A ros-responsive surface functional coating and preparation and application thereof

Abstract

The application relates to a ROS response type double-layer coating structure with a Fe-B (iron-boron) inorganic inner layer and a phenylboronic acid grafted sodium alginate (SA-PBA) outer layer on the surface of probiotic bacteria, and the technical core is as follows: chemical mechanism: Fe 3+ The Fe-B inorganic inner layer is generated by in-situ coordination of borate on the bacterial surface, and has ROS scavenging activity; the SA-PBA outer layer is assembled on the inner layer through a dynamic borate ester bond. Response principle: under a high ROS microenvironment, the borate ester bond of the SA-PBA outer layer is broken, and the coating responds and dissociates. Application scene: the coating is coated on the surface of an engineered IL-4 secreting lactobacillus paracasei, does not affect the bacterial activity and functional expression, and is used for acute lung injury and ARDS treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a ROS-responsive surface functional coating and its preparation and application. Background Technology

[0002] Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are common critical illnesses in clinical practice. Their core pathological features include uncontrolled inflammatory responses, excessive reactive oxygen species (ROS) accumulation, alveolar barrier disruption, and severe pulmonary edema, resulting in persistently high mortality rates. Excessive ROS not only directly causes oxidative damage to lung tissue but also activates inflammatory pathways such as NF-κB, inducing a massive release of pro-inflammatory factors and creating a vicious cycle of oxidative stress and inflammatory storm, a key factor contributing to the rapid progression of lung injury. Current clinical treatment primarily relies on supportive therapies such as mechanical ventilation and fluid management, lacking targeted interventions that can simultaneously block the oxidative stress and inflammatory cascade.

[0003] Probiotics, due to their high safety, ability to colonize locally, and ease of genetic engineering modification, have become potential carriers for local delivery and immune regulation in lung diseases. However, natural probiotics have significant limitations in application: first, they lack the ability to scavenge ROS, making it difficult for them to survive and colonize in lung tissue damaged by high oxidative stress; second, the bacterial cells have a short residence time in inflamed lung tissue and weak targeted enrichment ability; and third, single probiotics cannot simultaneously achieve multiple functions such as antioxidation, anti-inflammation, and response to the lesion microenvironment, resulting in limited therapeutic effects in acute lung injury and hindering clinical translation.

[0004] To enhance the survival and functionalization of probiotics, surface modification of materials has become an important improvement path. Existing bacterial coating materials are mostly designed solely to protect the bacteria, lacking responsiveness to the lesion microenvironment and failing to achieve targeted release of active ingredients. While some antioxidant coatings can scavenge free radicals, they are difficult to synergize with the immunomodulatory functions of engineered probiotics. Furthermore, most coating systems suffer from stringent preparation conditions, poor biocompatibility, and insufficient stability in the complex environment of the lungs, making it difficult to meet the application requirements of in vivo biological therapeutics.

[0005] In summary, there is an urgent need to develop a probiotic modified through synthetic biology to secrete anti-inflammatory cytokines such as IL-4, enabling targeted regulation of lung inflammation. Furthermore, this should be combined with a novel functional coating that possesses ROS-responsive dissociation, efficient scavenging of reactive oxygen species, and adaptability to engineered probiotic surface modifications. This would address the bottlenecks of poor survival, weak colonization, and limited functionality of probiotics in pulmonary oxidative stress environments, achieving a synergistic effect of antioxidant and immune regulation. This would provide a novel carrier and functionalized solution for inflammatory respiratory diseases such as acute lung injury. Summary of the Invention

[0006] To address the above deficiencies, this invention provides a ROS-responsive surface functional coating, its preparation, and its application. Details are as follows.

[0007] A ROS-responsive surface functional coating, comprising a two-layer composite structure, consisting of an Fe-B inorganic inner layer and a phenylboronic acid-grafted sodium alginate outer layer assembled via dynamic borate ester bonds, which are integrally coated on the surface of probiotic cells; the Fe-B inorganic inner layer is composed of Fe... 3+ The phenylboronic acid is in situ coordinated with borates on the surface of probiotics and has ROS scavenging activity; the outer layer of sodium alginate grafted with phenylboronic acid is covalently bound to the surface of Fe-B inorganic inner layer by dynamic boronic acid ester bonds, which can break the boronic acid ester bonds in a high ROS microenvironment, realizing the responsive dissociation of the coating; the coating does not affect the activity and functional expression of probiotic cells after coating.

[0008] The above-mentioned method for preparing ROS-responsive surface functional coatings involves four steps in sequence: constructing engineered probiotics, preparing sodium alginate grafted with phenylboronic acid, forming an in-situ Fe-B inorganic inner layer on the bacterial surface, and combining an outer layer of sodium alginate grafted with phenylboronic acid, thereby obtaining functionalized engineered probiotics coated with the coating.

[0009] The application of the above-mentioned ROS-responsive surface functional coatings in the preparation of therapeutic agents for acute lung injury or acute respiratory distress syndrome.

[0010] Preferred technical solution

[0011] ROS-responsive surface functional coatings

[0012] Furthermore, the phenylboronic acid grafted sodium alginate is prepared by an amidation reaction of sodium alginate and 3-aminophenylboronic acid catalyzed by EDC / NHS; the Fe-B inorganic inner layer is generated by in-situ mineralization of ferric chloride and borax on the surface of the cell.

[0013] Furthermore, the probiotic is an engineered Lactobacillus paracasei Lap_4 that can secrete IL-4; the engineered Lactobacillus paracasei uses Lactobacillus paracasei as a chassis strain and constitutively expresses and secretes mouse-derived IL-4 anti-inflammatory cytokine through recombinant plasmids.

[0014] Preparation method of ROS-responsive surface functional coating

[0015] A method for preparing the aforementioned ROS-responsive surface functional coating includes the following steps:

[0016] S1. Preparation of engineered Lactobacillus paracasei Lap_4 that secretes IL-4;

[0017] S2. Preparation of phenylboronic acid-grafted sodium alginate SA-PBA;

[0018] S3. An in-situ Fe-B inorganic inner layer is formed on the surface of Lactobacillus paracasei Lap_4 cells;

[0019] S4. By binding the SA-PBA outer layer with dynamic borate ester bonds, engineered probiotics coated with a ROS-responsive coating are obtained.

[0020] Furthermore, step S3 specifically involves: resuspending the bacterial cell in a Tris-HCl buffer solution at pH 8.0, adding ferric chloride solution and borax solution sequentially, vortexing to mix, and then generating an in-situ Fe-B inorganic inner layer on the bacterial cell surface.

[0021] Further, step S4 specifically involves adding SA-PBA solution to the bacterial solution coated with the Fe-B inorganic inner layer and reacting at 37°C and 500 rpm for 0.5 h to obtain a double-coated bacterial cell.

[0022] Furthermore, the preparation steps of SA-PBA are as follows: EDC, NHS and 3-aminophenylboronic acid are added to an aqueous solution of sodium alginate, the mixture is stirred at room temperature, and after the reaction is completed, it is dialyzed and lyophilized to obtain solid SA-PBA.

