Nanoparticles for autophagy-targeted degradation of tlr4 and cgas, and preparation and application thereof

Abstract

The application relates to the technical field of nanoparticles, and particularly discloses a nanoparticle for autophagy-targeted degradation of TLR4 and cGAS as well as preparation and application thereof. The application adopts an emulsification method to prepare a nanoparticle (named as CLEAR) for autophagy-targeted degradation of TLR4 and cGAS with polylactic acid (PLA) as a core. The surface of the double-targeted nanoparticle is grafted with a TLR4 targeting ligand and a cGAS targeting ligand, so that M1 macrophage-specific TLR4 and cGAS are synergistically exhausted. The CLEAR treatment realizes about 80% survival rate by synergistically silencing two key inflammatory pathways. Overall, the strategy developed based on the nanoparticle (CLEAR) for autophagy-targeted degradation of TLR4 and cGAS establishes a promising treatment paradigm for sepsis and exhibits clinical transformation potential.

Description

Technical Field

[0001] This application relates to the field of nanoparticle technology, and in particular to a nanoparticle for autophagy-targeted degradation of TLR4 and cGAS, its preparation and application. Background Technology

[0002] Sepsis is a fatal syndrome driven by host immune dysregulation, and currently lacks effective treatments due to the intertwining of cytokine storm and immunosuppression. The pathophysiology of this syndrome is characterized by a vicious cycle of cytokine storm and immunosuppression, primarily driven by abnormal macrophage polarization. At the heart of this immune dysregulation lies a shift towards a pro-inflammatory (M1) macrophage pattern, which exacerbates tissue damage through continuous cytokine release. Against this backdrop, macrophages, as key mediators of the sepsis response, are significantly activated via Toll-like receptor 4 (TLR4). Bacterial endotoxin-dependent TLR4 ligand activation can transduce the intracellular NF-κB cascade, thereby consolidating M1 macrophage polarization and amplifying systemic inflammation. Simultaneously, the cytoplasmic DNA sensor loop GMP-AMP synthase (cGAS) functions as a parallel inflammatory pathway: cytoplasmic DNA fragments originating from internalized pathogens or damaged organelles effectively activate STING signaling, inducing potent type I interferon (IFN-I) production, leading to multiple organ failure. The key issue is that while TLR4 or STING antagonists have shown efficacy in isolated inflammatory conditions, their use as monotherapy for sepsis is insufficient to achieve clinical remission. This inadequacy underscores the urgent need to develop strategies that simultaneously weaken the TLR4 and cGAS-STING signaling pathways within macrophages, a challenge that may be addressed through emerging protein degradation technologies.

[0003] Based on this paradigm, targeted protein degradation (TPD) platforms utilize endogenous cellular mechanisms to clear disease-related proteins. Among these, the ubiquitin-proteasome system (UPS) pathway employs heterobifunctional molecules to recruit E3 ubiquitin ligases, thereby promoting the degradation of intracellular targets; in particular, proteolytic targeted chimeras (PROTACs) are widely used. Although PROTACs have shown significant potential in degrading traditionally "undruggable" cytoplasmic proteins, their efficacy against membrane-bound proteins remains limited. As a complement to intracellular degradation strategies, lysosome-targeted chimeras (LYTACs) have emerged, inspired by the physiological principle of endogenous ligands triggering endocytosis and lysosomal transport of cell surface receptors. Initial LYTAC designs utilized well-defined receptors, such as the cation-independent mannose-6-phosphate receptor (CI-M6PR) and the desialylate glycoprotein receptor (ASGPR). Building upon this foundation, LYTACs can now utilize a variety of endocytosis-triggered receptors, including glucose transporter 1 (Glut1), sorting proteins, folate receptor α (FRα), and transferrin receptor (TfR). The ligand-binding domains of these receptors can lead to the specific internalization and lysosomal degradation of pathogenic proteins. Notably, nanoparticles enable receptor-independent endocytosis, a mechanism that significantly expands the therapeutic scope of LYTACs while simplifying their design and fabrication process.

