An in-situ injectable mRNA-hydrogel microsphere composite and a preparation method and application thereof

Abstract

The application provides an in-situ injectable mRNA-hydrogel microsphere complex, a preparation method and application thereof, and belongs to the technical field of biological medicine. The application constructs an injectable mRNA / sgRNA lipid nanoparticle-gel micro-complex, which is used for functionally reprogramming immune-suppressed macrophages in situ at a lesion, and aims to simultaneously strengthen the bactericidal capacity and restore the immune activation function. The encapsulation of the hydrogel microspheres makes the LNP stably stay at the lesion site, and the released phosphatidylserine modified LNP can realize selective delivery to macrophages through TAM receptors. The knockout of MerTK enhances the phagocytic capacity of macrophages and activates the pro-inflammatory signaling pathway, and the delivery of LL-37 mRNA realizes the continuous secretion of antibacterial peptides, thereby significantly enhancing the bactericidal effect. The in-vitro and in-vivo experimental results show that the LMP can realize precise positioning expression at the lesion site, significantly enhance the phagocytic activity, intracellular clearance capacity and antigen presentation function of macrophages, thereby overall inhibiting the progress of implant-related infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an injectable mRNA-hydrogel microsphere complex, its preparation method, and its application. Background Technology

[0002] Methicillin-resistant Staphylococcus aureus (MRSA)-induced implant-associated infections (IAIs) are among the most serious complications following orthopedic surgery. Macrophages are the core of the antimicrobial defense system, but their role is often limited in MRSA-related IAIs: they should be able to rapidly clear pathogens in the early stages and bridge specific immunity through antigen presentation, but this is weakened or even directly damaged by MRSA's multiple immune escapes and inflammatory manipulation. Therefore, it is necessary to simultaneously enhance the "offensive" (antimicrobial) and "defensive" (cellular protection / immune regulation) functions of macrophages.

[0003] However, currently used antibiotic treatments, immunomodulatory drugs, and implant antimicrobial coatings / carriers lack precise synergy across multiple antimicrobial and immune pathways, and struggle to maintain efficacy under lesion conditions such as low pH and high oxidative stress, thus failing to address the complex pathology of intralesional arterial disease (IAI). Therefore, there is an urgent need for in-situ programming tools that are stable in the lesion microenvironment and capable of synergistically regulating multiple pathways, to simultaneously endow macrophages with both "spear" and "shield" to efficiently intervene in MRSA-mediated IAI.

[0004] In routine bacterial infections, macrophages possess a comprehensive "arsenal": they take up pathogens through phagocytosis, rely on phagolysosomes for bactericidal action and autophagy for intracellular clearance, and secrete pro-inflammatory and chemokine-like factors to recruit neutrophils, NK cells, and T cells; simultaneously, they act as antigen-presenting cells to activate specific immunity. However, in MRSA-mediated immunoassay (IAI), this system is weakened by multiple mechanisms: MRSA inhibits phagocytosis through its capsule and Protein A, blocks phagosome-lysosome fusion, and clears ROS to evade immune killing; it secretes Panton-Valentine leukotoxin and α-toxin to directly damage macrophages; it releases superantigens to induce non-specific T cell activation and inflammatory storms; and it defends against immunity and drugs with biofilms and a low metabolic state. Meanwhile, pathogens can also hijack the Tyro3-Axl-MerTK (TAM) pathway of macrophages, inhibiting immune activation and further amplifying immunosuppression. Although macrophages can secrete antimicrobial peptides that are still effective against MRSA (such as LL-37), their upstream pathways are also deactivated in the context of immunosuppression, making it difficult for them to exert a limited and effective attack. In summary, in the pathological microenvironment of IAI, macrophages lack both a powerful "spear" (efficient bactericidal action) and a "shield" (self-protection and immune regulation), making it difficult to effectively suppress MRSA.

[0005] Gene editing technologies, exemplified by CRISPR-Cas9, hold immense potential for cellular functional reprogramming, and in vitro editing has already achieved breakthroughs in the treatment of various diseases. Compared to traditional gene tools, CRISPR-Cas9 offers significant advantages in multi-gene synergistic regulation: it relies on sgRNA to precisely locate genomic sites, supports single-vector multi-sgRNA or multi-vector parallel processing, enabling simultaneous knockout / knock-in / regulation of multiple genes, and is compatible with technologies such as RNA interference and mRNA delivery, reducing pathway crosstalk. With the rapid development of lipid nanoparticles (LNPs), gene editing is accelerating its in vivo application. LNPs possess excellent biocompatibility, low immunogenicity, and high delivery efficiency, protecting Cas9 mRNA and sgRNA from in vivo degradation and achieving targeted delivery, thereby significantly improving gene editing efficiency and reducing off-target effects.

[0006] In the complex pathological microenvironment, macrophages are often remodeled into an immunosuppressive state, characterized by decreased bactericidal efficacy and limited immune activation. This functional imbalance is particularly pronounced in implant-associated infections (IAIs), making it difficult for traditional antibiotics and immunomodulatory strategies to achieve sustained and multi-pathway immune control at the macrophage level. Therefore, developing a delivery system that integrates multi-gene / multi-pathway programming tools for macrophages to simultaneously enhance the relationship between the "spear" (efficient bactericidal action) and the "shield" (self-protection and immune regulation) to effectively suppress MRSA has become an urgent technical challenge. Summary of the Invention

[0007] This invention aims to solve the aforementioned technical problems by providing an injectable mRNA-hydrogel microsphere complex, its preparation method, and its applications. The technical objective of this invention is to address the limitations of existing antibacterial strategies, which are confined to extracellular sterilization or single immune regulation, making it difficult to effectively eliminate intracellular residual bacteria and restore host immune function. This invention provides a multi-gene / multi-pathway programming tool integrating a nanodelivery system and macrophages, while simultaneously strengthening the relationship between the "spear" (efficient sterilization) and the "shield" (self-protection and immune regulation), significantly improving the clearance efficiency of intracellular MRSA.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides a method for preparing an injectable mRNA-hydrogel microsphere complex, comprising the following steps: (1) DLin-MC3-DMA, distearate phosphatidylcholine, cholesterol, 2,3-dioleoyl-propyl-trimethylammonium and phosphatidylserine were mixed in the ethanol phase; LL-37 mRNA and Cas9 mRNA / sgMerTK were mixed in citrate buffer; mRNA-LNP was prepared by microfluidic technology. (2) Using GelMA solution as the aqueous phase and Span80 and liquid paraffin as the oil phase, droplets were prepared by microfluidic technology, and then GelMA microspheres were prepared by crosslinking with a photoinitiator; GelMA microspheres were mixed with dopamine solution to form a polydopamine coating in situ. (3) The GelMA microspheres coated with polydopamine were immersed in the mRNA-LNP suspension of step (1) to construct an LNP-hydrogel microsphere complex that can be injected in situ.

[0009] This invention achieves highly efficient in-situ multiplex gene engineering of macrophages by fusing CRISPR-Cas9 with mRNA LMP tools and optimizing the formulation, thereby resisting MRSA and inhibiting IAI. First, negatively charged porous gelatin methacrylamide microspheres were prepared using microfluidic technology. Then, five components were added (DC-cholesterol and phosphatidylserine (PS) and the PEG component was removed) to construct a weakly positively charged immunosuppressive macrophage-targeting LNP system. Finally, mRNA LMP was prepared through electrostatic assembly.

[0010] To achieve more effective gene editing and engineering, we designed a multi-gene delivery strategy, including a MerTK knockout system (Cas9 mRNA + MerTK sgRNA) and an antimicrobial peptide secretion system (signal peptide-LL37 mRNA), which enhanced the defense and attack mechanisms against MRSA, respectively. In vitro and in vivo experiments verified the immune enhancement and MRSA-killing effect of gene-edited engineered macrophages. Furthermore, we verified the function of macrophages as antigen-presenting cells (APCs) in activating T cells to achieve MRSA-specific immunity.

[0011] In summary, this invention achieves multi-gene regulation of macrophages in vivo through a novel mRNA LMP tool, giving macrophages both a "spear" and a "shield" against MRSA, and ultimately inhibiting the occurrence of IAI.

[0012] Studies have shown that immunosuppressive macrophages highly express TAM receptors and preferentially recognize phosphatidylserine (PS) residues on the surface of apoptotic cells, enhancing endocytosis-related cell death. Introducing PS onto the surface of nanoparticles allows for selective targeting of immunosuppressive macrophages. Based on this, selective knockout of TAM family members (such as MerTK) using CRISPR-Cas9 has the potential for macrophage reprogramming. Simultaneously, direct delivery of recoded antimicrobial peptide mRNA via LNP can bypass suppressed upstream signals, continuously inducing macrophages to secrete effector molecules (such as modified antimicrobial peptides) that remain highly effective against MRSA. In summary, the combination of Cas9 mRNA, TAM sgRNA, and antimicrobial peptide mRNA provides macrophages with an integrated multi-gene / multi-pathway programming tool, potentially enhancing both the "spear" (bactericidal) and the "shield" (self-protection and immune regulation), thereby effectively inhibiting MRSA-related IAI.

[0013] The inventiveness of this invention is further explained below: (1) The composite system proposed in this invention is not a simple superposition of existing delivery materials. In the LNP part, by introducing PS and removing PEG, and combining it with DOTAP, the system can simulate cell burial-related signals and be preferentially recognized and taken up by immunosuppressive macrophages, thereby achieving specific delivery to target cells. If a conventional LNP system containing PEG is used, the uptake efficiency of macrophages will be significantly reduced due to the shielding effect, resulting in the failure of mRNA delivery in the infection microenvironment. At the same time, relying solely on the LNP system makes it difficult to achieve effective retention at the infection site, and it is easily cleared quickly in the inflammatory microenvironment, making it difficult to maintain sustained expression. However, after introducing hydrogel microspheres, the porous structure can achieve local fixation and sustained release of LNP, thereby significantly prolonging the action time of the delivery system at the lesion site. Without this microsphere structure, the delivery system will spread rapidly, making it difficult to produce a stable therapeutic effect.

