obtain solid cerium oxide. The surface chemistry and crystal phase were evaluated using powder X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), respectively. The potential of CeO2 was determined using a Zetasizer in various dispersion media, including H2O, PBS, 10% FBS, and physiological saline. The morphology of CeO2 was observed using TEM. The size of CeO2 was analyzed using ImageJ software.

[0042] 2. Performance Testing

[0043] 2.1 Structural Characterization

[0044] Transmission electron microscopy (TEM) images show the morphology of CeO2, and ImageJ analysis indicates an average particle size of 2.80 ± 0.86 nm. Figure 1 China A Figure 1 (D).

[0045] The chemical valence state of cerium (Ce) was analyzed using X-ray photoelectron spectroscopy (XPS). 3+ / Ce 4+ Energy dispersive spectroscopy (EDS) analysis confirmed the presence of cerium, oxygen, and carbon by examining the ratios and surface oxygen species. Using the C 1s peak at approximately 285.3 eV as an internal standard, the O 1s peaks ranged from 530 to 533 eV, corresponding to different oxygen species. The significant peaks in the 880-920 eV range were characteristic of cerium 3d orbitals, confirming the presence of cerium. 4+ The dominant peak was observed. No other impurity peaks were detected, indicating high sample purity. Figure 1 Quantitative analysis showed that Ce 3+ and Ce 4+ They accounted for 38.26% and 61.74% of the total Ce, respectively. Figure 2 (A)

[0046] Four peaks were fitted from the O1s spectrum ( Figure 2 (B) Based on the results, the oxygen vacancy concentration was calculated to be 47.89%, calculated as follows: Ov (%) = [Ce (III) - O2- / (Ce(III)- O 2- +Ce(IV)- O 2- )]×100%.

[0047] X-ray diffraction (XRD) results show that, within the corresponding 2θ range, the diffraction peaks corresponding to the crystal planes (200), (220), (311), (331), (400), (420), and (222) can be clearly identified. Figure 1 (F), which is consistent with the typical diffraction behavior of cubic fluorite CeO2.

[0048] Zeta potential measurements showed that CeO2 exhibited different charges in different media: +5.04 mV in H2O, +1.40 mV in physiological saline, while -13.60 mV in fetal bovine serum (FBS), -4.79 mV in SBF, and -7.29 mV in phosphate-buffered saline (PBS). Figure 2 (C)

[0049] 2.2 Detection of LPS detoxification mechanism of CeO2 nanozyme

[0050] Spectroscopy was acquired using positive ion mode with DHB as the matrix (m / z 1400–2800). Figure 3 A significant peak at m / z 2189.559 was observed in the untreated LPS group. Figure 4 (A) corresponds to the LPS isotype containing three phosphate groups. This peak completely disappeared in the CeO2-treated group. Figure 4 The peak at m / z 1954.008 was observed, while a new peak appeared at m / z 1954.008. The mass difference (Δm / z ≈ 235.5 Da) was consistent with the theoretical value (3 × 80 Da), confirming the breaking of the PO bond. Other characteristic peaks (such as m / z 1365.603 and 1745.133) remained unchanged, indicating that CeO2 specifically catalyzed the dephosphorylation reaction without destroying the LPS core structure.

[0051] 2.3 Detection of the catalytic performance of CeO2 nanozymes

[0052] The LAL assay (LAL kit) was used to evaluate the LPS degradation activity of CeO2 at different concentrations and incubation times. The results showed that the percentage of residual LPS decreased in a concentration- and time-dependent manner. Figure 1 (G). Moreover, at a CeO2 concentration of 200 μg / mL, the residual LPS decreased to about 5% after 1 hour and almost 0% after 2 hours, indicating that efficient LPS inactivation was achieved through catalytic dephosphorylation.