[0004] Beyond established degradation strategies based on UPS and LYTAC, increasing research is turning to autophagy: an evolutionarily conserved pathway for the rapid delivery of cytoplasmic cargo to lysosomes. This approach utilizes selective autophagy receptors to promote lysosomal clearance of refractory intracellular targets. As a representative model, autophagy-targeting chimeras (AUTACs) specifically degrade insoluble protein aggregates and dysfunctional organelles, extending the therapeutic potential of TPD to previously untreatable diseases. This strategy continues to evolve, including supramolecular nano-AUTACs that induce K63-linked polyubiquitination to degrade targets, and AUTOTACs that directly bind to autophagy receptors (such as p62) to degrade proteins and their aggregates. Furthermore, ATTECs, as bifunctional molecules, can simultaneously bind cellular components and autophagy-associated proteins (such as microtubule-associated protein 1A / 1B light chain 3B, LC3B), enhancing phagocytosis of entire organelles and subsequent autophagic degradation. Recently, charge-mediated autophagy inducers have emerged as an alternative strategy, circumventing traditional receptor recruitment pathways. Despite these advances, challenges such as drug delivery barriers and off-target risks remain key obstacles to the effective treatment of sepsis with autophagy-based degradative agents. Summary of the Invention

[0005] The purpose of this application is to overcome the shortcomings of the prior art and provide a nanoparticle for autophagy-targeted degradation of TLR4 and cGAS, as well as its preparation and application.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a method for preparing nanoparticles that target the autophagy degradation of TLR4 and cGAS, comprising the following steps: S1. Polylactic acid is dissolved in dichloromethane and stirred to form an oil phase; distearate phosphatidylethanolamine-polyethylene glycol-amino, hexadecyltrimethylammonium bromide and polyvinyl alcohol are mixed to form an aqueous phase; S2. The oil phase is added to the aqueous phase for pre-emulsification, and then emulsified by ultrasonic homogenization to obtain an emulsion; the emulsion is dispersed in a polyvinyl alcohol solution and stirred and evaporated to obtain an unfunctionalized nanoparticle solution. S3. Mix the nanoparticle solution obtained in step S2 with a buffer solution containing the first and second targeting ligands, then centrifuge and ultrafilter to remove monomers, filter to remove bacteria, and obtain nanoparticles that autophagy-targeted degrade TLR4 and cGAS. The first targeting ligand specifically binds to the extracellular domain of a pro-inflammatory transmembrane receptor on the macrophage membrane, and the second targeting ligand specifically binds to an intracellular pattern recognition receptor on the macrophage cytoplasm.

[0007] In this application, an emulsification method is used to prepare nanoparticles (named CLEAR) with polylactic acid (PLA) as the core for autophagy-targeted degradation of TLR4 and cGAS. The surface of these dual-targeting nanoparticles is grafted with a first and a second targeting ligand, achieving M1 macrophage-specific synergistic depletion of TLR4 and cGAS. Through the first targeting ligand, CLEAR is internalized by the cell via nanoparticle-mediated endocytosis, directing TLR4 to endosomes and promoting its subsequent degradation. During lysosomal acidification, the abundant positive surface charge of CLEAR (provided by cetyltrimethylammonium bromide) acts as a proton pump and permeability enhancer, inducing endosome-lysosomal permeability rupture and further promoting the release of CLEAR into the cytoplasm. Once in the cytoplasm, the net positive charge on the CLEAR surface triggers autophagic flux, simultaneously binding cytoplasmic cGAS and TLR4, which are subsequently degraded via the autophagosome-lysosome pathway. This process enables the synchronous degradation of membrane proteins and cytoplasmic proteins, effectively disrupting inflammatory crosstalk between the TLR4-NF-κB and cGAS-STING signaling axes, thereby alleviating cytokine storm and M1 macrophage polarization.

[0008] Ultimately, in a mouse model of sepsis with cecal ligation and perforation, CLEAR treatment achieved approximately 80% survival by synergistically silencing two key inflammatory pathways. Overall, this strategy, developed based on autophagy-targeted TLR4 and cGAS nanoparticles (CLEAR), establishes a promising therapeutic paradigm for sepsis and demonstrates clinical translational potential.

[0009] In a preferred embodiment of the preparation method of the dual-targeted nanoparticles described in this application, in step S1, the ratio of polylactic acid to dichloromethane is (0.1~1.0) g: (5~20) mL.

[0010] In some specific embodiments, 0.3 g of polylactic acid was dissolved in 15 mL of dichloromethane.

[0011] Preferably, the mass-volume concentration of the polyvinyl alcohol solution is 1%~10% w / v (preferably 5% w / v).