[0014] (2) In terms of functional design, the MerTK gene knockout system and the antimicrobial peptide LL37 expression system are synergistically integrated. The former is used to relieve the immunosuppressive state of macrophages, and the latter is used to provide a sustained antimicrobial effect. The two achieve synergistic effects in the same delivery system. If only a single strategy is used, it is difficult to achieve both immune reconstitution and efficient sterilization at the same time, whether it is antimicrobial peptide delivery or gene editing.

[0015] (3) Existing antibacterial strategies are mostly limited to extracellular sterilization or single immune regulation, which are difficult to effectively eliminate intracellular residual bacteria and restore host immune function. However, this invention achieves sustained expression of antimicrobial peptide mRNA in macrophages through lipid nanoparticle-mediated mRNA delivery, thereby overcoming the limitations of traditional antimicrobial molecules that are difficult to enter cells and are easily degraded, and significantly improving the clearance efficiency of intracellular MRSA. On this basis, by knocking out MerTK through the CRISPR system, the immunosuppressive state of macrophages in the infection microenvironment is effectively reversed, and their phagocytic and pro-inflammatory functions are restored. At the same time, the antigen presentation function of macrophages is enhanced, T cell activation is promoted, and a specific immune response against MRSA is formed, showing a shift from simple treatment to the establishment of immune memory.

[0016] Furthermore, among the ionizable lipids, phospholipids, cholesterol, cationic lipids, and phosphatidylserine mentioned in step (1), ionizable lipids include, but are not limited to: Dlin-MC3-DMA, SM-102, ALC-0315, etc. Phospholipids include, but are not limited to: 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine (DSPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), etc. Cationic lipids include, but are not limited to: 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3-dioleoyloxypropyl)]-N,N,N-trimethylammonium (DOTMA), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), etc.

[0017] Furthermore, the weight percentages of the ionizable lipids, phospholipids, cholesterol, cationic lipids and phosphatidylserine mentioned in step (1) are 40~60%: 10~30%: 20~40%: 0~20%: 0~5%.

[0018] Furthermore, the weight ratio of LL-37 mRNA and Cas9 mRNA / MerTK sgRNA in step (1) is 1:1 to 20:1.

[0019] Furthermore, in step (2), the weight ratio of GelMA solution, Span80 and liquid paraffin is 1:0.01~0.2:5~20.

[0020] Furthermore, the concentration of the mRNA-LNP suspension in step (3) is 0.01–10 mg / mL.

[0021] A second objective of this invention is to provide an injectable mRNA-hydrogel microsphere complex prepared by the method described above.

[0022] A third objective of this invention is to provide the application of the injectable mRNA-hydrogel microsphere complex described above in the preparation of medicaments for treating implant-related infections.

[0023] The beneficial effects of this invention are as follows: This invention constructs an injectable mRNA / sgRNA lipid nanoparticle-gel microcomplex (LMP) for in-situ functional reprogramming of immunosuppressive macrophages at lesion sites, aiming to simultaneously enhance their bactericidal ability (“spear”) and restore immune activation function (“shield”). We identified MerTK as not only a key receptor mediating macrophage Tyro3-Axl-MerTK (TAM) pathway-dependent endocytosis, but also a core regulatory node driving immunosuppression. Encapsulation with hydrogel microspheres allows LNPs to remain stably at the lesion site, while the released phosphatidylserine-modified LNPs can selectively deliver to macrophages via the TAM receptor. MerTK knockout enhances macrophage phagocytosis and activates pro-inflammatory signaling pathways, while LL-37 mRNA delivery enables sustained secretion of antimicrobial peptides, thereby significantly enhancing the bactericidal effect. In vitro and in vivo experimental results show that LMPs can be precisely expressed locally at the lesion site, significantly enhancing macrophage phagocytic activity, intracellular clearance capacity, and antigen presentation function, thereby overall inhibiting the progression of implant-related infections. This invention proposes an immune engineering strategy based on in situ functional editing, providing a new approach for the treatment of refractory infections. Attached Figure Description

[0024] Figure 1Sequencing analysis and molecular validation of MerTK targets in macrophages under infected conditions; (A) Schematic diagram of in vitro and intracellular infection model of methicillin-resistant Staphylococcus aureus (MRSA) and sequencing; (B) Volcano plot of differentially expressed genes (DEGs) between infected and uninfected groups; (C) Heatmap of mRNA transcription profiles between infected and uninfected groups showing differential expression of genes involved in pro-inflammatory and anti-inflammatory activities; (D) GO and KEGG enrichment analysis of differentially expressed genes between infected and uninfected groups; (E, F) GESA assessment of representative immune signaling pathways in macrophages after infection; (G) Violin plot showing the effects of GESA on the immune signaling pathways in macrophages after infection. Functional scores of M1 pro-inflammatory macrophages in the control group and MRSA group; (H) Correlation analysis between MerTK gene expression level and M1 functional score; (I) Immunofluorescence staining to verify the upregulation of MerTK in macrophages after intracellular MRSA infection in vitro and immunohistochemical images of sections; red fluorescence represents MerTK, and blue represents DAPI-labeled nuclei; (J) Immunofluorescence staining to verify the upregulation of MerTK in the infected animal model; red fluorescence represents CD68 (macrophage marker), green fluorescence represents MerTK, and blue represents DAPI-labeled nuclei; (K) Western blot analysis of macrophage phenotypic changes after infection and MerTK intervention; (L) MerTK knockout enhances the ability of macrophages to clear intracellular MRSA; Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test; n = 3, **p < 0.01. Data are expressed as mean ± standard deviation.

[0025] Figure 2Characterization of the preparation, physicochemical properties, and intracellular delivery performance of LNPs; (A) Schematic diagram of the preparation process of lipid nanoparticles (LNPs); (B) Particle size distribution, PDI, and mRNA encapsulation efficiency of LNPs; (C) Zeta potential of LNPs; (D) Changes in particle size and PDI stability of LNPs stored at 4°C for 14 days; (E) Representative transmission electron microscopy (TEM) images of LNPs; (F) Tyndall effect of mRNA-LNPs; (G) TNS fluorescence curve of mRNA-LNPs; (H) Representative fluorescence image of EGFP protein expression after co-culturing EGFP mRNA-LNPs with Raw264.7 cells and BMDM for 48 hours; (I) mRNA release behavior of mRNA-LNPs under different pH conditions; (J) Confocal images of Raw264.7 cells and BMDM after treatment with Fr-RNAs, mRNA-LNPs, and PS / mRNA-LNPs; RNA was labeled with Cy5 (red), and cell nuclei were labeled with Hoechst. 33342 (blue) counterstaining; (K, L) flow cytometry analysis of cellular uptake efficiency of Fr-RNAs, mRNA-LNPs, and PS / mRNA-LNPs in Raw264.7 cells and BMDM; (M, N): representative confocal images of Raw264.7 cells and BMDM incubated with PS / mRNA-LNPs at 37°C for 1, 3, and 6 hours; cell nuclei stained with Hoechst 33342 (blue), endosomes / lysosomes stained with LysoTracker Green (green), and RNA labeled with Cy5 (red); fluorescence contour scans are shown on the right; scale bar, 20 μm; statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test; n = 3, **** p < 0.0001; data are expressed as mean ± standard deviation.

[0026] Figure 3 Preparation and characterization of LMP-mRNA; (A) Schematic diagram of the LMP-mRNA hydrogel microsphere preparation process; (B, C) Bright-field images of hydrogel microspheres; (D) Particle size distribution of hydrogel microspheres; (E) PDA-coated hydrogel microspheres modified with polydopamine (PDA) coating; (F, G) Scanning electron microscope (SEM) images of lyophilized microspheres; (H) Fluorescence images of LNP loaded on hydrogel microspheres; (I) Encapsulation efficiency of LNP in LMP and LMP-NC; (J) Cumulative release curves of LNP in LMP and LMP-NC; (K) Representative images of the degradation process of microspheres over time under an inverted microscope; (L) Degradation curve of microsphere weight over time; (M) XPS spectroscopy confirms the elemental composition of GM and LMP-NC; Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test; n = 3, data are expressed as mean ± standard deviation.

[0027] Figure 4 Construction and validation of MerTK gene knockout and mRNA-mediated LL37 expression in macrophages; (A) Schematic diagram of a lipid nanoparticle (LNP)-based mRNA / CRISPR co-delivery system, used to simultaneously achieve Cas9 / sgMerTK-mediated MerTK gene knockout and LL37 mRNA-mediated antimicrobial peptide expression in macrophages; (B, C) Western blot and quantitative analysis of MerTK protein expression; (D) q-PCR analysis of the relative mRNA expression level of MerTK in BMDM after specified treatment; (E) Representative fluorescence images of the control group and LNP-MTK- / - after in vitro incubation with BMDM; (F, G) Western blot and quantitative analysis of LL37 protein expression; (H) Quantitative analysis of LL37 secretion level in culture medium after LNP-LL37 treatment; (I) Representative fluorescence images of the control group and LNP-LL37 after in vitro incubation with BMDM; (JL) Western blot and quantitative analysis of LL37 and MerTK protein expression. Blot and quantitative analysis; (M) Representative fluorescence images of the control group, LNP-LL37, LNP-MTK- / - and LNP-LL37 / MTK- / - after in vitro incubation with BMDM; Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison test; n=3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no statistical significance; Data are expressed as mean ± standard deviation.