[0023] Furthermore, the method for constructing the engineered Lactobacillus paracasei Lap_4 is as follows: inserting the mouse IL-4 gene fused with the secretory signal peptide SPUSP45 into the pIB184 plasmid carrying the constitutive strong promoter P23 to construct the recombinant plasmid pIB184-SP-IL-4; amplifying the recombinant plasmid with Escherichia coli, electroporating it into Lactobacillus paracasei, and screening to obtain the engineered strain Lap_4 that can constitutively secrete IL-4.

[0024] Application of coatings

[0025] The aforementioned application of ROS-responsive surface functional coatings in the preparation of therapeutic agents for acute lung injury or acute respiratory distress syndrome.

[0026] Furthermore, the therapeutic agent can clear excess ROS in the lungs, inhibit the release of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, induce macrophages to polarize towards the M2 anti-inflammatory phenotype, reduce the wet-to-dry weight ratio of the lungs and repair alveolar structure, while regulating the lung microecology and metabolic homeostasis.

[0027] Furthermore, the therapeutic agent is suitable for LPS-induced or Pseudomonas aeruginosa-induced acute lung injury models, and has synergistic effects of antioxidation, anti-inflammation, immune regulation, antibacterial activity, and microecological remodeling.

[0028] like Figure 1As shown, by mediating the polarization of M1 to M2 macrophages through IL-4, ROS is cleared, IL-6 or TNF-α release is inhibited, oxidative damage to airway epithelium and local inflammation are reduced, and finally pulmonary edema and systemic inflammatory splenomegaly are relieved, thus achieving anti-inflammatory repair of the lungs.

[0029] like Figure 2 As shown, Lactobacillus paracasei Lap_4, which can secrete IL-4, was first constructed through gene editing, and then... 3+ By coordinating and assembling a ROS-sensitive sodium alginate composite coating on the bacterial cell surface, a functional engineered bacterial preparation M@Lap_4, capable of releasing IL-4 and targeting the inflammatory microenvironment of the lungs, was prepared. Figure 1 Its anti-inflammatory and repair mechanisms.

[0030] (i) The ROS-responsive surface functional coating provided by the present invention has excellent microenvironment response specificity, can rapidly dissociate in a pathological microenvironment with high reactive oxygen species, achieve targeted release of the coated bacteria from the lesion, avoid premature drug release in normal tissues, and significantly improve the specificity of the action site and bioavailability.

[0031] (ii) The coating has a high efficiency ROS scavenging ability, which can directly degrade excess reactive oxygen species in the lung injury site, interrupt the vicious cycle of "oxidative stress-inflammation cascade amplification", reduce oxidative damage to lung tissue from the source, and solve the technical defect that traditional probiotics do not have autonomous antioxidant capacity.

[0032] (III) The coating is achieved through mild biomimetic mineralization and dynamic chemical bond assembly. The preparation conditions are mild and the biocompatibility is high. It does not affect the activity of probiotics and the secretion function of IL-4. At the same time, it significantly improves the survival rate, colonization rate and residence time of engineered probiotics in the inflammatory environment of the lungs, breaking through the bottleneck of traditional probiotic delivery to the lungs being easily cleared.

[0033] (iv) The coating of the present invention can form a synergistic effect with engineered probiotics, realizing the dual function integration of anti-oxidation and anti-inflammatory immune regulation. It can significantly inhibit the release of pro-inflammatory factors such as TNF-α and IL-6, induce macrophages to polarize to the anti-inflammatory phenotype, reduce the wet-to-dry weight ratio of the lungs, and improve the pathological damage of lung tissue. The effect is significantly better than that of single probiotics or single antioxidant materials.

[0034] (v) The coating has good structural versatility and scalability, and can be applied to the surface modification of various probiotics and live biological therapeutic agents. It can endow bacteria with functions such as ROS response, anti-oxidation and targeted enrichment without complex modification, providing a modular and reproducible functional platform for inflammation-related respiratory diseases.

[0035] (vi) The coating of the present invention can reshape the homeostasis and metabolic balance of the lung microecology, significantly increase the abundance of beneficial bacteria and upregulate the level of protective metabolites, make up for the shortcomings of traditional treatment methods that neglect microecological regulation, and have the comprehensive advantages of long-term effect, stability and multiple targets, making it more suitable for clinical translational application. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the application principle of an embodiment of the present invention;

[0037] Figure 2 This is a schematic diagram of the coating-bound Lactobacillus paracasei (Lap_4) described in an embodiment of the present invention;

[0038] Figure 3 Strain identification and IL-4 expression verification diagram;

[0039] Figure 4 Characterization diagrams of coating morphology, particle size, potential, elemental composition, and antioxidant properties;

[0040] Figure 5 Quantitative analysis of iron loading and TEM-EDS elemental distribution characterization;

[0041] Figure 6 ROS-responsive dissociation fluorescence verification diagram of the coating;

[0042] Figure 7 Safety and pro-inflammatory factor detection of engineered bacterial cells;

[0043] Figure 8 Graphs showing in vitro anti-inflammatory and antioxidant effects;

[0044] Figure 9 Figure showing the efficacy of LPS-induced acute lung injury in mice;

[0045] Figure 10 In vivo efficacy detection in mice with Pseudomonas aeruginosa-induced acute lung injury;

[0046] Figure 11 Bacterial cell activity detection graph;

[0047] Figure 12 In vitro antibacterial performance test results;

[0048] Figure 13 Image of lung 16S rDNA microbial community sequencing analysis;

[0049] Figure 14 Pulmonary metabolomics PCA, clustering, and KEGG pathway enrichment map;

[0050] Figure 15 Differential metabolite heatmap;

[0051] Figure 16Macrophage inflammatory factors and polarization detection map;

[0052] Figure 17 Flow cytometry plot of macrophage M1 / M2 polarization;

[0053] Figure 18 Quantitative detection chart of IL-4 and pro-inflammatory factors in vivo and in vitro. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the reagents, reagent kits, consumables, and instruments used in the present invention are conventional reagents, reagent kits, consumables, and instruments in this technical field.

[0055] Microbial cultures of *Lactobacillus paracasei* (Lap), *Pseudomonas aeruginosa* PA14, and *Staphylococcus aureus* were all purchased from the China General Microbiological Culture Collection Center (CGMCC). *Lactobacillus paracasei* was cultured on MRS medium; *Pseudomonas aeruginosa* PA14 and *Staphylococcus aureus* were cultured on LB medium at 37°C under aerobic conditions. For routine subculturing and amplification, the strains were diluted 1:25 (4% ​​inoculum). Animal administration procedure: *Lactobacillus paracasei* culture (5 × 10⁸ CFU / mL) and *Pseudomonas aeruginosa* PA14 culture (1 × 10⁷ CFU / mL) in logarithmic growth phase were collected, centrifuged at 3000 rpm for 5 min, and the bacterial pellet was collected. The pellet was washed once with sterile phosphate-buffered saline (PBS), centrifuged again under the same conditions, and finally resuspended in sterile PBS for oral administration to mice.