[0053] 2.4 Coupling and Optimization of CeO2@PEG

[0054] The effects of three different linkers (HS-PEG-NHS, NHS-PEG-COOH, and EDC-NHS) on the CeO2 degradation activity of LPS were investigated. The results showed that the linker type had no significant negative impact on the activity, with NHS-PEG-COOH exhibiting the least interference. Figure 2 (E). Furthermore, the phosphatase-like activities of CeO2@PEG at different mass ratios (NHS-PEG-COOH: CeO2 = 100:0, 100:25, 100:50, 100:100) were tested. The results showed no significant difference in activity among the different mass ratios. Figure 2 (F), confirming that NHS-PEG-COOH is a suitable linker.

[0055] Coupling of CeO2@PEG: Different concentrations (0, 10, 25, 50, 100 μg / mL) of NHS-PEG-COOH were mixed with CeO2 (100 μg / mL) at a 1:1 volume ratio for 30 minutes to form CeO2@PEG. The highest binding efficiency (53.21%) was achieved when the mass ratio of CeO2 to NHS-PEG-COOH was 100:50. Figure 2 (G).

[0056] Example 2: Preparation and performance testing of XO3@CeO2

[0057] 1. X03 Filtering

[0058] Single colonies of PAO1 were inoculated into 20 mL of 2×LB medium and incubated until the bacterial culture reached an optical density of 0.3 at 600 nm (OD600 nm). Simultaneously, wastewater samples were centrifuged at 4 °C and 8000 rpm / min for 20 min. The supernatant was further filtered using a 0.22 μm filter. Then, 20 mL of treated water was added to the PAO1 culture, allowed to stand for 15 minutes, and then incubated overnight at 37 °C for enrichment. After incubation, the mixture was centrifuged and filtered. Then, 100 μL of serially diluted filtrate was separately mixed with 100 μL of PAO1 culture in logarithmic growth phase and added to 5 mL of LB medium containing 0.7% agar at 45 °C. After thorough mixing, the mixture was poured onto LB agar to form clear, individual plaques. Clear and well-defined plaques were selected. Each phage plaque needs to be co-incubated with PAO1 using the double agar plate method, and this process is repeated 3 times to obtain a series of purified phages, which are then morphologically characterized by TEM. Figure 1 (B)

[0059] 2. Preparation method of XO3@CeO2

[0060] In an ice-water bath, CeO2@PEG was reacted with a fixed concentration (1×10⁻⁶). 10 X03 (PFU / mL) was mixed with CeO2 for 2 hours to form X03@CeO2. Then, X03@CeO2 was salted out with 25% PEG-6k to remove unbound CeO2@PEG, and then resuspended in 100 μL PBS (pH 7.4). The concentration of Ce was quantitatively analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The morphology of X03@CeO2 was observed by TEM after negative staining with phosphotungstic acid.

[0061] Enzyme activity assays (CeO2, CeO2@NHS, and XO3@CeO2 containing the same concentrations of CeO2 (50, 100, 200 μg / mL), were mixed with 100 ng / mL LPS at a 1:1 ratio, and the enzyme activity after ligation was detected using the LAL method after 30 min) showed that neither linker modification nor phage conjugation significantly affected the LPS degradation activity of CeO2. Figure 1 In addition, in vitro experiments confirmed (using the plaque-forming unit assay to count the number of phages) that CeO2 and different concentrations of NHS-PEG-COOH had no significant inhibitory effect on the biological activity of phage XO3. Figure 1 Middle I, Figure 2 (D)

[0062] Based on these results, the optimal ratio for subsequent experiments was determined to be CeO2:NHS-PEG-COOH:XO3 = 2:1:2 (mg:mg: 10). 11 PFU). Transmission electron microscopy images confirmed the successful coupling of CeO2@PEG with XO3. Figure 1 (C)