[0028] Figure 5The effects of LNP-LL37 / MTK- / - on the phagocytic and bactericidal functions of macrophages; (A) Schematic diagram of a Transwell co-culture system simulating the chemotaxis of macrophages in conditioned medium; (B) Typical images of BMDM cells cultured in Transwell under different conditioned mediums; (C) Typical images of macrophages phagocytizing bacteria. (D) Green fluorescence indicates Staphylococcus aureus, red fluorescence indicates macrophages, and blue fluorescence indicates cell nuclei; (F) Representative flow cytometry image of macrophages phagocytizing and clearing bacteria; (G) Representative photograph of standard colony counts of Staphylococcus aureus phagocytosed by macrophages; (H) Analysis of the number of colonies formed after macrophages phagocytose bacteria; (I) Quantitative results of macrophage phagocytosis rate; (J) Analysis of the number of colonies formed by surviving bacteria after macrophage phagocytosis; (X) Bactericidal rate calculated based on the number of surviving bacteria after phagocytosis; (X) Live / dead fluorescence images of Staphylococcus aureus; SYTO9 staining (green) indicates live bacteria, PI staining (red) indicates dead bacteria; (XK, L) Live / dead flow cytometry and quantitative images of Staphylococcus aureus; (M) Evaluation of membrane permeability of Staphylococcus aureus after different treatments using ONPG hydrolysis method; (N) Quantitative analysis of the proportion of intracellular protein leakage in S. aureus after different treatments; (O) S. aureus after different treatments. Scanning electron microscopy (SEM) images of *S. aureus*, with arrows indicating morphological changes; (P) Transmission electron microscopy (TEM) images of *S. aureus* after different treatments, with arrows indicating morphological changes; Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test; n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no statistical significance. Data are expressed as mean ± standard deviation.

[0029] Figure 6(A) Schematic diagram of immune phenotype remodeling and immunomodulatory function mediated by engineered macrophages; (B) Typical scatter plot of macrophage surface markers CD86 (pro-inflammatory / immunoenhancing macrophage marker) and CD206 (anti-inflammatory / immunosuppressive macrophage marker) detected by flow cytometry; (C) Immunofluorescence staining images of BMDM after different treatments; green fluorescence represents iNOS (pro-inflammatory macrophage marker), red fluorescence represents Arg-1 (anti-inflammatory macrophage marker), and blue fluorescence represents the cell nucleus; (D, E) Quantitative analysis of iNOS and Arg-1 immunofluorescence staining; (FI) q-PCR results showing the expression levels of representative genes; (J, K) Representative flow cytometry analysis and quantitative results of dendritic cells (DCs); (L, M) Representative flow cytometry analysis and quantitative results of myeloid-derived suppressor cells (MDSCs); Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test; n =3, ***p < 0.001, ****p < 0.0001, ns indicates no statistical significance; data are expressed as mean ± standard deviation.

[0030] Figure 7 To illustrate the therapeutic effect of engineered macrophage strategy in an in vivo implant-associated infection model; (A) Schematic diagram of the establishment and treatment process of the rat implant infection model; (B) Macroscopic images of infected knee joints in rats of different treatment groups; (C) Mean circumference curves of infected knee joints in rats of different treatment groups; (D) Core body temperature changes in rats of different treatment groups; (E) Two-dimensional images of the distal femur of rats; (F, G) Three-dimensional reconstructed images of the distal femur of rats; (H) Bone mineral density (BMD); (I) Bone volume fraction (BV / TV); (J) Trabecular bone number (Tb.N); (K) Standard colony count plate photographs of implants and surrounding tissues; (L) Representative images stained with HE; (M) Representative images stained with Masson's trichrome; (N) Representative images stained with Gram; Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test; n = 3, p < 0.0001, ns indicates no statistical significance; Data are expressed as mean ± standard deviation.

[0031] Figure 8To assess the remodeling of the immune microenvironment surrounding the implant; (A) Representative immunofluorescence staining image of iNOS; (B) Representative immunofluorescence staining image of CD206; (C) Representative immunofluorescence staining image of CD4; (D) Representative immunofluorescence staining image of Foxp3; (E) Immunohistochemical image after staining with TNF-α antibody; (F) Immunohistochemical image after staining with IL-1β antibody; (G) Quantitative analysis of relative fluorescence intensity of iNOS; (H) Quantitative analysis of relative fluorescence intensity of CD206; (I) Quantitative analysis of relative fluorescence intensity of CD4; (J) Quantitative analysis of relative fluorescence intensity of Foxp3; Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test; n = 3, **p < 0.01, ****p < 0.0001; Data are expressed as mean ± standard deviation.

[0032] Figure 9 (A) Schematic diagram of mRNA therapy for implant-associated infection; (B, C) Representative flow cytometry analysis and quantitative results of pro-inflammatory M1-like macrophages (CD86⁺) in implant-draining lymph nodes (IDLNs) after different treatments; (D, E) Representative flow cytometry analysis and quantitative results of anti-inflammatory M2-like macrophages (CD206⁺) in IDLNs after different treatments; (F, G) Representative flow cytometry analysis and quantitative results of dendritic cells (DCs) in IDLNs after different treatments; (H, I) Representative flow cytometry analysis and quantitative results of myeloid-derived suppressor cells (MDSCs) in IDLNs after different treatments; (J) CD4⁺ T cells and CD8⁺ T cells in IDLNs after different treatments. Representative flow cytometry analysis of cells; (K) Quantitative analysis of the ratio of CD4⁺ T cells to CD8⁺ T cells (CD4⁺ / CD8⁺) in IDLNs after different treatments; (L, M) Serum IFN-γ (L) and TNF-α (M) levels detected by ELISA kits; (N, O) Serum IgG (N) and IgM (O) levels detected by ELISA kits; Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test; n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001; Data are expressed as mean ± standard deviation.

[0033] Figure 10Transcriptome sequencing reveals reprogramming of immune-related pathways in the infection microenvironment; (A) Principal component analysis (PCA) plot of differentially expressed genes (DEGs); (B) Volcano plot of differentially expressed genes (DEGs); (C) Heatmap of differentially expressed genes (DEGs); (D) Chord plot connecting enriched GO entries and upregulated genes; (E) KEGG pathway enrichment analysis of differentially expressed genes (DEGs); (FI) GSEA for analyzing enriched pathways related to positive regulation of the immune process.

[0034] Figure 11 To demonstrate the role of LMP-LL37 / MTK- / - in inducing antimicrobial immune memory and preventing relapse; (A) Schematic diagram of LMP-LL37 / MTK- / - immunotherapy in preventing implant infection relapse; (B, D) Representative flow cytometry atlases and quantitative analysis of CD4+ T cell-gated memory CD4+ T cells (high CD44, low CD62L); (C, E) Representative flow cytometry atlases and quantitative analysis of CD8+ T cell-gated memory CD8+ T cells (high CD44, low CD62L); (F, G) Quantitative analysis of bacterial load in the implant and surrounding tissues using colony-forming units (CFU); (H, I) Serum IFN-γ (H) and TNF-α (I) levels detected using ELISA kits; (J, K) Serum IgG (J) and IgM (K) levels detected using ELISA kits; Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test; n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001; data are expressed as mean ± standard deviation.

[0035] Figure 12 A schematic diagram illustrating the use of a "spear-shield" synergistic lipid-hydrogel microcomplex (LMP) for anti-infective immunotherapy; (A) Preparation of lipid nanoparticles (LNP) loaded with LL-37 mRNA and Cas9 / sgMerTK using microfluidic mixing technology; (B) Preparation of hydrogel microspheres and LNP loading on their surface to form a lipid-hydrogel microcomplex (LMP); (C) After intra-articular in situ injection, LMP continuously releases LNP and is endocytosed by macrophages. LL-37 expression directly enhances bacterial killing activity ("spear"), while MerTK knockout alleviates infection-induced immunosuppression and restores immune activation ("shield"). This synergistic innate immune reprogramming further promotes adaptive immune responses, thereby achieving efficient bacterial clearance and durable immune protection.

[0036] Figure 13 Principal component analysis (PCA) for RNA-seq.

[0037] Figure 14 To assess representative immune signaling pathways in macrophages after infection using GESA (A: positive regulation of immunity mediated by T cells; B: positive regulation of immunity mediated by B cells; C: defense response against bacteria).

[0038] Figure 15 The image shows the fluorescence of Raw264.7 after double staining with Calcein-AM / PI.

[0039] Figure 16 The image shows the fluorescence of BMDM double-stained with Calcein-AM / PI.

[0040] Figure 17 Semi-quantitative results of Calcein-AM / PI double staining fluorescence for Raw 264.7.

[0041] Figure 18 Semi-quantitative results of Calcein-AM / PI double staining fluorescence of BMDMs.

[0042] Figure 19 The results of CCK-8 assay in RAW264.7 cells under different treatments.

[0043] Figure 20 CCK-8 detection results of BMDM under different treatments.

[0044] Figure 21 The ratio of green to red in live / dead bacteria detection.

[0045] Figure 22 Quantitative analysis of intracellular DNA leakage in S. aureus after different treatments.

[0046] Figure 23 Changes in body weight of rats in different treatment groups.

[0047] Figure 24 (A) Trabecular separation (Tb.Sp); (B) Trabecular thickness (Tb.Th); (C) Bone surface area to bone volume ratio (BS / BV). Figure 25 For (A) quantitative analysis of standard colony counts in implants; (B) quantitative analysis of standard colony counts in tissues surrounding implants.

[0048] Figure 26 For the quantitative analysis of peripheral blood routine and liver and kidney function indicators; (A) white blood cells; (B) red blood cells; (C) PLT; (D) ALT; (E) AST; (F) ALB; (G) UA; (H) CREA; (I) BUN.

[0049] Figure 27Biocompatibility of different treatment groups in vivo; representative H&E staining images of the heart, liver, spleen, lungs and kidneys.

[0050] Figure 28 To detect the level of IL-6 in serum using an ELISA kit.

[0051] Figure 29 The ELISA kit was used to detect the level of IL-10 in serum.

[0052] Figure 30 GSEA was used to analyze enriched pathways associated with positive regulation of the immune process; (A) NOD-like receptor signaling pathway; (B) natural killer cells.

[0053] Figure 31 Representative images of HE staining.

[0054] Figure 32 Representative images of Giemsa staining.

[0055] Figure 33 To detect the level of IL-6 in serum using an ELISA kit.