[0056] Example 1

[0057] The construction of engineered Lactobacillus paracasei (Lap_4) secreting IL-4: Using Lactobacillus paracasei (Lap) as the chassis strain, synthetic biology techniques were employed to introduce the mouse-derived IL-4 anti-inflammatory cytokine gene into the bacterial cells via a recombinant plasmid, enabling the engineered bacteria to constitutively secrete IL-4, thereby obtaining an engineered probiotic with immunomodulatory functions.

[0058] 1.1 Target Gene Synthesis: The mouse IL-4 gene sequence was synthesized according to GeneID: 16189, and the lactobacillus secretion signal peptide SPUSP45 was fused to its 5' end to ensure that IL-4 can be secreted extracellularly to exert its function. The mouse IL-4 gene fragment was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0059] 1.2 Recombinant plasmid construction: Using pIB184 driven by the constitutive strong promoter P23 as the expression backbone, the signal peptide-IL-4 fragment was inserted into the multiple cloning site of the plasmid using the HieffClone® Plus cloning kit to construct the recombinant expression plasmid pIB184-SP-IL-4.

[0060] 1.3 Transformation and Validation of E. coli: The recombinant plasmid was heat-shocked and transformed into E. coli DH5α competent cells, plated on LB agar plates containing erythromycin resistance, and incubated overnight at 37°C; single clones were picked for PCR amplification and sequencing validation to ensure that the sequence was completely correct.

[0061] 1.4 Electroporation of Lactobacillus paracasei: The correctly sequenced recombinant plasmid was introduced into Lactobacillus paracasei competent cells for electroporation. Electroporation parameters: 2.0 kV, 25 μF, 200 Ω. After electroporation, MRS resuscitation medium was added immediately and the cells were incubated at 37°C for 2 h.

[0062] 1.5 Screening and identification of positive strains: Spread the revived bacterial culture on MRS solid plates containing 1‰ erythromycin and anaerobic culture at 37℃ for 24–36 h; pick resistant single colonies and perform colony PCR identification using specific primers.

[0063] 1.6 IL-4 secretion expression verification: Positive strains were inoculated into MRS liquid medium and cultured to the logarithmic phase. The supernatant was collected and the IL-4 protein concentration was detected using an ELISA kit to confirm that the engineered bacteria could stably and efficiently secrete IL-4.

[0064] 1.7. Effect Verification; such as Figure 3 As shown, to construct a multifunctional engineered strain, *Lactobacillus paracasei* (Lap) was selected as the chassis strain in this study. This bacterium colonizes the human gut and oral cavity and naturally possesses important immunomodulatory functions. First, the mouse-derived IL-4 gene was cloned into the pIB184 plasmid, and then introduced into *Lactobacillus paracasei* via electroporation. Specific primer identification results confirmed that the IL-4 gene had been successfully integrated into the strain's genome, as shown in the figure. Figure 3 a). Further results from enzyme-linked immunosorbent assay (ELISA) showed that the engineered strain Lap_4 had a significantly higher ability to secrete IL-4 in vitro. Figure 3 b; PCR results showed a specific band at the 698bp position, proving that the IL-4 gene was successfully integrated; ELISA results showed that the IL-4 concentration in the supernatant of the engineered bacteria was significantly higher than that in the empty vector control group, indicating that Lap_4 can stably secrete IL-4.

[0065] Example 2

[0066] Preparation of phenylboronic acid-grafted modified sodium alginate (SA-PBA): The amidation reaction between carboxyl groups and amino groups is catalyzed by EDC / NHS, and 3-aminophenylboronic acid is grafted onto the molecular chain of sodium alginate, endowing sodium alginate with the ROS-responsive boronate bond binding ability, so that it can undergo bond scission and dissociation in a high ROS environment.

[0067] 2.1. Specific steps:

[0068] Weigh 1.0 g of sodium alginate (SA), add 100 mL of deionized water, and stir at 50°C until completely dissolved;

[0069] Add 1.0 g of EDC and 0.4 g of 3-aminophenylboronic acid in sequence. After complete dissolution, react at 25°C and 400 rpm for 24 h;

[0070] Dialyze against pure water using a 3500 Da dialysis bag for 3 days to remove unreacted small molecules;

[0071] Freeze-dry for 24 h to obtain SA-PBA solid; before use, prepare a 5 mg / mL solution with pH=8.0 Tris-HCl.

[0072] 2.2. Effect verification; Infrared spectroscopy shows that a new absorption peak appears in the characteristic peak region of boronic acid, proving that phenylboronic acid is successfully grafted; it has good water solubility and stability, and can form boronate bonds with the Fe-B layer under mild conditions.

[0073] Example 3

[0074] Preparation of ROS-responsive surface functional coating (Fe-B / SA); Using Fe 3+ to undergo a metal-ligand coordination reaction with borate, an Fe-B inorganic antioxidant nanolayer is generated in-situ on the surface of the bacterial cell; then the SA-PBA outer layer is compounded through dynamic boronate bonds to form a ROS-responsive structure: stable in a normal environment, rapidly broken in a high ROS environment to release the bacterial cell. The characterization of the coating structure, morphology and composition is as Figure 4 shown. The original Lap is rod-shaped with a smooth surface; uniform and dense particles appear on the Lap / Fe-B surface; as shown in attached Figure 4 c: A continuous and complete polymer coating layer is formed on the Lap / Fe-B / SA surface. Particle size and Zeta potential: the particle size increases sequentially: Lap < Lap / Fe-B < Lap / Fe-B / SA; the potential is maintained at -20~-40 mV, with stable dispersion. XPS elemental analysis: characteristic peaks of C1s, Fe2p and B1s are detected, confirming the successful construction of the Fe-B coating. Fluorescent labeling and flow cytometry: FITC-SA-PBA shows uniform green fluorescence on the surface of the bacterial cell; the quantitative fluorescence intensity by flow cytometry is significantly increased, with a high coating rate. Antioxidant performance (DPPH): The free radical scavenging rate of Lap / Fe-B / SA reaches ~25%, which is significantly higher than that of uncoated bacteria.