[0063] 2. Performance Testing

[0064] 2.1 Antibacterial activity of XO3@CeO2

[0065] The antibacterial effect was evaluated using the plate count method. At high bacterial concentrations (1×10⁻⁶), the plate count was used. 9 Under conditions of CFU / mL, the single XO3 formulation, the physical mixture (XO3+CeO2), and the XO3@CeO2 composite formulation all exhibited excellent bactericidal efficiency, reducing the number of viable bacteria by 99.99% within 2 hours. In contrast, the clinical antibiotic tobramycin (TOB) showed only limited antibacterial effect at conventional concentrations (88.16% reduction in viable bacteria), a difference that was statistically significant compared to the other three formulations (P<0.01). The single CeO2 formulation showed only weak antibacterial activity under these conditions (24.68% reduction in viable bacteria). Figure 5 (A)

[0066] fluorescein diacetate (FDA) / propidium iodide (PI) live / dead staining (treated with different materials (CeO2, XO3, XO3@CeO2, XO3 + CeO2, PBS, Tobramycin) 2×10 9 After 1 hour of PAO1 bacterial suspension at CFU / mL, samples were collected and bacterial pellets were obtained by centrifugation (5000 rpm, 5 min). The pellet was washed once with PBS, resuspended in 990 μL PBS, then 10 μL of 2.5 mg / mL FDA was added for 10 min, followed by 10 μL of 500 μM PI, and incubated for another 10 min at room temperature in the dark. Afterwards, the samples were centrifuged (5000 rpm, 5 min) to obtain bacterial pellets, which were washed twice with PBS and once with H2O. 5 μL of the stained bacterial suspension was added to a glass slide, covered with a coverslip, and observed under a fluorescence microscope to confirm these findings. The CeO2@PEG and TOB groups showed weak red fluorescence signals, while the X03, X03+CeO2, and X03@CeO2 groups showed strong signals, indicating bacterial death. Figure 5 (D).

[0067] 2.2 Anti-biofilm activity of XO3@CeO2

[0068] Biofilms can protect the bacteria embedded within them from attacks by antibiotics and the host's immune system. This invention employs the crystal violet staining method (200 μL of LB liquid (containing 2% glucose) and 10 μL of fresh bacterial culture are added to a 96-well plate. The plate is incubated at 37 ℃ for 24 h to form a biofilm. 100 μL of supernatant is discarded, and 100 μL of the material is added. The plate is incubated at 37 ℃ for 12 h. 100 μL of supernatant is aspirated, and 10 μL of 25% glutaraldehyde is added for fixation. After fixation at room temperature for 30 min, the plate is dried in a 70 ℃ oven for 2 h. A 0.5 mg / mL aqueous solution of crystal violet is prepared, and 100 μL is added to each well. The plate is incubated for 30 min, washed with water until almost colorless (approximately 4-5 times), and all liquid is aspirated. 200 μL of 33% acetic acid is added, and extraction is performed in a clean bench / fume hood at room temperature for 30 min. The plate is shaken appropriately during extraction. 100 μL of the extracted material is placed in a new 96-well plate, and the absorbance at OD595 nm is measured using a microplate reader) to evaluate the biofilm removal efficiency. The results showed that the clearance rate of the X03 single-drug group was as high as 54.23%, which was significantly better than that of the TOB group (21.34%, P<0.001). Figure 5 (B)

[0069] Scanning electron microscopy (SEM) observations supported these quantitative analysis results. Phage treatment disrupted the biofilm structure, leading to bacterial lysis and leakage of contents. The X03@CeO2 group showed the most significant biofilm structural damage. Figure 5 (E).

[0070] 2.3 Inactivation effect of XO3@CeO2 on LPS

[0071] To evaluate the LPS regulation capability of this constructed system, the LPS content in the supernatant after bacterial treatment was measured. The LPS content in the supernatant of *Pseudomonas aeruginosa* treated with PBS was used as a control (set as 100%). The results showed that treatment with TOB or XO3 alone led to an increase in LPS levels in the supernatant, reaching 112.85% and 116.52% of the control group, respectively (P<0.05). Figure 5 (C)

[0072] Treatment with CeO2 alone resulted in a slight decrease in LPS levels, with the supernatant value being 87.21% of the control group. In contrast, when bacteria were treated with a physical mixture of XO3 and CeO2 (XO3 + CeO2@PEG) or a covalently coupled XO3@CeO2, the LPS levels in the supernatant decreased to 63.38% and 49.60% of the control group, respectively. The difference between the two groups suggests that covalent coupling may provide a more effective method for regulating LPS levels.