[0056] Figure 34 The ELISA kit was used to detect the level of IL-10 in serum. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example

[0058] I. Experimental Methods and Procedures 1. Preparation and characterization of lipid nanoparticles

[0059] LNPs were prepared using a microfluidic LNP preparation device (Suzhou Unigen Biotechnology Co., Ltd.). MC3, DSPC, cholesterol, DOTAP, and PS were precisely weighed and dissolved in anhydrous ethanol at a molar ratio of 50%:10%:38.5%:0.75%:0.75% to form the ethanol phase. LL37 mRNA, Cas9 mRNA, and sgRNA were dissolved in citrate buffer to form the aqueous phase. Then, the ethanol and aqueous phases were mixed at a volume ratio of 1:3 using the LNP device. The resulting mixture was diluted with PBS and transferred to an ultrafiltration tube (30 kDa) for centrifugation to remove ethanol, thus preparing mRNA-LNPs.

[0060] The particle size, polydispersity, and zeta potential of LNPs were evaluated using a Zetasizer Nano S system (Malvin Instruments, UK). First, the prepared LNPs were diluted to appropriate concentrations with PBS buffer. Samples for particle size and polydispersity measurements were placed in quartz cuvettes; the suspension for zeta potential measurements was placed in the Malvin sample cell. Measurements were performed at room temperature (approximately 25°C). Total mRNA and free mRNA content were determined using an RNA quantification kit (Nanjing Novizan Biotechnology Co., Ltd.). Encapsulation efficiency was calculated using the formula: EE (%) = (total mRNA − free mRNA) / total mRNA × 100%. The morphology of LNPs was observed using a transmission electron microscope (Hitachi, Japan). LNPs were stored in PBS at pH 7.4 at 4°C with gentle shaking on a shaker. Samples were taken at 1, 3, 7, 10, and 14 days to measure changes in particle size and PDI.

[0061] 2. In vitro mRNA-LNP transfection To evaluate in vitro transfection efficiency, an mRNA-LNP system loading EGFP mRNA was constructed. RAW264.7 cells in logarithmic growth phase and bone marrow-derived macrophages (BMDMs) were transfected at a rate of 3 × 10⁻⁶ mRNA. 5 Cells were seeded at a density of 10 cells / well in 6-well plates. After the cells adhered and stabilized, mRNA-LNP containing 1 μg of EGFP mRNA was added for transfection. After transfection, EGFP expression was observed using a fluorescence microscope to evaluate the in vitro transfection efficiency of mRNA-LNP.

[0062] 3. Preparation and characterization of hydrogel microsphere composite system First, methacrylamide gelatin (GelMA) was synthesized. 10 wt.% gelatin was dissolved in carbonate buffer (pH 9.0), and methacrylic anhydride was added under continuous stirring, with the reaction proceeding for 3 h. After the reaction, the resulting solution was dialyzed for 2 days to remove unreacted matter, and then freeze-dried to obtain GelMA hydrogel. An aqueous solution was prepared by dissolving GelMA and the photoinitiator LAP in deionized water; simultaneously, an oil solution was prepared by mixing paraffin oil and Span 80. The flow rate ratio of the aqueous and oil phases was controlled using a microfluidic device to form uniform droplets, and the cross-linking reaction was completed under ultraviolet light irradiation to prepare hydrogel microspheres. The obtained microspheres were freeze-dried and used in subsequent experiments.

[0063] To construct surface-functionalized microspheres, dopamine hydrochloride was dissolved in Tris-HCl buffer to prepare a 2 mg / mL dopamine solution. Lyophilized hydrogel microspheres were completely immersed in this solution and incubated on a shaker at room temperature for 24 h to allow a polydopamine coating to form in situ on their surface. After the reaction, the microspheres were repeatedly washed with sterile deionized water to remove unbound dopamine, yielding polydopamine-modified microspheres. Subsequently, these microspheres were immersed in an LNP-mRNA suspension and incubated at 4 °C for 24 h. LNP-mRNA adsorption on the microsphere surface was achieved through electrostatic interactions and hydrogen bonding, ultimately yielding the LMP complex system.

[0064] The physicochemical properties of LMP were systematically characterized. Microsphere size was measured using an inverted microscope. To observe morphological changes during degradation, LMP was immersed in PBS buffer (pH 7.4) containing 0.2 U mL⁻¹ collagenase and cultured in a constant-temperature shaker (37 °C, 60 rpm), with the collagenase-containing PBS solution replaced every 2 days. Samples were taken at predetermined time points, and the degradation of the microspheres was recorded using an inverted microscope. To quantitatively assess degradation behavior, samples were collected at different time points under the same conditions, and their residual weight was measured to calculate the mass loss of the microspheres. Furthermore, the morphology of the lyophilized microspheres was observed using scanning electron microscopy (SEM).

[0065] Further analysis was conducted on the loading capacity and release characteristics of LNPs (containing and without mRNA). To facilitate detection, a small amount of FITC fluorescent dye was introduced during LNP preparation. The diluted FITC solution was added to the ultracentrifuged LNP suspension, and after thorough mixing, a standard curve for LNPs was established using a microplate reader. Subsequently, lyophilized GM microspheres were added to the LNP suspension and incubated thoroughly, allowing LNPs to bind to the microsphere surface through physical adsorption. After the microspheres settled naturally, the absorbance of the supernatant was measured, and the loading efficiency of LNPs in LMPs was calculated based on the standard curve. To study the release behavior, LMPs were placed in PBS buffer (pH 7.4) and cultured with shaking at 37 °C. Release solutions were collected at different time points, and the cumulative release amount of LNPs was calculated based on the standard curve.

[0066] 4. Cell Culture and Processing RAW264.7 cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and maintained at a constant temperature of 37 °C and 5% CO2. Bone marrow-derived macrophages (BMDMs) were cultured in RPMI 1640 medium, also containing 10% FBS and 1% penicillin / streptomycin, with an additional 50 ng / mL of macrophage colony-stimulating factor (M-CSF, Novoprotein) to promote differentiation and survival. The medium was replaced with fresh medium every 48 h during culture. Cells were passaged when they reached approximately 80–90% confluence for subsequent experiments.

[0067] 5. Cell uptake detection BMDM and RAW264.7 cells were mixed at a ratio of 1×10⁻⁶. 5 Cells were seeded per well in 24-well plates and incubated overnight at 37°C with 5% CO2 to allow cell adhesion. The following day, Fr-RNA, LNP-mRNA, and PS / LNP-mRNA containing Cy5-labeled mRNA were added, and incubation continued at 37°C with 5% CO2 for 4 h. After incubation, cells were washed three times with PBS, and Cy5 signal was detected by flow cytometry. Cells were then observed using a confocal laser scanning microscope (CLSM), with nuclei stained with DAPI (blue) before CLSM analysis (CLSM 780, Carl Zeiss).

[0068] 6. Endolysosomal escape BMDM and RAW264.7 cells were treated with Cy5-labeled mRNA-PS / LNP-mRNA for 1, 3, and 6 h (37°C, 5% CO2), respectively. LysoTracker Green and Hoechst 33342 were then added to the cells according to the reagent instructions, and incubation was continued at 37°C for 30 min. Cells were washed with PBS and fixed with 4% paraformaldehyde, then observed under CLSM.

[0069] 7. Establishment and functional evaluation of a macrophage intracellular infection model To establish an in vitro intracellular infection model, macrophages were seeded in 6-well plates and cultured to a suitable confluence. The macrophages were then incubated with different treatment groups for 24 hours. GFP-labeled MRSA was added to the macrophage wells at a multiplicity of infection (MOI) of 20, and the cells were incubated at 37°C for 2 hours. The suspension was then replaced with fresh medium supplemented with 200 μg / mL gentamicin, and the cells were incubated at 37°C for another 2 hours to eliminate extracellular bacteria. The medium was then discarded, and the cells were washed, fixed, and stained with DiI. Images of all cells were acquired using a confocal laser scanning microscope (CLSM), and the phagocytic level of bacteria was determined and quantified by flow cytometry. Furthermore, after removing the gentamicin medium, 1 mL of 0.1% Triton X-100 solution was added to each well to promote macrophage membrane penetration, thereby allowing phagocytosed bacteria to leak out. The solutions were collected, and bacterial counting was performed using serial dilution and diffusion plate methods. Colony counting was performed after 24 hours of incubation.

[0070] II. Experimental Results and Discussion 1. Sequencing analysis and molecular validation of MerTK targets in macrophages under infected conditions To systematically evaluate changes in local immune characteristics after bacterial infection and the potential role of MerTK in intracellular MRSA infection, we first constructed an intracellular MRSA infection model using the classic gentamicin protection assay, and then performed transcriptome RNA sequencing (RNA-seq) analysis on macrophages from both infected and uninfected groups. Figure 1 (A). Principal component analysis (PCA) showed clear clustering and significant separation between groups, indicating reliable sequencing data quality and clear differences in infection-induced transcriptional characteristics. Figure 13 Volcano plot analysis identified 459 upregulated genes and 965 downregulated genes, among which MerTK was significantly upregulated in the infected group. Figure 1 (B) Meanwhile, differential gene heatmaps showed that multiple anti-inflammatory genes were significantly elevated after MRSA infection, while pro-inflammatory genes were generally downregulated (B). Figure 1 The presence of C in the middle of the spectrum suggests that infection induces macrophage remodeling towards an immunosuppressive phenotype. Further enrichment analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that... Figure 1 In the middle D), infection-related differentially expressed genes were significantly enriched in key pathways such as antigen processing and presentation, Toll-like receptor signaling pathway, NF-κB signaling pathway, and macrophage endocytosis.