[0075] To quantitatively determine the iron loading of the Fe-borax coating, the total iron content was determined using the o-phenanthroline colorimetric method: after acid digestion and reduction treatment, the quantification was performed using the characteristic absorption peak of the ferrous iron-o-phenanthroline complex at 512 nm. The method was based on a standard curve with excellent fit (coefficient of determination (R²)). 2 The bacterial concentration (OD) can be calculated from 0.998. 600 When the iron content is 0.1%, the iron loading per 1 mL of bacterial culture is (3.18 pm 0.022 nmol); Appendix Figure 5 Figure a shows the colorimetric gradient diagram for iron loading determination using the o-phenanthroline colorimetric method. The lower figure shows the standard curve (coefficient of determination (R²)) at a wavelength of 512 nm. 2 =0.998); Transmission electron microscopy-energy dispersive spectroscopy (TEM-EDSmapping) further verified the spatial distribution of iron and the integrity of the composite structure: Fe-B mineralization framework was visible inside the sodium alginate-phenylboronic acid ester (SA-PBA) layer (circled area), and Fe-borax complex was enriched on the local surface of the cell (rectangular area), proving that the mineralized shell was fully assembled. Figure 5 b. Structure and Elemental Characterization: The top image shows TEM / STEM images, labeled with the internal Fe-BOX layer (circular region) and the surface-bound complex (rectangular region), scale bar: 1 μm; the bottom image shows EDS spectra, verifying the elemental composition of the mineralized coating. Under a fluorescence microscope, a uniform, bright green fluorescent ring is clearly visible around the bacterial cells, visually demonstrating the uniform coating of the SA-PBA mineralized shell. This mineralized biohybrid interface introduces ROS-sensitive phenylboronic acid ester bonds, which can rapidly dissociate under oxidative stimulation, releasing the internally encapsulated engineered probiotics. Figure 6 The overall characterization included colorimetric quantitative detection of iron ions, morphological analysis by transmission electron microscopy / energy dispersive spectroscopy (TEM / EDS), and fluorescence verification of ROS-triggered coating dissociation characteristics.

[0076] Lactobacillus paracasei (Lap) was cultured overnight in MRS medium containing 1‰ erythromycin; the bacterial cells were washed with sterile PBS and resuspended in Tris-HCl buffer (pH=8) to adjust the OD of the bacterial culture. 600 Adjust the value to 0.2. Take 0.9 mL of OD. 600A bacterial suspension with a pH of 0.2 was mixed with 1 μL of 100 mg / mL ferric chloride (FeCl3) solution and shaken for 20 s. Then, 1 μL of 100 mg / mL borax solution was added, and the mixture was shaken again for 20 s. The mixture was washed with PBS and resuspended in 0.9 mL of Tris-HCl buffer, followed by the addition of 0.1 mL of 20 mg / mL SA-PBA solution. The mixture was stirred at 37 °C and 500 rpm for 0.5 h. After the reaction, the cells were washed twice with PBS to obtain a pale yellow precipitate, which was then dispersed in sterile PBS for later use. The bacterial cells without SA-PBA coating were named Lap / Fe-B; the final product after secondary SA-PBA coating was named Lap / Fe-B / SA.

[0077] The microstructures of Lap, Lap / Fe-B, and Lap / Fe-B / SA were observed using transmission electron microscopy (TEM, Hitachi HT7700) and field emission scanning electron microscopy (SEM, Quanta 250FEG, operating voltage 10kV). After freeze-drying, the Lap / Fe-B / SA samples were subjected to XPS analysis using a Thermo Fisher Scientific ESCALAB 250Xi X-ray photoelectron spectroscopy system (150W power, 1253.6eV excitation energy). The average hydrodynamic particle size and zeta potential of Lap, Lap / Fe-B, and Lap / Fe-B / SA were determined by dynamic light scattering (DLS) using a Zetasizer nanoparticle size potentiometer (NanoZS, model ZEN3600).

[0078] 3.1 Specific steps:

[0079] Take Lap_4 bacterial culture, centrifuge at 3000 rpm for 5 min, wash with sterile PBS, and resuspend in Tris-HCl at pH 8.0 to OD. 600 =0.2;

[0080] Add 1 μL of 100 mg / mL FeCl3 solution and vortex for 20 seconds;

[0081] Add 1 μL of 100 mg / mL borax solution, vortex for 20 s, and an Fe-B inner layer is formed in situ.

[0082] After washing with PBS, 0.1 mL of 5 mg / mL SA-PBA solution was added, and the mixture was reacted at 37 °C and 500 rpm for 0.5 h.

[0083] Wash twice with PBS to obtain engineered probiotics Lap / Fe-B / SA coated with a ROS-responsive functional coating.

[0084] 3.2. Effect verification: TEM / SEM showed that a uniform and continuous double-layer coating structure was formed on the surface of the bacteria; XPS confirmed that Fe and B elements were present on the surface of the bacteria, proving that the coating was successfully assembled; the zeta potential and particle size gradually increased, proving that the layer-by-layer coating was successful.

[0085] Example 4

[0086] Verification of coating ROS response dissociation performance: Under high ROS environment, the borate ester bonds in the coating undergo oxidative breakage, the SA outer layer disintegrates, the Fe-B layer is exposed and ROS is cleared, and the internal engineered probiotics are released at the same time.

[0087] 4.1 Specific steps:

[0088] The Lap / Fe-B / SA ratio was divided into two groups:

[0089] Control group: PBS buffer;

[0090] Simulated pathology group: PBS solution containing 100 μM H2O2;

[0091] Incubate at 37°C in the dark for 30 minutes;

[0092] TEM and DLS were used to observe changes in particle size and morphology.

[0093] 4.2 Effect verification: Control group: coating intact, particle size stable; High ROS group: coating rapidly dissociates, shell cracks, particle size significantly decreases, proving that it has precise ROS response dissociation capability.

[0094] Example 5

[0095] ROS removal capability test of coating (DPPH method); DPPH free radicals have characteristic absorption at 520nm. After being removed, the absorbance decreases, and the degree of decrease directly reflects the antioxidant capacity.

[0096] 5.1 Specific steps:

[0097] Prepare a DPPH working solution containing 0.5 mg / 12 mL ethanol;

[0098] Take Lap, Lap / Fe-B, and Lap / Fe-B / SA bacterial suspensions (OD) 600 =1.5) 1mL each, centrifuge and collect the precipitate;

[0099] The precipitate was resuspended in 1 mL of DPPH working solution and incubated at 37 °C in the dark for 30 min.

[0100] Centrifuge and collect the supernatant, measure the absorbance at 520 nm, and calculate the free radical scavenging rate.

[0101] Weigh 0.5 mg of DPPH powder and dissolve it in 12 mL of anhydrous ethanol to prepare a DPPH working solution. Take 1 mL of OD... 600 =1.5 Lap, Lap / Fe-B, and Lap / Fe-B / SA bacterial suspensions were centrifuged to collect the bacterial precipitate; the precipitate was completely dispersed in 1 mL of DPPH working solution and thoroughly mixed. The system was incubated at 37℃ in the dark for 0.5 h, then centrifuged, and the supernatant was collected. The absorbance value was measured at 520 nm using a UV-Vis spectrophotometer.

[0102] 5.2 Effect Verification: The Lap group had almost no scavenging ability; the Lap / Fe-B group showed improved scavenging rate; the Lap / Fe-B / SA group achieved a free radical scavenging rate of approximately 25%, proving that the coating has excellent antioxidant activity.