[0012] In a preferred embodiment of the preparation method of the dual-targeted nanoparticles described in this application, in step S2, the volume ratio of the oil phase to the water phase is 1:(2~10); preferably, the volume ratio of the oil phase to the water phase is 1:5.

[0013] This application limits the concentrations of polylactic acid, polyvinyl alcohol, and the oil-water volume ratio within the aforementioned ranges. This ensures the formation of an oil-in-water microdroplet system during emulsification, preventing demulsification or particle aggregation, thereby obtaining nanoparticle cores with uniform particle size and good dispersibility. Exceeding these ranges may result in excessively high initial emulsion viscosity, preventing homogenization, or insufficient surfactant leading to excessively large particle size.

[0014] In a preferred embodiment of the preparation method of the dual-targeted nanoparticles described in this application, in step S1, the volume ratio of distearate phosphatidylethanolamine-polyethylene glycol-amino and hexadecyltrimethylammonium bromide is (0.1~10):(10~0.1).

[0015] Based on the autophagy-inducing ability of positively charged nanoparticles, dual-targeting nanoparticles were designed. This application first synthesizes an ungrafted, unfunctionalized nanoparticle solution (unCLEAR) using biocompatible polylactic acid (PLA) as the core. Nanoparticles are then prepared via emulsification combined with CTAB (cation source) and DSPE-PEG-NH2, followed by solvent evaporation to remove dichloromethane (DCM).

[0016] In a preferred embodiment of the preparation method of the dual-targeted nanoparticles described in this application, the volume ratio of distearate phosphatidylethanolamine-polyethylene glycol-amino and hexadecyltrimethylammonium bromide is (2~6):(4~8); preferably 2:8.

[0017] In the technical solution of this application, the surface charge intensity of the dual-targeting nanoparticles provided by this application determines their cellular behavior. Disteazylephosphatidylethanolamine-polyethylene glycol-amino is responsible for providing -NH2, which reacts with -COOH in the two ligands to graft the two ligands. Hexadecyltrimethylammonium bromide is responsible for providing the surface positive charge, incubating the nanoparticles' "endosome escape" and "autophagy induction" functions. The disteazylephosphatidylethanolamine-polyethylene glycol-amino and hexadecyltrimethylammonium bromide of this application adopt the above-mentioned volume ratio, which can enable the nanoparticles to connect a sufficient number of ligands while retaining the positive charge that enables "endosome escape" and "autophagy induction".

[0018] In a preferred embodiment of the preparation method of the dual-targeted nanoparticles described in this application, in step S2, the pre-emulsification speed is 10000~20000 rpm, and the pre-emulsification time is 1~5 min; And / or, the ultrasonic power for ultrasonic homogenization is 100~500W, and the homogenization time is 1~5min; And / or, the stirring rate for stirring and evaporation is 100~500 rpm, and the stirring time is 18~30h.

[0019] The application employs the aforementioned pre-emulsification conditions to form emulsion droplets from the oil and water phases, and then uses ultrasonic homogenization emulsification conditions to further atomize the droplets into uniformly sized droplets.

[0020] In some specific embodiments, in step S2, the pre-emulsification speed is 15000 rpm and the pre-emulsification time is 1 min; And / or, the ultrasonic power for ultrasonic homogenization is 300W, and the homogenization time is 5 minutes; And / or, the stirring rate for evaporation is 300 rpm, and the stirring time is 24 h.

[0021] This application employs the aforementioned pre-emulsification and ultrasonic homogenization emulsification to prepare dual-targeting nanoparticles with polylactic acid as the core. The surface of the dual-targeting nanoparticles is grafted with a first targeting ligand and a second targeting ligand, thereby achieving M1 macrophage-specific synergistic depletion of TLR4 and cGAS, and thus better treating sepsis.

[0022] In a preferred embodiment of the preparation method of the dual-targeting nanoparticles described in this application, in step S3, the pro-inflammatory transmembrane receptor is selected from the Toll-like receptor family, the interleukin receptor family, the tumor necrosis factor receptor family, or the scavenger receptor family; the intracellular pattern recognition receptor is selected from the intracellular free nucleic acid sensing receptor or the NOD-like receptor family. Preferably, the first targeting ligand is a TLR4 targeting ligand, selected from at least one of TLR4-IN-C34-C2-COOH, Eritoran, and single-chain antibody fragments that specifically bind to the extracellular region of TLR4; The second targeting ligand is a cGAS targeting ligand, selected from at least one of cGAS-IN-1, RU.521, RU.322, CU-32, CU-76, and A151 oligonucleotides.