[0073] 2.4 Cell-protective effect of X03@CeO2 on LPS-induced THP-1 cells

[0074] The steps are as follows: First, 2 weeks of THP-1 cells were seeded into each well of a 96-well plate, and different concentrations of LPS (0, 100, 200, 400, 800 ng / mL) were added and incubated for 12 hours to explore the appropriate concentration for modeling. 800 ng / mL LPS was used for subsequent cell experiments to establish the model.

[0075] Cells were seeded in 96-well plates and incubated overnight, after which the culture medium was aspirated. To investigate the protective effect of X03@CeO2 on LPS-stimulated THP-1 cells, different compositions of materials were added to PBS, X03, TOB, CeO2@PEG, X03+CeO2, and X03@CeO2 in the presence of 800 ng / mL LPS. After co-incubation for 12 hours, cell viability was detected using the CCK-8 assay.

[0076] To investigate the therapeutic effect of X03@CeO2 on LPS-stimulated THP-1 cells, cells were seeded in 96-well plates overnight. After adhesion, the culture medium was aspirated, and then 800 ng / mL of LPS was added. After incubation for 12 hours, the LPS-containing medium was aspirated, and culture medium containing different components was added: PBS, X03, TOB, CeO2@PEG, X03+CeO2, and X03@CeO2. After culturing for 12 hours, the culture medium was aspirated, and cell viability was detected using the CCK-8 assay.

[0077] Lipopolysaccharide (LPS) can induce macrophage activation, excessive inflammatory response, and cell death. This study used two administration routes to evaluate the protective and therapeutic effects of X03@CeO2.

[0078] Protective mode (co-incubation): Compared with the control group, LPS treatment (PBS group) significantly reduced cell viability to 48% (P<0.001). After co-incubation with different formulations, cell viability significantly recovered (all P<0.001). The CeO2@PEG group recovered to 95.2%, the XO3+CeO2@PEG group recovered to 90.3%, and the XO3@CeO2 group showed the best recovery, reaching 108.70%. Figure 6 (A)

[0079] LPS treatment reduced cell viability to 40% (P<0.0001). Subsequent treatments with different agents significantly improved cell viability (all P<0.0001). Cell viability recovered to 71% in the CeO2@PEG group, 73.8% in the XO3+CeO2@PEG group, and 75.7% in the XO3@CeO2 group. Figure 6 (B)

[0080] The survival rates of the X03 and TOB groups were very close to those of the PBS group, indicating that the X03 and TOB groups had no significant effect on LPS-stimulated THP-1 cells.

[0081] 2.5 Anti-inflammatory effects of XO3@CeO2

[0082] To evaluate the anti-inflammatory potential of this system, the concentrations of IL-6 and TNF-α in the cell supernatant were quantitatively detected using an Elabscience kit. The results showed that, compared with the control group, LPS stimulation significantly upregulated the levels of IL-6 and TNF-α by 7.22-fold (P<0.05) and 2.07-fold (P<0.001), respectively. Figure 4 C, Figure 4 (D).

[0083] After intervention with different formulations, the groups exhibited different patterns: in the CeO2@PEG group, X03+CeO2@PEG group, and X03@CeO2 group, IL-6 and TNF-α levels decreased to near normal values, indicating that these formulations may effectively reduce cellular inflammation by enzymatically cleaving LPS. Compared with the PBS group, the X03 group showed a slight increase in TNF-α and a slight decrease in IL-6, but the difference was not statistically significant, indicating that X03 alone had no significant regulatory effect on LPS-induced inflammation. After treatment with tobramycin (TOB), TNF-α levels decreased by 3.55-fold (P < 0.05), while IL-6 levels showed only a non-statistically significant decrease. Figure 6 C in the middle Figure 6 (D).