[0103] Example 6

[0104] In vitro verification of the synergistic effects of anti-inflammatory and antioxidant agents: engineered bacteria secrete IL-4 to inhibit the NF-κB pathway and downregulate pro-inflammatory factors; the coating removes ROS and blocks the vicious cycle of oxidation-inflammation, and the two work together to achieve anti-inflammatory and antioxidant effects.

[0105] Safety evaluation of engineered bacteria M@Lap_4: To assess the biosafety of the engineered bacteria constructed in this study, different multiples of infection (MOIs) were first set, and Lap_4 and M@Lap_4 were co-cultured with A549 lung epithelial cells, respectively. Figure 7 As shown, Figure 7 a. The viability of A549 cells treated with M@Lap_4 at different multiples of infection (MOI) was detected using the CCK-8 assay; the CCK-8 cell viability assay results showed that neither strain damaged cell viability; Figure 7 The relative mRNA expression levels of inflammatory cytokines IL-6(b), IL-1β(c), and TNF-α(d) in A549 cells after M@Lap_4 intervention were quantitatively detected by RT-qPCR (n=3). Subsequently, real-time quantitative PCR (RT-qPCR) was used to detect the potential impact of the engineered bacteria on cellular inflammatory responses. Gene transcription level results showed that the engineered bacteria intervention did not upregulate the expression of key pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. Error bars represent standard deviations, and statistical analysis was performed using the two-tailed Student's test (*p<0.05; **p<0.01; ***p<0.001).

[0106] like Figure 8 As shown; ( Figure 8 a- Figure 8 b) RT-PCR detection of the engineered strain's effect on the pro-inflammatory factor IL-6 ( Figure 8 a) and TNF-α Figure 8 b) The inhibitory effect on expression levels; Figure 8 c) Relative expression level of IL-22 after intervention with engineered strains (N=3); (df) Quantitative detection of inflammatory factor IL-1β in each group using ELISA kits. Figure 8 d) IL-6 Figure 8 e), TNF-α Figure 8 f) protein secretion level; Figure 8 g) Intracellular ROS levels in different treatment groups were detected using a DCFH-DA fluorescent probe combined with fluorescence microscopy. Cell nuclei were stained with DAPI. Scale bar: 50 μm. Figure 8 h) Flow cytometry was used to quantitatively analyze the intracellular ROS levels of A549 cells under different treatment conditions (N=3). Error bars represent standard deviations. Statistical analysis was performed using the two-tailed Student's test (*p<0.05; **p<0.01; ***p<0.001).

[0107] Cellular Antioxidation and Supplementary Experimental Methods for RT-qPCR:

[0108] The antioxidant capacity of M@Lap_4 cells was detected using a reactive oxygen species (ROS) assay kit (Beyotime, catalog number S0033S) combined with DCFH-DA fluorescence staining. A549 cells were seeded at a rate of 1 × 10⁵ cells / well in six-well plates and cultured until adherence. After adherence, the culture medium was changed: the control group was treated with DMEM containing PBS, and the model group was treated with DMEM containing a final concentration of 10 μg / mL LPS, and incubated for 12 h. The original medium was discarded, and fresh DMEM was added. Sterile PBS, 100 μg / mL Lap_4, and 100 μg / mL M@Lap_4 were added respectively, and the cells were cultured for another 12 h. After treatment, the cells were washed with PBS, and DCFH-DA working solution was added. The cells were incubated at 37°C in the dark for 20 min to complete ROS staining; after staining, the cells were rinsed three times with PBS. The nuclei were then counterstained with Hoechst nuclear staining solution (Beyotime, catalog number C1011) for 25 min, followed by rinsing three times with PBS. Representative fluorescence images of each group were acquired using a Zeiss SSLSM998 laser scanning confocal microscope. RT-qPCR experiments: Total RNA was extracted from A549 cells using TransZolUp (version 2.0) reagent and diluted to an appropriate concentration; cDNA was synthesized using the EvoM-MLV reverse transcription kit with genomic DNA removal function. qPCR amplification was performed on a Roche LightCycler 96 real-time PCR instrument using SYBR Green Premix ProTaq HS real-time PCR premix. Thermal cycling program: 95℃ pre-denaturation for 30 s; followed by 40 amplification cycles: 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s. Fluorescence signals were acquired at the end of each cycle.

[0109] 6.1 Specific steps:

[0110] A549 lung epithelial cells were plated and adhered to the plate. After 12 hours of stimulation with 10 μg / mL LPS, an inflammation model was established.

[0111] Add PBS, Lap_4, and Lap / Fe-B / SA respectively and continue culturing for 12 h;

[0112] RT-qPCR and ELISA were used to detect IL-6, TNF-α, and IL-1β.

[0113] The DCFH-DA fluorescent probe was used to detect intracellular ROS levels.

[0114] 6.2. Effect verification: IL-6, TNF-α, and IL-1β were significantly downregulated in the Lap / Fe-B / SA group, with a much stronger inhibitory effect than that of single-Lap_4; the intracellular ROS fluorescence intensity was significantly reduced, basically returning to the level of normal cells; proving that the coating and engineered bacteria achieve synergistic anti-inflammatory and antioxidant effects.

[0115] Example 7

[0116] The coating enhances the colonization and survival of probiotics. It protects bacteria from immune clearance and oxidative stress damage, and improves their survival rate, adhesion, and colonization time in an inflammatory lung environment.

[0117] 7.1 Specific steps:

[0118] Lap_4 and Lap / Fe-B / SA were incubated in an oxidizing environment containing H2O2.

[0119] Serial dilutions were applied to plates, and the number of viable bacteria was counted.

[0120] Observe the adhesion ability of bacteria on the surface of airway epithelial cells.

[0121] 7.2 Effect verification: The survival rate of Lap / Fe-B / SA was significantly improved in the oxidative environment; the number of adhesions was significantly higher than that of the uncoated group; proving that the coating can enhance the lung colonization ability of probiotics.