[0071] Consistently, gene set enrichment analysis (GSEA) further revealed that, compared with the uninfected group, the infected group showed a significant overall downregulation trend in multiple key immune pathways, including antigen processing and presentation, NF-κB signaling pathway, positive regulation of T cell-mediated immunity, positive regulation of B cell-mediated immunity, and the bacterial defense response. Figure 1 E and F in the middle, Figure 14 These results indicate a decline in antigen presentation and pro-inflammatory immune function of macrophages after intracellular infection, which may be related to immune escape and immunosuppressive states. Furthermore, we hypothesized whether this immunosuppression was related to activated MerTK in the macrophage endocytosis pathway. To this end, we first performed a GSVA scoring analysis on the M1 pro-inflammatory gene set (…). Figure 1 (G), the results showed a significant decrease in M1 functional score after infection. Subsequently, we performed a correlation analysis between MerTK expression level and M1 functional score (G). Figure 1 (H). The results showed a significant negative correlation between MerTK and M1 functional scores, suggesting that elevated MerTK may lead to a decrease in pro-inflammatory macrophages, thereby promoting immunosuppression.

[0072] Next, the above findings were validated using immunofluorescence, immunohistochemistry, and Western blot (WB) experiments. Representative immunofluorescence and immunohistochemical images show ( Figure 1 In both in vitro MRSA-infected macrophages and in in vivo infected tissue sections from animal sources, the expression level of MerTK was significantly higher than that in the uninfected control group. Western blotting results further confirmed this. Figure 1 Compared with the control group, the expression of pro-inflammatory marker CD86 in macrophages was significantly decreased after intracellular MRSA infection, while the expression of anti-inflammatory / immunosuppressive related molecules CD206 and PD-L1 was significantly upregulated.

[0073] To clarify the role of MerTK in infection-induced immunophenotypic changes, we knocked down MerTK using small interfering RNA (siRNA). The results showed that after MerTK knockdown, CD86 expression was significantly restored, while the expression of CD206 and PD-L1 was significantly reduced, suggesting that MerTK activation is closely related to infection-induced macrophage immunosuppressive phenotype.

[0074] Furthermore, we established an intracellular MRSA infection model using a gentamicin protection assay, and after MerTK knockdown, we plated the residual MRSA in macrophages. The results showed that, compared with the control group, the number of colony-forming units (CFU) in the MerTK knockout group was significantly reduced, indicating that MerTK knockout significantly enhances the macrophage's ability to clear intracellular MRSA. Figure 1 (L).

[0075] These results indicate that MRSA infection significantly activates the MerTK pathway in macrophage endocytosis, subsequently mediating the suppression of pro-inflammatory function and the formation of an immunosuppressive phenotype, thereby weakening macrophages' ability to clear intracellular bacteria. This phenomenon suggests that the MerTK-mediated signaling axis may be an important molecular basis for the imbalance of macrophage immune function in the infection microenvironment.

[0076] Previous studies have shown that the accumulation of anti-inflammatory macrophages in an infectious state can promote the formation of an immunosuppressive microenvironment and is closely related to adverse clinical outcomes. Given the dual key role of macrophages in bacterial clearance and adaptive immune initiation, we hypothesize that targeting MerTK to reverse the infection-induced immunosuppressive phenotype of macrophages may restore their antibacterial function, thus becoming a potential therapeutic strategy for combating bacterial infections.

[0077] 2. Preparation, physicochemical properties, and intracellular delivery performance characterization of LNP In this study, we introduced cationic components to meet this requirement. Based on the above assumptions, we introduced DC-cholesterol and PS into the traditional LNP formulation to construct an LNP system composed of five components. This system has a weakly positive surface charge and an intrinsic targeting ability for immunosuppressive macrophages.

[0078] Specifically, DLin-MC3-DMA (MC3), distearate phosphatidylcholine (DSPC), cholesterol, 2,3-dioleoyl-propyltrimethylammonium (DOTAP), and PS were mixed in an ethanol phase; LL-37 mRNA and Cas9 mRNA / sgMerTK were mixed in a citrate buffer phase; subsequently, microfluidic technology was used to mix the two phases to prepare mRNA-LNP ( Figure 2 (A). We then measured the particle size and polydispersity index (PDI) of the LNPs using dynamic light scattering (DLS). The results are as follows... Figure 2 As shown in Figure B, the LNP particle size is 104.2 nm, and the PDI is 0.118, indicating that the LNP particle size distribution is relatively uniform. We further measured the encapsulation efficiency of the LNP using the RNA-Quant kit, finding that the mRNA encapsulation efficiency was 92.93% (…). Figure 2 (B) We also measured the Zeta potential of empty LNPs and LNPs encapsulating mRNA. The results showed that the Zeta potential of both empty LNPs and mRNA-LNPs was weakly greater than 5, exhibiting a weakly positive potential, which is conducive to binding to the negatively charged cell membrane. Figure 2 (C). We further evaluated the stability of mRNA-LNP. Figure 2 As shown in Figure D, mRNA-LNP exhibited negligible differences in particle size and PDI after 14 days of storage at 4°C, indicating excellent stability. This was demonstrated by transmission electron microscopy (TEM). Figure 2 In the middle E), we found that LNPs exhibited regular, uniform spherical particles. Under laser irradiation, a significant Tyndall effect could be observed in the mRNA-LNP solution. Figure 2 (F). pKa represents how pH affects the surface charge and stability of LNPs, which is a decisive factor in determining the lysosomal escape behavior of LNPs within cells. Therefore, we measured the pKa of mRNA-LNPs to be 6.45 using fluorescence titration. Figure 2 The pKa range (G) corresponds to the optimal pKa range required for efficient nucleic acid transfection. Furthermore, transfection of mouse macrophages (Raw264.7) and rat primary bone marrow-derived macrophages (BMDM) with green fluorescent protein (GFP) mRNA showed that mRNA-LNP had high transfection efficiency. Figure 2 (H). To observe the release of mRNA from LNP, we exposed mRNA-LNP to serum to mimic the circulation of LNP in vivo. Figure 2 As shown in the results in section I, we observed that the level of mRNA release from LNP continuously increased over time at pH 6.0. This result indicates that mRNA-LNP has the ability to accelerate the release of mRNA from the cytoplasm.

[0079] Next, we investigated the endocytosis and lysosomal escape of mRNA-LNP in macrophages. First, mRNA was labeled with a red fluorescent probe (Cy5), and then delivered into macrophages via LNP. We found that LNP could efficiently deliver Cy5-labeled mRNA to Raw264.7 and BMDM. Notably, when PS was modified in LNP, we observed a significant enhancement in the endocytosis of LNP by Raw264.7 and BMDM. Figure 2 (J). Flow cytometry results further indicated that PS-modified LNPs could more effectively deliver genes to macrophages (J). Figure 2 (K, L). For example Figure 2As shown in M ​​and N, in macrophages treated with PS / mRNA-LNP, the overlap between the Cy5-labeled red fluorescence and the lysosomal green fluorescence gradually decreased with prolonged incubation, indicating that LNP can escape from lysosomes and enter the cytoplasm. In summary, PS / mRNA-LNP possesses favorable physicochemical properties, efficient macrophage delivery capability, and significant lysosomal escape characteristics, making it suitable as an mRNA delivery vector targeting infection-associated immunosuppressive macrophages.

[0080] 3. Preparation and characterization of the LMP-mRNA complex system To improve the stability and sustained-release effect of LNP in the infected microenvironment after local injection, this study prepared an LMP mixed system by loading LNP onto gelatin methacrylamide (GelMA) microspheres (GM) to achieve long-term retention and slow release after local injection.

[0081] First, we prepared GM using a mature microfluidic-photocrosslinking technique. In short, a GelMA solution was used as the aqueous phase, and Span80 / liquid paraffin as the oil phase. The shear force of the paraffin oil channels caused the GelMA solution to form droplets, which were then photocrosslinked under UV irradiation to form solidified microspheres. By adjusting the flow rate ratio of the aqueous and oil phases, the diameter of the microspheres could be controlled within a specific range. Next, to enhance the surface adhesion and LNP loading capacity of the microspheres, we coated the microsphere surface with dopamine under alkaline conditions to form an in-situ polydopamine (PDA) coating. Finally, GM was immersed in an mRNA-LNP suspension. The PDA promoted the interaction of van der Waals forces and hydrogen bonds, adsorbing mRNA-LNP onto the microsphere surface, thus constructing an LMP-mRNA complex system. Figure 3 (A)

[0082] Optical microscopy revealed that the hydrogel microspheres exhibited good dispersibility and a regular spherical shape. Figure 3 (B and C). The average diameter of the microspheres is 190 μm, and the diameter distribution is relatively concentrated. Figure 3 (D). Scanning electron microscopy (SEM) was used to observe the microstructure of the microspheres, revealing that they exhibit a porous network structure, which is beneficial for LNP loading. Figure 3 (F, G). Figure 3 As can be seen from the image, a PDA coating was successfully applied to the surface of the GM microspheres. Furthermore, using FITC-labeled mRNA to locate the LNPs, we found that the LNPs were successfully loaded onto the microsphere surface. Figure 3 (H). Next, we tested the encapsulation efficiency of the microspheres. (e.g., H). Figure 3As shown in Figure I, the encapsulation efficiencies of both LMP and LMP-NC exceeded 90%, demonstrating good loading efficiency. To evaluate the release effect of GM microspheres on LMP, we immersed the microspheres in PBS solution at 37°C and pH 7.5, and measured the absorbance of the supernatant at different time points using a UV spectrophotometer to quantify the release efficiency of LMP and LMP-NC. Results ( Figure 3 The results (J) showed that LMP and LMP-NC were released rapidly on day 1, followed by slower but sustained release in later stages. Simultaneously, we evaluated the biodegradability of the microspheres using an inverted microscope. Figure 3 (Middle K). Over time, the microspheres degraded significantly. The degradation curves observed by gravimetric measurements showed results similar to those observed under an inverted microscope (Middle K). Figure 3 (L).