[0084] 2.6 Inhibit the generation of reactive oxygen species (ROS)

[0085] First, seed 2×10⁶ cells / well in a 24-well plate. 5 THP-1 cells in each well were induced to differentiate and adhere using medium containing PMA. After 12 hours, the medium was aspirated, and the cells were washed three times with PBS. Subsequently, different components were added to medium containing 200 ng / mL LPS: PBS, X03 (1×10⁻⁶). 11 PFU / mL), TOB (1.5 μg / mL), CeO2@PEG (500 μg / mL), X03 (1×10 11 PFU / mL)+CeO2(500 μg / mL), X03@CeO2(1×10 11 Cells were cultured for 12 hours with PFU / mL (500 μg / mL) and no treatment as the control group. The culture medium was then aspirated, and the cells were washed three times with PBS. DCFH-DA (10 µM) was added to serum-free 1640 medium, and the cells were incubated at 37 °C in the dark for 30 min. The cells were then washed three more times with PBS to remove excess dye, and observed under an upright fluorescence microscope. ImageJ was used for quantitative analysis of fluorescence intensity.

[0086] The results showed that LPS stimulation led to a significant increase in green fluorescence intensity, indicating an increase in intracellular ROS levels ( Figure 6 (E). In contrast, the fluorescence signals of the CeO2@PEG, XO3+CeO2@PEG, and XO3@CeO2 groups were comparable to those of the untreated control group. This visual observation was further validated by quantitative analysis. Figure 10 (B)

[0087] The above results indicate that X03@CeO2 can effectively restore cellular redox balance. This effect stems from the synergistic effect of LPS degradation (blocking the source of reactive oxygen species generation) and CeO2's inherent antioxidant enzyme mimicry activity (directly scavenging existing reactive oxygen species).

[0088] 2.7 Inhibitory effect of X03@CeO2 on LPS-induced TLR4 activation in THP-1-derived macrophages

[0089] First, seed 2×10⁶ cells / well in a 24-well plate. 5 THP-1 cells in each well were induced to differentiate and adhere using medium containing PMA. After 12 hours, the medium was aspirated, and the cells were washed three times with PBS. Subsequently, different components were added to medium containing 200 ng / mL LPS: PBS, X03 (1×10⁻⁶). 11 PFU / mL), TOB (1.5 μg / mL), CeO2@PEG (500 μg / mL), X03 (1×10 11 Cells were cultured for 12 hours with PFU / mL + CeO2 (500 μg / mL) and X03@CeO2 (500 μg / mL). Afterward, the culture medium was aspirated, and cells were gently washed 2-3 times with pre-cooled PBS to remove residual medium. 4% paraformaldehyde (PFA) was added, and the cells were fixed at room temperature for 15-20 minutes. The cells were washed 3 times with PBS for 5 minutes each time to thoroughly remove the fixative. PBS solution containing 0.1% Triton X-100 was added, and the cells were incubated at room temperature for 10-15 minutes. The cells were washed 3 times with PBS for 5 minutes each time. PBS solution containing 5% BSA (bovine serum albumin) was added, and the cells were blocked at room temperature for 1 hour. TLR4 primary antibody was added, and the cells were incubated overnight at 4°C to ensure complete antibody binding. The samples were removed from 4°C and allowed to return to room temperature. The cells were washed 3 times with PBS for 5 minutes each time to remove unbound primary antibody. The fluorescent secondary antibody was diluted with PBS or blocking buffer. The secondary antibody must be stored and handled in the dark. Add secondary antibody and incubate in a humidified chamber protected from light at room temperature for 1-2 hours. Wash three times with PBS to remove unbound secondary antibody. Add DAPI dye and incubate for 30 minutes, then rinse rapidly with PBS 1-2 times to remove excess DAPI. Observe under an upright fluorescence microscope. ImageJ was used for quantitative analysis of fluorescence intensity.