[0023] In some specific embodiments, the TLR4 targeting ligand is TLR4-IN-C34-C2-COOH; and the cGAS targeting ligand is cGAS-IN-1.

[0024] In the technical solution of this application, the pro-inflammatory transmembrane receptors of the M1 macrophage membrane can be used as the first target site, and the target ligands can specifically bind to the extracellular domain of the receptor. The core receptors applicable to the membrane surface include multiple families: the Toll-like receptor family uses TLR2, TLR4, and TLR5 expressed on the membrane surface, with TLR4-IN-C34-C2-COOH, Eritoran, and OPN-305 as target ligands; the interleukin receptor family uses IL-1R1 as the core, with Anakinra as the target binding; the tumor necrosis factor receptor family targets TNFR1, using single-domain antibodies and WP9QY peptides; the scavenger receptor family targets CD36, achieving specific binding with the help of SSO.

[0025] Intracellular pattern recognition receptors can serve as secondary target sites. After nanoparticles escape through endosomes or lysosomal membranes, corresponding targeting ligands can bind to cytoplasmic targets and mediate their entry into autophagosomes. Intracellular nucleic acid sensing receptors are mainly cGAS, STING, and RIG-I. Inhibitors such as cGAS-IN-1, RU.521, CU series inhibitors, and A151 oligonucleotides can be used to target and inhibit cGAS, while H-151 specifically inhibits STING. The NOD-like receptor family targets NLRP3, NOD1, and NOD2, and potent inhibitors such as MCC950 and CY-09 are commonly used to exert their targeting effects.

[0026] If there is a domain spatial mismatch blind zone, TAK-242 and CLI-095, which target the intracellular TIR domain of TLR4, and VIPER peptide, which targets intracellular interactions, cannot be used as the first surface targeting ligands. Large molecular nanoparticles cannot penetrate the cell membrane to reach the intracellular domain, making it difficult to achieve targeting and easy to be cleared by the body. The first targeting ligand should be a substance that binds to the extracellular region of the receptor.

[0027] If agonists with contradictory pharmacological effects are selected, such as LPS and MPLA for surface targeting and long-chain dsDNA and G3-YSD for intracellular targeting, their potent pro-inflammatory effects will superimpose and induce a cytokine storm, causing shock and death in animals, thus failing to achieve the purpose of anti-inflammatory treatment.

[0028] If ligands lacking carboxyl, amino, or other modifiable functional groups are used, covalent grafting cannot be completed through amidation condensation and click chemistry, making it difficult to synthesize and prepare dual-target nanoparticles.

[0029] If mannose and IL-4R targeting peptides are used, they are prone to misdirecting onto anti-inflammatory M2 macrophages, damaging repair cells and failing to target pathogenic M1 macrophages, ultimately leading to treatment failure and hindering tissue repair. In this application, TLR4 targeting ligands and cGAS targeting ligands are key pattern recognition receptors in the pathogenesis of sepsis. A therapeutic drug is formed by modifying unfunctionalized nanoparticle solutions (unCLEAR) with TLR4 and cGAS targeting ligands. Specifically, the dual-targeting nanoparticles of this application are formed by forming an amide bond between the amino group of distearate-phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH2) and the carboxyl group of the TLR4 / cGAS targeting ligands.

[0030] Experiments showed that FTIR spectroscopy confirmed successful ligand coupling through characteristic amide bond signals, and ¹H nuclear magnetic resonance (NMR) analysis verified the effective incorporation of TLR4 and cGAS targeting ligands. These results confirmed the successful construction of dual-targeting nanoparticles with precisely designed positive charges. The dual-targeting nanoparticles obtained in this application have good storage stability and excellent system biosafety.

[0031] In a preferred embodiment of the preparation method of the dual-targeted nanoparticles described in this application, in step S3, the mass ratio of the first targeting ligand to the second targeting ligand is (0.5~1.2):(0.5~1.0); preferably, the mass ratio of the first targeting ligand to the second targeting ligand is 0.8:0.7.

[0032] In a preferred embodiment of the preparation method of the dual-targeted nanoparticles described in this application, in step S3, the buffer solution comprises MES buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide.