[0122] Example 8

[0123] In vivo therapeutic effects of LPS-induced ALI model in mice; such as Figure 9 As shown, ( Figure 9 a) Animal experiment grouping and intervention protocol: Mice were divided into a blank control group (PBS), an LPS model group, a dexamethasone positive drug group (DXMS), a primitive probiotic Lap intervention group, an engineered Lap_4 intervention group, and a functionally modified engineered M@Lap_4 intervention group (n=6); Figure 9 b and Figure 17Flow cytometry results showed that both Lap_4 and M@Lap_4 significantly increased the proportion of CD206+ / CD86+ repair macrophages, with a regulatory effect superior to that of the positive control drug dexamethasone; Figure 9 c) Immunofluorescence staining was used to detect lung ROS levels in different intervention groups; immunofluorescence staining directly confirmed that M@Lap_4 could effectively inhibit the massive generation of reactive oxygen species in the lungs induced by LPS stimulation; Figure 9 d) H&E-stained pathological sections of mouse lung tissue 24h and 36h after intervention; observation of hematoxylin-eosin (H&E) pathological sections of lung tissue showed that: the LPS model group had severe lung tissue damage, manifested as alveolar dilation, capillary congestion, massive infiltration of neutrophils, alveolar wall thickening and even rupture; after intervention with engineered bacteria, various pathological damages were significantly alleviated, and the repair effect of M@Lap_4 gradually increased with the extension of intervention time; Figure 9 e) Statistical analysis of the wet / dry weight (W / D) ratio of lung tissue in each group of mice; the lung wet / dry weight ratio (W / D) can directly reflect the degree of pulmonary edema, and this ratio increased significantly after LPS modeling; Lap_4, M@Lap_4 and dexamethasone can all reduce the lung wet / dry weight ratio, among which M@Lap_4 has the most prominent improvement effect; Figure 9 f) Statistical results of spleen weight in each group of mice; LPS-induced splenomegaly could be alleviated by dexamethasone and two engineered bacteria, while wild-type Lap showed no significant improvement; Figure 9 g) Dynamic monitoring and statistical analysis of mouse body weight. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001). There were no significant differences in mouse body weight among the groups, indicating that the intervention method had no systemic toxicity. Figure 9 a: Experimental timeline and administration regimen; M@Lap_4 significantly increased the proportion of CD206+M2 macrophages, superior to dexamethasone; immunofluorescence showed a significant decrease in ROS fluorescence intensity in the lungs; H&E staining showed intact alveolar structure and a significant reduction in inflammatory infiltration, with the therapeutic effect continuously enhanced from 24h to 36h; the lung wet-to-dry weight ratio was significantly reduced, and pulmonary edema was improved; the spleen coefficient decreased, and systemic inflammation was suppressed; there was no difference in mouse body weight, indicating good safety.

[0124] To elucidate the mechanism of action of the strains, culture supernatants from RAW264.7 macrophages and A549 lung epithelial cells were collected for analysis. Quantitative results confirmed that both Lap_4 and M@Lap_4 can stably and efficiently secrete interleukin-4 in vitro (see attached image). Figure 18 a– Figure 18b). Bronchoalveolar lavage fluid (BALF) from mice was collected for in vivo quantitative analysis. Results showed that both Lap_4 and M@Lap_4 could colonize the lung lesion microenvironment in large quantities; intervention with engineered bacteria significantly downregulated the expression levels of core pro-inflammatory factors such as IL-6, IL-1β, and TNF-α in lung tissue (see attached). Figure 18 c– Figure 18 f).

[0125] Supplement to the general animal experimental protocol: All animal operations in this study strictly followed the guidelines of the Shanghai Municipal Laboratory Animal Management Committee. The experimental protocol was ethically reviewed and approved by the Laboratory Animal Center of Shanghai Chengxi Biotechnology Co., Ltd. (ethics approval number: CX052501097). Pentobarbital sodium was used for intraperitoneal anesthesia during animal operations, and various measures were taken throughout the process to minimize pain and stress in the mice.

[0126] Experimental animals: Male C57BL / 6J mice aged 6–8 weeks were purchased from Vital River Laboratory Animal Technology Co., Ltd. (China). All mice were housed in the animal housing of Shanghai Chengxi Biological, under a constant temperature of 24±2℃ and relative humidity of 40%–70%, with a 12-hour light-dark cycle. After acclimatization, mice were randomly assigned to experimental groups using a random number method.

[0127] Experimental Groups: Six groups were established: a control group received phosphate-buffered saline (PBS) via nasal instillation; the remaining groups were modeled with acute lung injury (ALI) via nasal instillation of lipopolysaccharide (LPS). Modeled mice were further divided into five subgroups: LPS model group, dexamethasone (DXMS) positive drug control group, wild-type Lap intervention group, Lap_4 intervention group, and M@Lap_4 intervention group. Administration Method: All therapeutic agents were delivered to the mouse lungs via nasal instillation (Note: Nasal instillation is a classic alternative to lung administration in rodents, but it is not directly equivalent to clinical nebulized inhalation). After ALI modeling, corresponding interventions were administered according to the group. Mice were euthanized 36 hours after modeling, and bronchoalveolar lavage fluid (BALF) was collected for metagenomics and metabolomics analysis. Lung tissue was dissected, fixed, and sectioned for hematoxylin-eosin (H&E) pathological staining and reactive oxygen species (ROS) fluorescence staining. At the same time, the weight of the lungs and spleen is measured to calculate the organ index, and the wet-to-dry weight ratio of lung tissue is measured to assess the degree of pulmonary edema.

[0128] Histopathology and ROS detection methods:

[0129] Histopathological examination: Lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned. The sections were dewaxed, rehydrated in a gradient, and then stained with hematoxylin and eosin (H&E). After dehydration and mounting, the sections were scanned using the PANNORAMICSCANII digital slide scanner from 3DHISTECH, Hungary, for pathological morphological observation.

[0130] Detection of reactive oxygen species (ROS) in lung tissue: The level of ROS in lung tissue was determined using a dihydroethidium (DHE) superoxide anion detection kit. Frozen sections were equilibrated at room temperature for 30 min, then incubated in the dark for 3 min to complete nuclear staining. After rinsing, DHE working solution was added, and the sections were incubated in a humidified chamber at room temperature in the dark for 1 h. After thorough rinsing, the sections were mounted with anti-fluorescence quenching mounting medium and stored at 4°C in the dark. Fluorescence images were acquired using a Hamamatsu NanoZoomer S360 digital slide scanning system (Japan), and fluorescence intensity was quantitatively analyzed using ImageJ software.

[0131] In summary, the above in vivo experimental results demonstrate that this engineered strain possesses potent anti-inflammatory and lung tissue protective effects in the LPS-induced acute lung injury model, and can significantly alleviate lung inflammatory damage.

[0132] Example 9

[0133] The in vivo efficacy of Pseudomonas aeruginosa-induced ALI model, such as Figure 10 As shown, ( Figure 10 a) Animal experimental intervention protocol: Mice were divided into a blank control group (PBS), a model group, a dexamethasone positive control group (DXMS), a Lap intervention group, a Lap_4 intervention group, and an M@Lap_4 intervention group (n=6); Figure 10 b) Immunofluorescence staining was used to detect ROS levels in the lungs of mice in each treatment group; Figure 10 c) H&E staining pathological images of mouse lung tissue on day 2 and day 5 of intervention; Figure 10 d) Statistical analysis of the wet / dry weight (W / D) ratio of lung tissue in mice of each treatment group; Figure 10 e) Spleen weight statistics of mice in each treatment group. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001). Bacterial infection model intervention protocol; M@Lap_4 significantly cleared lung ROS; H&E showed significant repair of lung tissue pathological damage, with improved efficacy from 2d to 5d; lung wet-to-dry weight ratio significantly decreased, with better effect than dexamethasone; splenomegaly significantly alleviated, and systemic inflammation was controlled.