[0090] The results showed that LPS stimulation led to a significant increase in TLR4 expression. Figure 6 (In the middle F), strong red fluorescence signals were observed under a fluorescence microscope. The red fluorescence intensity of the CeO2@PEG, XO3+CeO2, and XO3@CeO2 groups was comparable to that of the normal control group. This indicates that these formulations can effectively inhibit TLR4 activation by degrading LPS and thus blocking the initiation of downstream inflammatory signaling cascades.

[0091] 2.8 Biocompatibility of XO3@CeO2

[0092] With X03@CeO2 concentration (up to 1000 μg / mL; 1×10⁻⁶), the concentration of X03@CeO2 is approximately 1000 μg / mL. 11 THP-1 cells co-incubated with X03@CeO2 for 24 hours showed no significant cytotoxicity. Hemolysis assays confirmed that X03@CeO2 did not induce erythrocyte lysis, indicating its suitability for intravenous administration. Figure 7 China A Figure 7 (B)

[0093] Routine blood tests (white blood cells, red blood cells, PLT) and biochemical indicators (AST / ALT, CRE, BUN) showed no significant differences between the X03@CeO2 group and normal mice. H&E staining of major organs showed no obvious damage, confirming good biocompatibility. Figure 7 C- Figure 7 (Middle K).

[0094] Application Example 1: Animal Experiments

[0095] 1. The in vivo therapeutic effect of sepsis

[0096] To evaluate the therapeutic effect of X03@CeO2, 100 μL of Pseudomonas aeruginosa (1×10⁻⁶) was administered via intraperitoneal injection (ip). 8 A severe sepsis mouse model was established in 8-week-old male C57BL / 6 mice using CFU / mL. Figure 8 (A)

[0097] 2. Survival rate and clinical manifestations

[0098] The survival rate in the PBS control group was only 12.86% ( Figure 8 The robustness of the severe sepsis model was verified by the PBS group (PbA1). Treatment with tobramycin (TOB, 26.94%) or CeO2 alone (22.77%) did not significantly improve survival compared to the PBS group (p>0.05), indicating that single-modality strategies (whether bactericidal or anti-endotoxin agents alone) are insufficient. Phage X03 alone improved survival to 58.89% (p<0.01 compared to the TOB / CeO2 group), but the large standard deviation suggests inconsistency in efficacy. The X03@CeO2 complex showed the highest survival (87.50%), significantly superior to the PBS group (p<0.001) and the physical mixture X03+CeO2 group (53.33%, p<0.01).

[0099] Systemic inflammation during sepsis often leads to significant weight loss due to dehydration, reduced food intake, and accelerated catabolism. Weight changes were monitored with initial body weight as baseline (0). Mice in the PBS-treated group experienced a sustained weight loss of 0.43 grams after 24 hours. Figure 10 (A) Although TOB and CeO2 provided some relief, the X03@CeO2 treatment group experienced the least weight loss (0.03 g).

[0100] Furthermore, body temperature is a key indicator for assessing sepsis progression. Healthy mice maintained a stable body temperature (36.9–38.4°C). The X03@CeO2 group exhibited the most stable thermoregulation, maintaining a body temperature between 36.5–37.9°C throughout the observation period, which was statistically superior to all other groups. Figure 10 (B)

[0101] 3. Bacterial load, LPS clearance rate, and cytokine levels

[0102] To assess the effectiveness of bacterial clearance, blood samples were collected 6 hours post-treatment for colony counting. The bacterial load in the PBS group was as high as 2266.67 CFU / mL. X03@CeO2 treatment significantly reduced the bacterial count to 120.00 CFU / mL, which was significantly lower than that in the PBS group (P<0.001), the TOB group (1113.33 CFU / mL, P<0.01), and the X03-only group (553.33 CFU / mL, P<0.05). Figure 8 (D). This suggests that CeO2 modification may synergistically enhance the in vivo bactericidal activity of XO3.