[0033] In a preferred embodiment of the preparation method of dual-targeted nanoparticles described in this application, in step S3, the molecular weight cutoff of centrifugal ultrafiltration is 50~500 kDa. The centrifugation rate is 1000~5000×g, and the centrifugation time is 20~40min; In some specific embodiments, the molecular weight cutoff for centrifugal ultrafiltration is 100 kDa; The centrifugation rate was 3000 × g, and the centrifugation time was 30 min.

[0034] This application also provides dual-targeting nanoparticles obtained by the above-described method for preparing dual-targeting nanoparticles.

[0035] Experiments have shown that the dual-targeting nanoparticles provided in this application possess the ability to autophagic flux and degrade target proteins. The dual-targeting nanoparticles employ a two-phase mechanism to degrade different proteins: firstly, utilizing the endocytic properties of the nanoparticles, the dual-targeting nanoparticles specifically bind to TLR4 and transport it to the endosome-lysosome, degrading TLR4. The nanoparticles then escape from the lysosome through the proton sponge effect. After entering the cytoplasm, they bind to target proteins through surface ligands and induce autophagic flux using cationic properties, ultimately achieving autophagic-lysosomal degradation of cGAS and TLR4 in the cytoplasm. This inhibits the TLR4 and cGAS pathways, improving the therapeutic effect of sepsis.

[0036] In vitro experiments have shown that the dual-targeting nanoparticles provided in this application can effectively degrade cGAS and inhibit STING phosphorylation, reducing pSTING levels by 88% compared to baseline. Furthermore, they can alleviate excessive inflammation by synergistically downregulating cGAS-STING signaling and reprogramming macrophage functional states.

[0037] In vivo experiments showed that the dual-targeting nanoparticles provided in this application significantly improved the survival rate of the cecal ligation and perforation (CLP) model 168 hours after surgery, significantly improved the clinical score of sepsis in mice, and accelerated the recovery of normal body temperature, demonstrating effective protective efficacy against sepsis in mice. Furthermore, serum cytokine analysis showed that the dual-targeting nanoparticles provided in this application effectively inhibited the elevated levels of IL-6, TNF-α, and IL-1β in sepsis-affected mice. The dual-targeting nanoparticles significantly reduced TNF-α, IL-1β, and IL-6 levels in M1 macrophages and liver tissue. Histopathological and biochemical analyses of the kidneys also confirmed that the dual-targeting nanoparticles provided in this application have the ability to reduce renal inflammatory damage. This indicates that the dual-targeting nanoparticles achieve improved survival through a multimodal protective mechanism that synergistically inhibits systemic inflammation, alleviates organ-specific immune dysregulation, and protects multiple organ functions, thus improving the pathological development of sepsis.

[0038] Furthermore, the dual-targeting nanoparticles of this application also exert a protective effect by enhancing bacterial clearance and reducing sepsis-induced organ damage, and reduce sepsis-induced lung inflammation by targeting and degrading cGAS and regulating the cGAS-STING pathway.

[0039] This application also provides the application of the above-mentioned dual-targeting nanoparticles in the preparation of sepsis products.

[0040] In some specific embodiments, the product includes a therapeutic agent.

[0041] Compared with the prior art, this application has the following beneficial effects: This application provides a nanoparticle for autophagy-targeted degradation of TLR4 and cGAS, its preparation, and its application. The dual-targeting nanoparticle (CLEAR) provided in this application can inhibit two signaling pathways: activation of Toll-like receptor 4 (TLR4) and synergistic amplification of cytokine storm by the cyclic GMP-AMP synthase (cGAS)-STING signal transduction. This dual-targeting nanoparticle targeting M1 macrophages achieves synergistic degradation of cell membrane protein TLR4 and cytoplasmic protein cGAS through a biphasic mechanism: initially, the nanoparticles drive TLR4 from the cell membrane to lysosomes for degradation through endocytosis, and then escape from the lysosomes through the positively charged "proton pump" effect, further triggering autophagy to degrade cGAS and TLR4 in the cytoplasm through positive charge. The dual-targeting nanoparticles can continuously inhibit the STING-dependent interferon response, thereby blocking the inflammatory cascade. In a mouse sepsis model, treatment with the dual-targeting nanoparticles can alleviate cytokine storm, reduce pro-inflammatory macrophage aggregation, enhance bacterial clearance capacity, and significantly improve survival outcomes. By integrating the degradation of target proteins at different locations on the cell into a single nanoparticle, dual-targeting nanoparticles overcome the spatial limitations of traditional targeted protein degradation strategies, establishing a transformative paradigm for the treatment of inflammatory diseases centered on macrophages. Attached Figure Description