[0134] Example 10

[0135] viable bacterial activity testing and in vitro antibacterial performance verification, such as Figure 11As shown, the activity of the coated modified strain M@Lap_4 was basically the same as that of the original Lap_4, and there was no significant difference in the colony count (CFU) on agar plates, proving that the modification process did not damage the activity of the strain. Figure 12 As shown, coating modification can also promote the proliferation of IL-4 engineered bacteria, which is beneficial for the colonization of the strain in the lung mucosa and the long-term therapeutic effect. The upper figure shows that M@Lap_4 has a significant antibacterial effect on Pseudomonas aeruginosa PA14. The lower figure shows that M@Lap_4 has a significant antibacterial effect on Staphylococcus aureus. The antibacterial ability is significantly enhanced after coating modification, which can reduce the risk of secondary lung infection.

[0136] Example 11

[0137] To systematically evaluate the impact of engineered bacteria on the lung microbiota, this study performed 16S rDNA sequencing analysis on bronchoalveolar lavage fluid from mice in different treatment groups. Lipopolysaccharide (LPS) stimulation caused severe imbalance in the lung microbiota, significantly reducing microbial diversity; only 22 independent operational taxa (OTUs) were identified. Figure 13 As shown; ( Figure 13 a) The effects of different interventions on the composition of the lung microbiota in mice; Figure 13 b) Cluster analysis results of lung microbiota; M@Lap_4 can efficiently restore the microbial diversity damaged by LPS-induced lung injury, with particularly significant repair effects; the above results suggest that M@Lap_4 can improve lung tissue damage by regulating lung microbiota homeostasis. Figure 13 c) Relative abundance distribution of lung flora in mice of each treatment group; bar height represents the abundance level of the flora; species composition analysis showed that the four dominant bacterial genera in mouse lungs were *Acinetobacter*, *Pseudomonas*, *Acidovorax*, and *Bradyrhizobium*. Figure 13 d) Differential microbial species analysis between the engineered bacteria intervention group and the model group; M@Lap_4 intervention can specifically downregulate the relative abundance of potentially pathogenic genera such as *Acidobacterium* and *Oligotrophomonas*, while reducing the abundance of *Dubosiella*, which is associated with inflammatory states; Figure 13 e) Statistical analysis of the abundance differences of Lactobacillus spp. in the lungs of mice in each group (n=5); More importantly, the iron-borax nano-coating combined with the surface modification process of sodium alginate can significantly increase the colonization of beneficial Lactobacillus in the lungs ( Figure 13(d, 13e) confirms that material modification technology can effectively help probiotics colonize stably in the damaged lung micro-ecosystem. Error bars represent standard deviations, and statistical analysis was performed using a two-tailed Student's test (*p<0.05; **p<0.01; ***p<0.001). LPS caused a sharp drop in bacterial diversity; M@Lap_4 significantly restored bacterial diversity; species abundance distribution showed downregulation of pathogenic bacteria and upregulation of beneficial bacteria; M@Lap_4 reduced harmful bacteria such as *Acidobacterium* and *Oligotrophomonas*; and significantly increased the colonization abundance of *Lactobacillus*.

[0138] 16S rRNA sequencing experimental method: Following the kit instructions, microbial genomic DNA was extracted from bronchoalveolar lavage fluid samples using the Omega Bio-tek EZNA Soil DNA Extraction Kit (USA). DNA integrity, concentration, and purity were assessed by 1.0% agarose gel electrophoresis and a Thermo Fisher NanoDrop 2000 spectrophotometer (USA). The V3–V4 variable regions of the 16S rRNA gene were amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) in a Bio-Rad T100 gradient PCR instrument (USA).

[0139] 20μL PCR amplification system: 4μL 5×FastPfu buffer, 2μL 2.5mM dNTP mixture, 0.8μL each of forward and reverse primers (5μM), 0.4μL FastPfu high-fidelity DNA polymerase, and 10ng template DNA.

[0140] PCR amplification program: 95℃ pre-denaturation for 3 min; 27 cycles: 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s; 72℃ final extension for 10 min, and product stored at 4℃.

[0141] The amplified products were separated and recovered by 2% agarose gel electrophoresis, purified using a Chinese YuHua PCR purification kit, and the DNA concentration was measured using a Thermo Fisher Scientific Qubit 4.0 real-time fluorescence instrument. After constructing the sequencing library, paired-end sequencing (2×300bp reads) was performed on the Illumina NextSeq 2000 sequencing platform. Subsequent bioinformatics analysis was performed according to standard methods reported in published literature.

[0142] Example 12

[0143] Lung metabolomics regulation, such as Figure 14 As shown; ( Figure 14 a) The effects of different intervention methods on the composition of lung microbiota-related metabolites in mice; Figure 14b) Cluster analysis results of lung metabolic profiles in each group; Figure 14 c) Differential metabolite distribution characteristics among different treatment groups; Figure 14 d) KEGG pathway enrichment analysis of differentially enriched metabolites. The x-axis represents the pathway enrichment rate, and the y-axis represents the name of the enriched KEGG pathway. Bubble size indicates the number of differentially enriched metabolites in each pathway, and bubble color represents the statistical significance (p-value) of the pathway enrichment. PCA showed that M@Lap_4 regressed the metabolite profile towards the normal group; metabolite clustering was clear, and the intervention group was significantly distinguishable from the model group; protective metabolites such as indole-3-butyric acid (IBA) and fumaric acid were significantly upregulated; KEGG was enriched in immune repair pathways such as purine metabolism and arginine biosynthesis.

[0144] like Figure 15 As shown in the heatmap, M@Lap_4 can reverse lipid metabolism disorders and downregulate inflammation-related metabolites.

[0145] Metabolomics Detection Methods: Metabolomics analysis was performed at Shanghai Meiji Biotechnology Co., Ltd. using a UHPLC-QExactive liquid chromatography-mass spectrometry system. The chromatographic column was a Waters ACQUITY HSST3 column (100 mm × 2.1 mm, particle size 1.8 μm). Mobile phase system: Phase A was a water / acetonitrile mixture containing 0.1% formic acid (volume ratio 2:98); Phase B was pure acetonitrile containing 0.1% formic acid. Flow rate: 0.40 mL / min; column temperature: 40℃; injection volume: 5 μL.

[0146] A QExactive mass spectrometer with an electrospray ionization (ESI) source was used in series with a liquid chromatography system to acquire data in both positive and negative ion modes. The optimized mass spectrometry parameters were as follows: ion source temperature 400℃; sheath gas flow rate 40 arb; auxiliary gas flow rate 10 arb; spray voltage: -2800V for negative ion mode, 3500V for positive ion mode; secondary mass spectrometry fragmentation used a stepped collision energy, with normalized collision energies set to 20V, 40V, and 60V respectively. Data acquisition was performed in data-dependent mode (DDA), with a mass-to-charge ratio (m / z) scan range of 70–1050.