[0103] Serum lipopolysaccharide (LPS) residual clearance rate was measured 24 hours later. The LPS residual clearance rate in the sepsis model group (PBS-treated mice) was set as the baseline (100.00%), reflecting the large accumulation of LPS in untreated sepsis. The positive drug control group (TOB-treated group) showed limited LPS clearance capacity, with a residual clearance rate of 65.95%, indicating that traditional antibiotics have a weak detoxification effect on LPS. In single-group treatment, the residual clearance rate in the CeO2-treated group was 37.45%, while the residual clearance rate in the XO3-treated group was 49.75%. Both groups showed moderate detoxification effects, but the efficacy of the X03 monotherapy group was significantly inferior to that of the CeO2 group; the residual clearance rate of the physical mixture group (X03+CeO2) was 12.36%, indicating that its detoxification effect was better than that of the single-component group; the core experimental group (X03@CeO2 treatment) had the highest LPS clearance efficiency, with a residual clearance rate as low as 2.60%—even lower than that of the healthy control group (7.74%), confirming its significant advantage in clearing LPS in septic mice. Figure 8 (C)

[0104] Serum cytokine levels (IL-6 and TNF-α) were analyzed by ELISA. Figure 8 China E- Figure 8 (F). Sepsis-induced IL-6 levels increased significantly by 37.17-fold (P<0.001). X03@CeO2 treatment restored IL-6 levels to normal (54.86 pg / mL), which was not significantly different from the healthy control group (52.03 pg / mL), but significantly better than the TOB group and the X03+CeO2 group. TNF-α showed a similar trend, with X03@CeO2 reducing levels to 7.20 pg / mL (compared to the PBS group, P<0.001), and even slightly lower than the healthy control group (17.07 pg / mL, P<0.05).

[0105] 4. Hematology and Biochemical Analysis

[0106] 24-hour peripheral blood routine examination revealed characteristic changes of severe sepsis in the PBS group: total white blood cell (WBC), absolute neutrophil count (Nue#), and absolute lymphocyte count (Lym#) were all significantly decreased (P<0.01, P<0.001, P<0.001, respectively), while the percentage of neutrophils increased. The absolute monocyte count (Mon%) remained stable, but its percentage passively increased, suggesting that sepsis was in an immunosuppressive phase. Platelet count (PLT) was significantly decreased due to bone marrow suppression and consumptive coagulation dysfunction (P<0.001), while the red blood cell count remained stable. The short-term stability of the red blood cell count was attributed to the long lifespan of red blood cells and the absence of significant erythropoiesis inhibition during the acute phase.

[0107] X03@CeO2 intervention effectively reversed these hematological abnormalities. White blood cell (WBC), neutrophil (Nue#), lymphocyte (Lym#), monocyte percentage (Mon%), and PLT count all returned to near-normal levels (P>0.05 compared to the control group), indicating that this system has the ability to regulate immune homeostasis and repair coagulation function. Figure 9 China A- Figure 9 (Middle F).

[0108] 5. Histopathological examination

[0109] Liver: The PBS group showed severe liver damage, characterized by significantly elevated serum ALT, AST, and AST / ALT ratio (P<0.001), accompanied by hepatocellular edema and cholestasis (H&E staining). While TOB and CeO2 treatments provided some improvement, residual granulomas and edema remained. XO3@CeO2 treatment restored liver function indicators and histological findings to normal. Figure 9 G- Figure 9 Middle I, Figure 8 (G).

[0110] Heart: Mice treated with PBS exhibited myocardial edema, degeneration (loss of striations), and interstitial inflammation. Monotherapy with TOB and X03 reduced inflammation but left focal damage. The X03@CeO2 treatment group showed the lowest degree of inflammation and nearly normal myocardial structure. Figure 8 (H).

[0111] Spleen: As a central immune organ, the spleen of PBS mice exhibits typical immune dysfunction: extensive lymphocyte apoptosis in the white pulp region, manifested as nuclear pyknosis and karyolysis, and blurred red and white pulp boundaries. These apoptotic features are clearly visible under 40x magnification (indicated by red arrows). X03@CeO2 treatment preserved the white pulp structure, lymphocyte arrangement, and clear boundaries, indicating superior immunoprotective effect. Figure 8 (H).