[0083] In addition, we analyzed the samples using X-ray photoelectron spectroscopy (XPS). Figure 3 As shown in Figure M, the P2p characteristic peak in LMP-NC is significantly enhanced, which may be attributed to the phospholipid components in LNP and the phosphorus element introduced by the mRNA, further confirming the successful introduction of LNP into the GM microsphere system. Notably, this study used non-coding EGFP mRNA as the model nucleic acid to characterize LNP, thus avoiding interference from protein expression-related factors on physicochemical parameters such as particle size, zeta potential, encapsulation efficiency, and release behavior, ensuring that the measured results reflect the inherent physicochemical properties of LNP. The results indicate that the introduction of GM microspheres did not significantly affect the structural integrity or physicochemical properties of LNP. Considering the cellular uptake process, LNP needs to be released from the GM microspheres and internalized by cells in a nano-suspension state. Similar GM-liposome / nanoparticle complexes have been characterized in our previous work.

[0084] Finally, to systematically evaluate the in vitro biocompatibility of LMP-mRNA, its effects on cell survival and proliferation were detected using the live / dead staining and cell counting kit-8 (CCK-8). The experiment was set up with a control group, LMP-NC, LMP-LL37, LMP-MerTK⁻ / ⁻, and LMP-LL37 / MerTK⁻ / ⁻ groups, which were co-cultured with Raw264.7 cells and rat bone marrow-derived macrophages (BMDMs), respectively. Cell survival was observed using fluorescence microscopy at 24 h, 48 h, and 72 h. The live / dead staining results showed ( Figure 15 , Figure 16 No significant cell death was observed in any of the groups at different time points; semi-quantitative fluorescence analysis also revealed no significant differences between the groups. Figure 17 , Figure 18 The CCK-8 test results show that ( Figure 19 , Figure 20The trends in optical density values ​​at 24 h, 48 h, and 72 h were consistent across all groups, and the differences were not statistically significant (P > 0.05). These results collectively indicate that LMP-mRNA has good in vitro biocompatibility and no adverse effects on cell proliferation.

[0085] In summary, LMP-mRNA demonstrates significant advantages as a local injection delivery platform in terms of morphological characteristics, loading efficiency, and controllable release performance. It also exhibits good biocompatibility and is expected to prolong the residence time of LNP in the infection microenvironment and achieve continuous and stable mRNA delivery.

[0086] 4. Construction and validation of mRNA-mediated LL37 expression and MerTK gene knockout in macrophages We constructed an mRNA / CRISPR co-delivery system based on lipid nanoparticles (LNPs) and systematically evaluated its ability to mediate MerTK gene knockout and LL37 antimicrobial peptide expression in macrophages. Figure 4 (A)

[0087] First, we evaluated the silencing efficiency of MerTK protein levels by LNP delivery of Cas9 / sgMerTK. We encapsulated Cas9 mRNA with three designed MerTK sgRNAs (sgRNA1, sgRNA2, sgRNA3) individually or in combination (1+2+3) into LNPs and co-incubated them with BMDM for 24 h. Western blot analysis (WB) was used to detect changes in MerTK expression. The results showed ( Figure 4 In experiments B and C), sgRNA1, sgRNA2, sgRNA3, and sgRNA (1+2+3) all effectively knocked out MerTK, with knockout efficiencies exceeding 50%. Quantitative analysis revealed that sgRNA2 exhibited the highest knockout efficiency. Therefore, we selected sgRNA2 for subsequent experiments. Furthermore, qPCR showed that ( Figure 4 Treatment with LNP-mRNA / sgMerTK effectively reduced the relative mRNA expression of MerTK, suggesting that CRISPR-mediated gene editing has high efficiency in macrophages. Immunofluorescence results also confirmed this. Figure 4 Treatment with LNP-mRNA / sgMerTK significantly reduced the fluorescence signal of MerTK on the surface of macrophages, further confirming the successful knockout of MerTK in macrophages.

[0088] Subsequently, we evaluated LNP-mediated LL37 mRNA delivery and protein expression. After 24 h of in vitro incubation of BMDM with LNP encapsulating LL37 mRNA, Western blot results showed that LL37 protein was significantly upregulated in the LNP-LL37 treatment group ( Figure 4 Quantitative analysis further confirmed that its expression level was significantly higher than that of LNP-NC and the control group ( ). Figure 4 (G) confirmed that the delivery system could induce efficient expression of the target protein. Furthermore, to assess the secretion efficiency of LL37, we used ELISA to detect the LL37 content in the culture supernatant. The results showed that the LL37 concentration gradually increased over time at 24 h, 48 h, and 72 h. Figure 4 The presence of H in the image suggests that LL37 delivered by LNP achieves sustained and stable secretion in macrophages. Immunofluorescence staining also showed a significant increase in the fluorescence signal of LL37 in the LNP-LL37 treatment group. Figure 4 (I), which further verifies its effective expression.

[0089] Finally, we further co-encapsulated LL37 mRNA and Cas9 / sgMerTK in the same LNP to evaluate whether this platform could simultaneously achieve antimicrobial peptide expression and immunosuppressive signal desuppression in a single macrophage. Western blot results showed that co-delivery significantly reduced MerTK protein levels while upregulating LL37 expression. Figure 4 The quantitative analysis of the J-series further confirmed this "dual-function regulatory" effect. Figure 4 LL37 and MerTK signaling were simultaneously enhanced and decreased within the same cell, according to immunofluorescence results. Figure 4 The M indicates that this mRNA-LNP platform can perform the dual tasks of functional protein expression and targeted gene editing in macrophages in parallel.

[0090] The above results demonstrate that the LNP-based mRNA / sgRNA co-delivery system can not only achieve efficient CRISPR-mediated MerTK gene knockout in macrophages, but also induce stable expression and sustained secretion of the antimicrobial peptide LL37. This synergistic strategy of "gene editing + functional protein expression" provides a highly integrated engineered approach for reshaping the immune phenotype of macrophages in the infection microenvironment, and lays the foundation for subsequent evaluation of its anti-infective therapeutic effects.

[0091] 5. Effects of LNP-LL37 / MTK on phagocytic and bactericidal functions of macrophages First, to evaluate the migration ability of engineered macrophages, we conducted a Transwell migration assay. Figure 5 (A). The results showed that, compared with the control group, LNP-LL37 / MTK⁻ / ⁻ treatment significantly enhanced the migration ability of macrophages ( Figure 5 (B). Subsequently, we further investigated the effects of different treatments on the phagocytic function of macrophages. Macrophages from different treatment groups were co-cultured with GFP-labeled bacteria and analyzed by confocal microscopy and flow cytometry. Confocal imaging results showed that the bacterial fluorescence signal in macrophages of the LNP-LL37 / MTK⁻ / ⁻ group was significantly enhanced ( Figure 5 The presence of C in the middle of the cell line suggests increased phagocytic activity; flow cytometry results also show that the phagocytic rate in this group is significantly higher than that in the control group. Figure 5 (D). Further, MRSA was co-cultured with macrophages from different treatment groups for 1 h, followed by cell lysis and quantitative analysis. The results showed that the phagocytic levels in each group were consistent with the confocal results, with the LNP-LL37 / MTK⁻ / ⁻ group exhibiting significantly increased phagocytic capacity (D). Figure 5 (F, G).

[0092] To assess the bactericidal ability of macrophages, we used a standard colony counting method to detect the number of surviving bacteria in cells from different treatment groups. The results showed that, compared with the initial number of bacteria phagocytosed, the number of surviving bacteria in the LNP-LL37 / MTK⁻ / ⁻ group was significantly reduced. Figure 5 (E, H). Quantitative analysis further showed that the bactericidal efficiency of the LNP-LL37 / MTK⁻ / ⁻ group was significantly higher than that of the control group (E, H). Figure 5 (I). Subsequently, we performed live / dead staining on bacteria from different treatment groups. The results showed that a large number of dead bacteria stained red with propidium iodide (PI) were observed in the LNP-LL37 / MTK⁻ / ⁻ group. Figure 5 J, Figure 21 The number of dead bacteria was lower in the LNP-LL37 / MTK⁻ / ⁻ group, while other groups had fewer dead bacteria. Flow cytometry analysis further confirmed that the PI positivity rate was highest in the LNP-LL37 / MTK⁻ / ⁻ group. Figure 5 (K, L).

[0093] To investigate whether the enhanced bactericidal effect is related to bacterial membrane damage, we used the o-nitrophenyl-β-D-galactopyranoside (ONPG) method, combined with protein and DNA leakage detection, to assess changes in bacterial membrane permeability. The results showed that the ONPG signal was significantly increased in the LNP-LL37 / MTK⁻ / ⁻ group ( Figure 5 The presence of M (mean squared up) indicates increased bacterial membrane permeability. Simultaneously, the protein and DNA content in the culture supernatant of this group was significantly higher than in other groups. Figure 5 N, Figure 22Morphological analysis further validated the above results. Scanning electron microscopy (SEM) observation showed that the bacterial cell wall structure was significantly deformed after LNP-LL37 / MTK⁻ / ⁻ treatment. Figure 5 Transmission electron microscopy (TEM) results showed that the bacterial cell membranes of the control group were intact, smooth, and had clear boundaries, while the bacterial cell membranes of the LNP-LL37 / MTK⁻ / ⁻ group showed obvious damage or even rupture, with blurred boundaries and a large amount of cell debris. Figure 5 (China P).

[0094] The above results demonstrate that LNP-LL37 / MTK⁻ / ⁻ treatment significantly enhances the antibacterial effect of macrophages at multiple functional levels. This treatment not only promotes macrophage migration, facilitating their rapid recruitment to the infection focus, but also significantly improves phagocytic efficiency and intracellular clearance of bacteria, thus forming a synergistically enhanced phagocytic-bactericidal function. Notably, a large number of apoptotic and damaged bacterial fragments are important sources of bacterial antigens, which can be effectively taken up by dendritic cells and promoted cross-presentation, potentially enhancing subsequent adaptive immune responses. In summary, LNP-LL37 / MTK⁻ / ⁻ comprehensively enhances the antibacterial function of macrophages by promoting macrophage migration, enhancing phagocytic activity, and inducing bacterial membrane disruption and intracellular clearance, providing strong experimental evidence for cell therapy strategies based on engineered macrophages for implant-related infections.