[0042] Figure 1 This is a schematic diagram for the treatment of sepsis (where, Figure 1 In section A, CLEAR (also known as CLEAR) is a nanoparticle prepared by emulsification / solvent evaporation method for autophagy-targeted degradation of TLR4 and cGAS. Figure 1 (B in the diagram illustrates the mechanism of CLEAR in treating sepsis). Figure 2 Figure 1 shows the characterization and biosafety results of CLEAR (where, Figure 2 A in the diagram represents the fabrication process of CLEARs; Figure 2 China B to Figure 2 E represents the Zeta potential (ζ), hydrodynamic diameter (Dh), and polydispersity index (PDI) distributions of unCLEARs (B and C) and CLEARs (D and E); Figure 2 F is a graph showing the changes in ζ-potential and Dh distribution of CLEARs during an 8-week storage period; Figure 2 China G to Figure 2In the middle, I represents the representative transmission electron microscopy (TEM) image (G), Fourier transform infrared (FTIR) spectrum (H), and ¹H nuclear magnetic resonance (NMR) spectrum (I) of unCLEARs and CLEARs; Figure 2 China J to Figure 2 N represents the in vitro biosafety assessment of CLEARs: calcein-AM (live cells) / propidium iodide (dead cells) fluorescence staining images (J), flow cytometry contour plots (K), and the viability of RAW264.7 cells (L) and HUVEC cells (M) treated with CLEARs for 24 hours, as well as the hemolysis rate (N) of different concentrations of CLEARs. Figure 2 From O to Figure 2 The biocompatibility of CLEARs in vivo is as follows: After intravenous injection of CLEARs (20 mg / kg) –1 Representative H&E staining images (O) of the heart, liver, spleen, lung, and kidney of C57BL / 6J mice (four times a week) and iNOS fluorescence images (P) of lung tissue. FTIR spectra (H), NMR spectra (I), and images (G, J, O, P) represent three independent experiments. Data are expressed as mean ± standard deviation, n=3. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc multiple comparison test. ns indicates no significant difference. Figure 3 Figure 1 shows the degradation efficiency and mechanism of TLR4 and cGAS (where, Figure 3 Figure A shows the cell uptake of RAW264.7 cells after co-culturing with CLEARs for different time periods using flow cytometry. Figure 3 China B and Figure 3 In the middle, C shows representative immunofluorescence images (B) and fluorescence colocalization curves (C) of CLEARs-NR and lysosomes (LAMP1) after co-culturing in RAW264.7 cells for 1, 2, and 4 hours. Figure 3 In the middle D, Western blot analysis of LC3B and p62 protein expression was performed after CLEARs treatment for 6 hours. Figure 3 China E and Figure 3 In Figure F, the in vitro expression analysis of TLR4(E) and cGAS(F) proteins after 6 hours of different treatments is performed using Western blot. Figure 3 China G to Figure 3 The image shows flow cytometry pseudocolor images (G and I) and quantitative analysis (H and J) of TLR4+ (G and H) or cGAS+ (I and J) fluorescence in RAW264.7 cells after 6 hours of different treatments. Figure 3 K and Figure 3In the image, L represents the representative immunofluorescence image (K) and fluorescence colocalization curve (L) of CLEARs-NR, lysosomes (LAMP1), and autophagosomes (LC3B) after co-culturing in RAW264.7 cells for 6 hours. Data are expressed as mean ± standard deviation, n=3. Statistical significance was determined using one-way ANOVA and Tukey's post-hoc multiple comparison test. p<0.05； ***p<0.0001; ns indicates no significant difference). Figure 4 The figure shows the in vitro anti-inflammatory effects of CLEARs (where, Figure 4 A in the diagram represents CLEARs in vitro treatment of sepsis; Figure 4 China B and Figure 4 In the middle (C), the mean fluorescence intensity (MFI) of RAW264.7 cells under Ec-lys stimulation at different time points is quantified (B), and compared with untreated cells at 10 hours (C). Figure 4 In the middle D, Western blot analysis of cGAS, pSTING, and STING proteins was performed on RAW264.7 cells after co-culturing with Ec-lys and CLEARs for 6 hours. Figure 4 China E and Figure 4 F represents the IFN-β level in the supernatant of RAW cells stimulated with Ec-lys (E) or LPS (F) and treated with CLEARs for 6 hours. Figure 4 China G and Figure 4 In the middle, H represents a representative flow cytometry pseudocolor image (G) and quantitative analysis (H) of cGAS+ fluorescence in RAW264.7 cells after 6 hours of different treatments. Figure 4 From I to Figure 4 K represents the polarization phenotype assessment of RAW264.7 macrophages after 24 hours of treatment: flow cytometry pseudocolor image (I), quantitative analysis (J), and CD86 immunofluorescence staining (K). Figure 4 From L to Figure 4 In the mean square root (P), the mRNA levels of macrophage polarization markers (Tnfα, Nos2, Il6, Il1b) and the cGAS pathway gene Ifnb were observed in RAW264.7 macrophages. Data are expressed as mean ± standard deviation, n=3. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc multiple comparison test. * p<0.05; ** p<0.001, *** p<0.0005, **** p<0.0001, ns indicates no statistically significant difference). Figure 5 A graph showing the in vivo efficacy results of CLEARs in treating sepsis (wherein, Figure 5 A in the middle is a schematic diagram of the treatment timeline; Figure 5The survival rate curve for 168 hours is shown in Figure B (n=10). Figure 5 C and Figure 5 In the middle D, longitudinal monitoring of sepsis score (C) and rectal temperature (D) within 168 hours postoperatively (n=10); Figure 5 China E and Figure 5 F represents the sepsis score (E) and rectal temperature (F) at different time points (48, 72, and 96 hours); Figure 5 G represents weight change within 168 hours post-surgery (n=10). Figure 5 From H to Figure 5 J represents the serum cytokine levels 24 hours post-surgery (n=5): TNFα (H), IL1β (I), and IL6 (J). Figure 5 K represents the lung wet / dry weight ratio (pulmonary edema assessment) 24 hours postoperatively (n=5); Figure 5 From L to Figure 5 The values ​​in section U represent specific inflammation analysis of lung tissue (L to P) and liver tissue (Q to U) 24 hours postoperatively (n=5): flow cytometry analysis included representative contour plots (L and Q) and quantitative analysis (M and R) of CD86+ cells in the CD45+CD11b+F4 / 80+ gated population; and ELISA results of TNFα (N and S), IL1β (O and T), and IL6 (P and U) in the tissue homogenate supernatant. Figure 5 V represents serum biomarkers of organ dysfunction at 24 hours postoperatively (n=5): myocardial (CK-MB), renal (urea, creatinine), hepatic (LDH, AST, ALT), and systemic infection marker (PCT). Data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc multiple comparison test. *p<0.05; **p<0.001, ***p<0.0005, ****p<0.0001, ns indicates no statistically significant difference). Figure 6 Figure showing the results of CLEARs promoting bacterial clearance and inflammation reduction in sepsis (wherein, Figure 6 Image A shows a representative scanning electron microscope (SEM) image of CLEARs adsorbed on the surface of Escherichia coli. Figure 6 The results in Figure B represent the bacterial colony-forming units (CFU) in blood, lung, and liver tissue homogenates 24 hours post-surgery. Figure 6 C to Figure 6 The middle section (E) shows representative H&E staining of the liver (C) and lungs (D) 24 hours post-surgery, along with corresponding quantitative lung tissue pathological scores (E). Figure 6 China F to Figure 6In the middle section, H represents representative H&E staining of the liver (F) and lungs (G) at 168 hours postoperatively, and the corresponding quantitative lung histopathological scores (H). Data are expressed as mean ± standard deviation, n=4. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc multiple comparison test. ****p<0.0001, ns indicates no significant difference). Figure 7 The diagram shows the results of CLEARs degrading cGAS and inhibiting the cGAS-STING pathway in vivo (where, Figure 7 China A to Figure 7 Image J shows representative immunofluorescence staining of lung sections 24 hours post-surgery: co-staining of cGAS (red) and F4 / 80 (green) (A), co-staining of pSTING (red) and F4 / 80 (green) (D), and single staining of iNOS (red) (I). Scale bar: 100 μm (main image), 10 μm (inset); quantitative measurements of intracellular cGAS⁺ area in F4 / 80⁺ cells in lung sections (B), total F4 / 80⁺ area (C), quantitative measurements of intracellular pSTING⁺ area in F4 / 80⁺ cells (E), and quantitative measurements of iNOS⁺ area (J) (n=6). Figure 7 China F to Figure 7 H represents the IFN-β levels in serum (F), lung tissue (G), and liver tissue (H) 24 hours post-surgery (n=5). Figure 7 Middle K to Figure 7 The middle section (Q) represents representative immunofluorescence staining of lung sections one week post-surgery: co-staining of cGAS (red) and F4 / 80 (green) (K), co-staining of pSTING (red) and F4 / 80 (green) (M), and single staining of iNOS (red) (P). Scale bar: 100 μm (main image), 10 μm (inset). Quantitative measurements of intracellular cGAS⁺ area (L), intracellular pSTING⁺ area (N), total F4 / 80⁺ area (O), and iNOS⁺ area (Q) in lung sections (n=6). Data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc multiple comparison test. * p < 0.05; ** p < 0.001, *** p < 0.0005, **** p < 0.0001, ns indicates no significant difference). Figure 8 A graph showing the stability results of CLEARs during an 8-week storage period; Figure 9 The hydrogen nuclear magnetic resonance (¹H NMR) spectra of unCLEARs and CLEARs are shown. Figure 10 A diagram illustrating the biosafety assessment of CLEARs; Figure 11Figure 1. Western blot analysis of TLR4 and cGAS proteins in vitro expression after CLEARs were treated for 24 hours. Figure 12 Figure showing the results of an EC-lys-induced sepsis cell model; Figure 13 The figure shows the results of Western blot analysis of pSTING and STING proteins in RAW264.7 cells after 6 hours of different treatments. Figure 14 Figure 1 shows the weight monitoring results of mice in the sham-operated group, sepsis group, and treatment group; Figure 15 for Figure 4 Gating strategy diagram for flow cytometry analysis of lung macrophages in the middle 1; Figure 16 for Figure 4 Gating strategy diagram for flow cytometry analysis of hepatic macrophages in the middle q region; Figure 17 A graph showing the results of validating CLEARs' ability to alleviate inflammatory damage in the context of kidney function assessment. Figure 18 A diagram illustrating the gating strategy for flow cytometry analysis of 17A mid-renal macrophages; Figure 19 This is a representative image of colony-forming units (CFU) detected in renal tissue homogenate 24 hours post-surgery. Figure 20 A representative H&E staining image of spleen tissue; Figure 21 A representative H&E staining image of cardiac tissue; Figure 22 A representative H&E staining image of kidney tissue; Figure 23 The image shows the results of IFN-β level detection in kidney tissue 24 hours after surgery (n=5). Detailed Implementation