[0112] Lungs: The lungs are highly sensitive to sepsis. Mice in the PBS group developed diffuse interstitial inflammation, alveolar septal thickening, and hemorrhage. TOB and CeO2 failed to effectively alleviate the inflammation. XO3 treatment reduced septal thickening but did not reduce hemorrhage. The physical mixture group showed serous exudate. XO3@CeO2 treatment maintained alveolar structure, with no exudate and mild inflammation, very similar to the healthy control group. Figure 8 (H).

[0113] Kidneys: Given the acute nature of the 24-hour model, kidney damage was not yet clinically apparent. H&E staining showed no tubular degeneration, necrosis, or casts. Serum creatinine (CRE) and urea (UREA) levels did not differ significantly between groups, indicating that renal function remained stable in this early stage. Figure 10 C- Figure 10 (D).

[0114] In summary, this invention proposes a complementary "kill-detoxification" synergistic strategy and constructs an XO3@CeO2@PEG hybrid system. The role of nanozymes in the composite system is redefined; the primary function of CeO2 is not to assist in killing, but rather as a "detoxification unit."

[0115] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A polyethylene glycol-based X03 phage-cerium oxide nanozyme complex, characterized in that: The composite is X03@CeO2.

2. The complex according to claim 1, characterized in that: The linker for CeO2 and XO3 in the composite is one of NHS-PEG-COOH, and the ratio of CeO2, linker, and XO3 is CeO2:linker:XO3 = 2 mg: 1 mg: 2 × 10⁻⁶ mg. 11 PFU.

3. The method for preparing the complex according to claim 1, characterized in that: Includes the following steps: S1. Preparation of CeO2 nanozymes via hydrothermal method; Coupling of S2.CeO2@PEG: NHS-PEG-COOH and CeO2 were mixed in an ice-water bath at a volume ratio of 1:1 for 30 min to form CeO2@PEG; Preparation of S3.XO3@CeO2: In an ice-water bath, CeO2@PEG was mixed with 1×10 10 PFU / mL screened X03 was mixed for 2 hours to form X03@CeO2; then X03@CeO2 was salted out with 25% PEG-6k to remove unbound CeO2@PEG, and finally resuspended in 100 μL PBS.

4. The preparation method according to claim 4, characterized in that: The CeO2 nanozyme prepared in step S1 has an average particle size of 2.80 ± 0.86 nm, and the CeO2 nanozyme contains Ce 3+ and Ce 4+ They accounted for 38.26% and 61.74% of the total Ce, respectively, with an oxygen vacancy concentration of 47.89%.

5. The application of the complex according to claim 1, characterized in that: The complex is used to prepare a product for treating sepsis caused by multidrug-resistant Gram-negative bacteria, and a limited component of the product includes X03@CeO2.

6. The application according to claim 5, characterized in that: In the complex, CeO2 is the detoxification unit and XO3 is the bactericidal unit. When the bactericidal unit lyses bacteria, its covalently linked detoxification unit can simultaneously degrade the lipopolysaccharide released by the lysis, i.e., LPS, thereby blocking the activation of the TLR4-NF-κB inflammatory signaling pathway mediated by LPS at the source.

7. The application according to claim 5, characterized in that: The product achieved a 99.99% kill rate against Pseudomonas aeruginosa within two hours, with a concentration of 1×10⁻⁶ Pseudomonas aeruginosa. 9 CFU / mL.

8. The application according to claim 5, characterized in that: When treating sepsis, the product can increase the survival rate of mouse models infected with lethal doses of Pseudomonas aeruginosa to over 85%.

9. The application according to claim 5, characterized in that: After use, the product can reduce the residual LPS in the plasma of sepsis model animals to below 3%.

10. The application according to claim 5, characterized in that: The product includes at least one of pharmaceutical compositions, reagents, and kits.

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