[0095] 6. Engineered macrophage-mediated immunophenotypic remodeling and immunomodulatory functions MRSA can evade macrophage clearance through various strategies, allowing phagocytosed bacteria to survive intracellularly and inducing macrophages to acquire an immunosuppressive phenotype (e.g., decreased pro-inflammatory function and upregulation of anti-inflammatory factors), thus forming an "immunosuppressive zone" at the site of infection and hindering pathogen clearance. Therefore, reversing the immunosuppressive phenotype to a pro-inflammatory / immunoenhancing phenotype to restore the bactericidal capacity of macrophages is a key strategy for clearing intracellular MRSA. Figure 6 (A)

[0096] To evaluate the immunomodulatory effects of engineered macrophages, we established an intracellular infection model of macrophages using a gentamicin protection assay and systematically analyzed the changes in the immune status of macrophages under different treatments. Flow cytometry analysis showed that under intracellular infection conditions, macrophages exhibited a significant immunosuppressive phenotype, with a significant upregulation of CD206 expression. After treatment with LNP-LL37 or LNP-MTK- / -, CD206 expression was significantly decreased, with the LNP-LL37 / MTK- / - treatment group showing the most significant decrease (Figure 6B). Correspondingly, LNP-LL37 / MTK- / - treatment significantly increased the expression ratio of the pro-inflammatory phenotype marker CD86, raising it from 10.4% in the infection control group to 33%. Immunofluorescence staining further confirmed that in the intracellular infection model, LNP-LL37 / MTK- / - treated macrophages showed significantly enhanced iNOS expression and significantly decreased Arg-1 expression. Figure 6 (C), quantitative analysis further supports the corresponding results ( Figure 6 (D, E). Furthermore, qPCR analysis showed that, compared with other treatment groups, the LNP-LL37 / MTK- / - treatment resulted in the highest transcriptional levels of pro-inflammatory genes (TNF-α, IL-6, and iNOS). Figure 6 The expression of the anti-inflammatory gene Arg-1 was lowest (Figure 6, I). These results consistently indicate that under intracellular infection conditions, LNP-LL37 / MTK- / - can effectively reverse infection-induced macrophage immunosuppression, remodeling it from an immunosuppressive phenotype to an immunostimulatory phenotype, thereby restoring and enhancing its pro-inflammatory response and antibacterial effect.

[0097] Beyond the inherent reprogramming of macrophages, we next investigated the broader immunomodulatory effects of engineered macrophages on other myeloid cell populations involved in immune activation. Given that the initiation of adaptive immune responses heavily depends on functional APCs, we further examined the effects of engineered macrophages on DC maturation and MDSC proportions. After co-incubating bone marrow-derived DCs (BMDCs) with supernatants from different treatment groups for 24 h, flow cytometry analysis showed a significantly higher DC maturation rate in the LNP-LL37 / MTK- / - group (control group 8.31% vs LNP-LL37 / MTK- / - 26.4%). Figure 6 (J, K). Meanwhile, LNP-LL37 and LNP-MTK- / - can reduce the MDSC ratio respectively, while LNP-LL37 / MTK- / - results in the lowest MDSC ratio (J, K). Figure 6(L, M). These data indicate that engineered macrophages can not only autologously reverse the immunosuppressive phenotype, but also reshape the infection microenvironment and promote the initiation of an adaptive immune response against Staphylococcus aureus by promoting APC maturation and inhibiting immunosuppressive myeloid cells.

[0098] 7. The therapeutic effect of engineered macrophage strategy in an in vivo implant-associated infection model Intracellular pathogen infection is one of the main causes of persistent and recurrent implant infections in clinical practice. Given the excellent antibacterial and immunomodulatory capabilities of LNP-LL37 / MTK- / - in vitro, we were prompted to further investigate its in vivo anti-infective ability using rat implant infection. Schematic diagram (7A) illustrates the construction of the implant infection model and the related treatment process.

[0099] Two weeks after infection, both the control group and the LMP-NC group showed obvious abscesses and local inflammatory reactions, while the abscesses in the LMP-LL37 / MTK- / - group were significantly reduced. Figure 7 The presence of B indicates that it has excellent in vivo anti-infective effects. Meanwhile, knee swelling in the LMP-LL37 / MTK- / - group was significantly relieved ( Figure 7 (C), and the core body temperature was significantly higher than the other four groups ( Figure 7 (D), while there was no significant difference in body weight among the five groups of rats ( Figure 23 Next, we will display the two-dimensional images of the femoral micro-CT scan. Figure 7 In the control group (E), significant bone loss was observed around the implant in both the infected control group and the LMP-NC group, while the LMP-LL37 / MTK- / - group retained a more intact bone structure. We further analyzed the microstructural changes and bone remodeling of the distal femur using three-dimensional reconstruction. Figure 7 As shown in F and G, the LMP-LL37 / MTK- / - group had a larger bone volume and a significantly greater number of trabeculae around the implant compared to the control group. Quantitative analysis revealed that the LMP-LL37 / MTK- / - group had significantly higher bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) than the control group, while trabecular separation (Tb.Sp) and the ratio of bone surface area to bone volume (BS / BV) were significantly lower. Figure 7 HJ, Figure 24 Finally, we subjected the removed implant to ultrasonic agitation and abraded the surrounding tissue for routine colony counting. The results are as follows: Figure 7 Zhong K, Figure 25As shown, while the LMP-LL37 group and the LMP-MTK- / - group could reduce the bacterial load to some extent, the effect was not ideal. The LMP-LL37 / MTK- / - group had the lowest colony count, indicating the most thorough bacterial clearance. Furthermore, we performed routine blood tests on the peripheral blood of rats. The results showed no abnormal increases in peripheral blood routine tests or liver and kidney function indicators. Figure 26 The results indicate that there were no systemic inflammatory adverse reactions after treatment, demonstrating good biocompatibility and safety. Overall, the hydrogel microspheres significantly enhanced the retention and efficacy of LNP-mRNA in vivo, enabling precise and sustained treatment of infection sites.

[0100] In addition, we performed pathological analysis on the infected tissues. First, hematoxylin and eosin (H&E) staining showed significant bone destruction and extensive inflammatory cell infiltration in the control and LMP-NC groups, indicating typical bone infection, while the LMP-LL37 / MTK- / - group showed effective control (…). Figure 7 In addition, the results of Masson's trichrome staining (L). Figure 7 The results (M) showed that significant collagen deposition was observed in the LMP-LL37 / MTK- / - group, confirming its excellent efficacy in treating implant-related infections. Finally, Gram staining results ( Figure 7 Consistent with previous standard colony counts, the LMP-LL37 / MTK- / - group showed almost no bacterial residue. Furthermore, H&E staining of the heart, liver, spleen, lungs, and kidneys revealed no significant pathological abnormalities after treatment, highlighting the good systemic safety of this local administration strategy. Figure 27 ).

[0101] In summary, the LMP-LL37 / MTK- / - local delivery system based on hydrogel microspheres demonstrated significant antibacterial activity, inhibited infection-related bone damage, and promoted local tissue repair in a rat implant infection model, while maintaining good biocompatibility, confirming its feasibility and effectiveness as a potential strategy for treating implant-related infections.

[0102] 8. Remodeling of the immune microenvironment surrounding the implant After successfully evaluating the in vivo anti-infective efficacy, we further investigated the effect of LMP-LL37 / MTK- / - on the remodeling of the local immune microenvironment at the site of infection. Firstly, as... Figure 8 As shown in Figures A and G, compared with other groups, the number of iNOS-positive cells in the tissues of the LMP-LL37 / MTK- / - treatment group was significantly increased; correspondingly, the number of CD206-positive cells was significantly reduced. Figure 8 The presence of B and H in the samples suggests a significant change in the macrophage phenotype within the infection focus.

[0103] Next, we stained the tissue sections with CD4+ T cells. The results are as follows: Figure 8 As shown in C and I, the infiltration of CD4+ T cells was significantly increased in the LMP-LL37 / MTK- / - group, indicating that the LMP-LL37 / MTK- / - group activated an adaptive immune response after treatment. Furthermore, we stained the regulatory T cell marker Foxp3 (…). Figure 8 (D, J) The results showed that Foxp3 was significantly reduced in the LMP-LL37 / MTK- / - group, indicating that LMP-LL37 / MTK- / - can effectively enhance T cell immunity while reducing the production of suppressor T cells, thus lowering the immunosuppressive threshold and facilitating antibacterial immunity. Correspondingly, immunohistochemical results showed that TNF-α and IL-1β were significantly upregulated in the LMP-LL37 / MTK- / - group ( Figure 8 The presence of E and F in the middle of the spectrum further supports the establishment of an immune-enhancing microenvironment.

[0104] In summary, LMP-LL37 / MTK- / - significantly enhances the clearance of implant-related infections and promotes the formation of adaptive immunity by synergistically promoting macrophage pro-inflammatory polarization, enhancing CD4⁺ T cell immunity, inhibiting regulatory T cell generation, and upregulating local pro-inflammatory cytokines, thereby reshaping the immune microenvironment of infection foci at multiple levels.

[0105] 9. Immune remodeling and systemic immune response at the site of infection Given the immunostimulatory effects demonstrated by LMP-LL37 / MTK- / - in in vitro experiments, we further evaluated its impact on in vivo immune responses in a rat implant-associated infection model. Figure 9 (A). Flow cytometry analysis of infected draining lymph nodes (IDLN) showed that, compared with the control group, the proportion of pro-inflammatory M1-like macrophages was significantly increased in the LMP-LL37 / MTK- / - treatment group. Figure 9 In the middle B and C), the proportion of anti-inflammatory M2-like macrophages was significantly decreased ( Figure 9 (D, E). Simultaneously, the maturation rate of dendritic cells (DCs) increased from 5.40% in the control group to 15.6% in the LMP-LL37 / MTK- / - group ( Figure 9 (F, G). These results indicate that LMP-LL37 / MTK- / - alters the macrophage phenotypic distribution in IDLNs in vivo and increases the maturation level of DCs.