[0147] Liquid chromatography-mass spectrometry (LC-MS) data analysis employed ProgenesisQI software (Waters Biotechnology, Milford, USA) to preprocess the raw LC-MS data, generating a 3D data matrix in CSV format. The matrix contained sample information, metabolite IDs, and corresponding peak intensities. Internal standards and known interference signals (including noise, column bleed signals, and reagent-derived peaks) were removed, followed by peak redundancy removal and alignment. Metabolite annotation was performed by searching the Human Metabolomics Database (HMDB, http: / / www.hmdb.ca), the Metlin Metabolite Database (https: / / metlin.scripps.edu), and the MJDB metabolite database (Shanghai Meiji Biotechnology Co., Ltd., China). The processed data matrix was uploaded to the Meiji Cloud Platform (https: / / cloud.majorbio.com) for subsequent statistical analysis.

[0148] Example 13

[0149] To further explore the molecular mechanism of the anti-inflammatory effect of the strain, this study constructed an in vitro inflammation model using RAW264.7 macrophages. Lipopolysaccharide stimulation strongly activated the inflammatory response, and pro-inflammatory factors such as IL-6, TNF-α, and IL-1β were significantly upregulated (see appendix). Figure 16 a, Figure 16 b、 Figure 16 d); Both Lap_4 and M@Lap_4 interventions significantly downregulated the aforementioned pro-inflammatory factors, with M@Lap_4 intervention showing better results. Simultaneously, the expression of the anti-inflammatory marker cytokine interleukin-10 (IL-10) was significantly upregulated (see appendix). Figure 16 c).

[0150] To clarify whether this anti-inflammatory phenotypic shift is mediated by macrophage polarization regulation, flow cytometry was used to detect macrophage surface markers CD86 (marker of M1 macrophages) and CD206 (marker of M2 macrophages). Flow cytometry results showed that both Lap_4 and M@Lap_4 significantly reduced the M1 / M2 polarization ratio, with M@Lap_4 exhibiting the most significant regulatory effect (see attached). Figure 16 e). The above results indicate that M@Lap_4 can regulate macrophage polarization and promote the transformation of pro-inflammatory M1 macrophages into reparative M2 macrophages.

[0151] Liquid chromatography-mass spectrometry (LC-MS) data analysis employed ProgenesisQI software (Waters Biotechnology, Milford, USA) to preprocess the raw LC-MS data, generating a 3D data matrix in CSV format. The matrix contained sample information, metabolite IDs, and corresponding peak intensities. Internal standards and known interference signals (including noise, column bleed signals, and reagent-derived peaks) were removed, followed by peak redundancy removal and alignment. Metabolite annotation was performed by searching the Human Metabolomics Database (HMDB, http: / / www.hmdb.ca), the Metlin Metabolite Database (https: / / metlin.scripps.edu), and the MJDB metabolite database (Shanghai Meiji Biotechnology Co., Ltd., China). The processed data matrix was uploaded to the Meiji Cloud Platform (https: / / cloud.majorbio.com) for subsequent statistical analysis.

[0152] All experimental data were obtained from at least three biological replicates per group, and results are expressed as **mean ± standard deviation (SD)**. For normally distributed data, statistical differences between groups were assessed using either independent samples t-tests or one-way ANOVA, depending on the experimental conditions.

[0153] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A ROS-responsive surface functional coating, characterized in that: The coating is a bilayer structure formed by the assembly of an inorganic Fe-B inner layer and a sodium alginate SA-PBA outer layer grafted with phenylboronic acid through dynamic borate ester bonds. The Fe-B inorganic inner layer is composed of Fe 3+ It coordinates with borate in situ on the surface of probiotic cells to form ROS scavenging activity; The SA-PBA outer layer is covalently connected to the Fe-B inorganic inner layer surface through the borate ester bond, and the borate ester bond breaks under high ROS microenvironment, realizing the responsive dissociation of the coating; The coating can be applied to the surface of probiotic cells without affecting cell activity and functional expression.

2. The ROS-responsive surface functional coating according to claim 1, characterized in that: The SA-PBA was prepared by an amidation reaction of sodium alginate (SA) and 3-aminophenylboronic acid via EDC / NHS catalytic reaction. The Fe-B inner layer is formed by in-situ mineralization of FeCl3 and borax on the surface of the bacterial cell.

3. The ROS-responsive surface functional coating according to claim 1, characterized in that: The probiotic is an engineered Lactobacillus paracasei Lap_4 that secretes IL-4; The Lap_4 uses Lactobacillus paracasei as a chassis and constitutively expresses and secretes mouse-derived IL-4 anti-inflammatory cytokine through recombinant plasmids.

4. A method for preparing a ROS-responsive surface functional coating as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Preparation of engineered Lactobacillus paracasei Lap_4 that secretes IL-4; S2. Preparation of grafted modified sodium alginate SA-PBA; S3. An in-situ Fe-B inorganic inner layer is formed on the surface of Lap_4 cells; S4. By binding the SA-PBA outer layer with borate ester bonds, engineered probiotics coated with a ROS-responsive coating are obtained.

5. The preparation method according to claim 4, characterized in that... In step S3, the bacterial cells are resuspended in Tris-HCl buffer at pH 8.0, and FeCl3 solution and borax solution are added sequentially. After vortexing, Fe-B inner layer is generated in situ. In step S4, SA-PBA solution was added, and the reaction was carried out at 37°C and 500 rpm for 0.5 h to obtain double-layer coated Lap / Fe-B / SA.

6. The preparation method according to claim 4, characterized in that: The preparation steps of SA-PBA are as follows: EDC, NHS and 3-aminophenylboronic acid are added to an aqueous solution of sodium alginate, the mixture is stirred at room temperature, dialyzed and freeze-dried to obtain SA-PBA solid.

7. The ROS-responsive surface functional coating according to claim 3, characterized in that, The method for constructing the engineered Lactobacillus paracasei Lap_4 is as follows: The mouse IL-4 gene fused with the secretory signal peptide SPUSP45 was inserted into the pIB184 plasmid carrying the constitutive strong promoter P23 to construct the recombinant plasmid pIB184-SP-IL-4. After amplification by Escherichia coli, the sample was electroporated to Lactobacillus paracasei, and Lap_4, which can constitutively secrete IL-4, was obtained through screening.

8. The use of the ROS-responsive surface functional coating according to any one of claims 1-3 in the preparation of a therapeutic agent for acute lung injury or acute respiratory distress syndrome.

9. The application according to claim 8, characterized in that: The formulation is used to clear excess ROS in the lungs, inhibit pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, induce macrophage polarization towards the M2 anti-inflammatory phenotype, reduce the wet-to-dry weight ratio of the lungs, repair alveolar structure, and regulate the lung microecology and metabolic homeostasis.

10. The application according to claim 8, characterized in that: The formulation is suitable for LPS-induced or Pseudomonas aeruginosa-induced acute lung injury models, and has synergistic effects of antioxidation, anti-inflammation, immune regulation, antibacterial activity, and microecological remodeling.

Images