[0106] We further evaluated the levels of MDSCs. The results showed that the LMP-LL37 / MTK- / - group significantly inhibited MDSC infiltration, suggesting that it helps restore the immune competitiveness of the infection site. Figure 9(H, I). In chronic bacterial infection, APCs are in a frustrated and exhausted state, characterized by immunosuppression of macrophages and immaturity of dendritic cells. The state transformation of APCs indicates that LMP-LL37 / MTK- / - can effectively alter the immunosuppressive phenotype of macrophages in the infection state and promote the maturation of dendritic cells, thus promoting the formation of adaptive immunity.

[0107] The greater the infiltration of CD4+ T cells, the stronger the humoral immunity; however, inhibition of CD4+ T cell activation limits the eradication of implant-infected bacteria. As expected, significantly higher levels of CD4+ T cell infiltration were observed in rats in the LMP-LL37 / MTK- / - group. Figure 9 These results highlight the importance of LMP-LL37 / MTK- / - in altering the balance between suppressive and activating immune cells to enhance humoral immune efficacy. Furthermore, in rats treated with LMP-LL37 / MTK- / -, pro-inflammatory cytokines such as IFN-γ, TNF-α, and IL-6 were significantly upregulated in the infection microenvironment. Figure 9 L, M, Figure 28 ), while IL-10 decreased significantly ( Figure 29 These results further demonstrate that LMP-LL37 / MTK- / - can elicit a strong antibacterial immune response in the immunogenic infection microenvironment. In drug-resistant bacterial infections, antibody responses are considered a key protective factor, forming the basis for bacterial neutralization. However, antibody production is significantly hindered in the presence of intracellular infection. In this study, we observed significantly elevated serum IgG and IgM levels in rats in the LMP-LL37 / MTK- / - group. Figure 9 (N, O). Overall, LMP-LL37 / MTK- / - demonstrates strong immunomodulatory and antibacterial potential in the treatment of implant-associated drug-resistant bacterial infections by synergistically regulating innate and adaptive immune responses to construct a hyperresponsive immune state conducive to bacterial clearance.

[0108] 10. Transcriptome sequencing reveals the reprogramming of immune-related pathways in the infection microenvironment. To fully understand the antibacterial mechanism of LMP-LL37 / MTK- / -, we then performed transcriptomic analysis on macrophages treated with LMP-LL37 / MTK- / -. The results of PCA showed that ( Figure 10 The results (A) showed good consistency in intragroup bias, confirming the reliability of the RNA sequencing results. Figure 10 As shown in Figure B, compared with the control group (PBS), LMP-LL37 / MTK- / - treatment resulted in the upregulation of 1165 genes and the downregulation of 1362 genes, with a fold change of 2 and a P-value less than 0.05. Figure 10The heatmap showed that the infection microenvironment within macrophages in the LMP-LL37 / MTK- / - treatment group underwent significant changes. Transcription of pro-inflammatory cytokines (Csf3, IL-1β, IL-6, Tnf), antigen-presenting co-stimulatory molecules (CD40, CD80), and T cell chemokines (Ccl4, Cxcl10) was significantly increased, while immunosuppressive factors (Tdo2, Arg1, Lag3, etc.) were significantly decreased, indicating a reversal of the local immunosuppressive microenvironment. Simultaneously, GO analysis showed… Figure 10 LMP-LL37 / MTK- / - therapy was significantly enriched in differentially expressed genes, particularly in pathways related to the immune system, inflammation, and antimicrobial responses. Furthermore, KEGG pathway analysis revealed enrichment in the IL-17 signaling pathway and the Toll-like receptor signaling pathway. These findings suggest a close association between LMP-LL37 / MTK- / - therapy and immune-related pathways and pro-inflammatory responses. Figure 10 (E). GESA results further show ( Figure 10 China FI, Figure 30 Following LMP-LL37 / MTK- / - treatment, immune-related activation pathways were significantly enriched, including the IL-17 signaling pathway, NF-κB signaling pathway, T-cell receptor signaling pathway, B-cell receptor signaling pathway, and NOD-like receptor signaling pathway. This further supports the activation of the host immune response against bacterial infection. These results indicate that LMP-LL37 / MTK- / - can generate a robust immune response and effectively clear bacterial infection. In conclusion, both innate and adaptive immune responses are activated after LMP-LL37 / MTK- / - treatment, resulting in a potent antibacterial immune response.

[0109] 11. LMP-LL37 / MTK - / - Induces antibacterial immune memory and prevents relapse To explore the potential of LMP-LL37 / MTK- / - in activating antimicrobial immune memory and preventing Staphylococcus aureus recurrence, we conducted a reinfection experiment in a rat implant infection model. Figure 11 Rats were treated differently for the first 14 days. After the initial infection was controlled, bacteria were reinjected around the implant on day 14. On day 14 after the bacterial re-challenge, we used flow cytometry to assess whether LMP-LL37 / MTK- / - could effectively induce an immune memory response. The results showed that, compared with the control group, the proportion of CD4+ effector T cells (high expression of CD44, low expression of CD62L) in the spleen of the LMP-LL37 / MTK- / - group was significantly increased. Figure 11(B and D). Notably, the proportion of CD8+ effector memory T cells (high expression of CD44 and low expression of CD62L) in the spleen of the LMP-LL37 / MTK- / - group was also significantly higher than that in the control group (B and D). Figure 11 (C, E). Next, we performed a quantitative analysis of the bacterial load after reinfection. Colony-forming unit (CFU) analysis of the removed implant and surrounding tissue showed that the bacterial load in the LMP-LL37 / MerTK- / - group was significantly lower than that in the control group (CFU). Figure 11 Middle F, G). Histological analysis also confirmed that neutrophil infiltration and bacterial count were extremely low in the LMP-LL37 / MerTK- / - group ( Figure 31 , Figure 32 This indicates that the pathological remission was almost complete after the secondary infection. We further evaluated the systemic immune response. After reinfection, the release of pro-inflammatory cytokines in the serum of animals in the LMP-LL37 / MerTK- / - treatment group was significantly increased ( Figure 11 H, I and Figure 33 ), while the anti-inflammatory cytokine IL-10 was significantly reduced ( Figure 34 This indicates that the clearance-type inflammatory response was rapidly activated after re-exposure to the pathogen. Simultaneously, humoral immunity analysis showed that serum IgG and IgM levels in the LMP-LL37 / MerTK- / - group were significantly higher than in all other groups (J and K in Figure 11), indicating that a strong protective antibody response had been established.

[0110] In summary, these results demonstrate that LMP-LL37 / MerTK- / - not only effectively clears short-term infections but also induces potent and durable antimicrobial immune memory. This durable immune protection significantly reduces the risk of Staphylococcus aureus reinfection, highlighting the potential of this engineered macrophage-based strategy in preventing recurrence of implant-related infections.

[0111] III. Conclusion This invention constructs an injectable lipid nanoparticle-hydrogel microcomposite system (LMP-LL37 / MTK). - / - This platform enables in situ genetic programming of macrophages for the treatment of implant-related infections. By coupling sustained expression of the antimicrobial peptide LL-37 with CRISPR-Cas9-mediated MerTK gene knockout, it simultaneously enhances the bactericidal activity of macrophages and alleviates infection-induced immunosuppression, achieving systematic reprogramming of macrophage function. Figure 12 LMP-LL37 / MTK - / -This invention significantly improves bacterial clearance efficiency, reshapes the local and systemic immune microenvironment, inhibits infection-related bone destruction, and induces durable antimicrobial immune memory, thereby providing effective protection against reinfection. It establishes a gene-programmed macrophage immunotherapy strategy, offering a promising alternative for the long-term control and recurrence prevention of refractory implant-related infections, overcoming the limitations of traditional antibiotic treatment paradigms.

Claims

1. A method for preparing an injectable mRNA-hydrogel microsphere complex, characterized in that, Includes the following steps: (1) Ionizable lipids, phospholipids, cholesterol, cationic lipids and phosphatidylserine were mixed in the ethanol phase; LL-37 mRNA and Cas9 mRNA / MerTK sgRNA were mixed in citrate buffer; mRNA-LNP was prepared using microfluidic technology; (2) Using GelMA solution as the aqueous phase and Span80 and liquid paraffin as the oil phase, droplets were prepared by microfluidic technology, and then GelMA microspheres were prepared by cross-linking with a photoinitiator; GelMA microspheres were mixed with dopamine solution, and the pH was adjusted to neutral or slightly alkaline to form a polydopamine coating in situ. (3) The GelMA microspheres coated with polydopamine were immersed in the mRNA-LNP suspension of step (1) to construct an LNP-hydrogel microsphere complex that can be injected in situ.

2. The method according to claim 1, characterized in that, The ionizable lipids mentioned in step (1) include: Dlin-MC3-DMA, SM-102 or ALC-0315; the phospholipids include 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine or 1,2-distearyl-sn-glycerol-3-phosphatidylcholine; the cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane, N-[1-(2,3-dioleoyloxypropyl)]-N,N,N-trimethylammonium or 1,2-dimyristoyl-3-trimethylammonium propane.

3. The method according to claim 1, characterized in that, The weight percentages of ionizable lipids, phospholipids, cholesterol, cationic lipids and phosphatidylserine in step (1) are 40-60%: 10-30%: 20-40%: 0-20%: 0-5%.

4. The method according to claim 1, characterized in that, The weight ratio of LL-37 mRNA and Cas9 mRNA / MerTK sgRNA in step (1) is 1:1 to 20:

1.

5. The method according to claim 1, characterized in that, In step (2), the weight ratio of GelMA solution, Span80 and liquid paraffin is 1:0.01~0.2:5~20.

6. The method according to claim 1, characterized in that, In step (3), the concentration of the mRNA-LNP suspension is 0.01–10 mg / mL.

7. An injectable mRNA-hydrogel microsphere complex prepared by the method according to any one of claims 1-6.

8. The use of the injectable mRNA-hydrogel microsphere complex of claim 7 in the preparation of a medicament for treating implant-associated infections.

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