[0043] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0044] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0045] In the following embodiments, the cell lines and cultures include the following: Human umbilical vein endothelial cell line HUVEC was purchased from the American Type Culture Collection (ATCC), and mouse macrophage line RAW264.7 and human macrophage line THP1 were purchased from the National Laboratory Cell Resource Sharing Platform (Shanghai, China). All cell lines were identified by short tandem repeat (STR) analysis. Morphological and growth characteristics were monitored regularly using an inverted microscope. Mycoplasma contamination was ruled out at receipt and during routine culture.

[0046] Cells were cultured under standard conditions (37°C, 5% CO2, humidified environment) using a Forma™ Steri-Cycle™ i160 incubator (Thermo Scientific, USA). All cells were cultured in the following media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S): HUVEC cells were cultured in DMEM / F-12 medium (Corning, USA), RAW264.7 cells were cultured in high-glucose Dalberks modified Eagle medium (DMEM; 4.5 g / L glucose, Gibco, USA), and THP-1 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Corning, USA).

[0047] Example 1: A nanoparticle for autophagy-targeted degradation of TLR4 and cGAS and its preparation method This embodiment provides a method for preparing nanoparticles that target the autophagy degradation of TLR4 and cGAS, including the following steps: S1. Dissolve polylactic acid (PLA) (0.3 g) in dichloromethane (DCM, 15 mL) and stir magnetically (500 rpm, 24 h) to form an oil phase; mix hexadecyltrimethylammonium bromide (CTAB) (15 mM), distearate phosphatidylethanolamine-polyethylene glycol-aminoDSPE-PEG-NH2 (7.5 mM) and polyvinyl alcohol (PVA) (5% w / v) to form an aqueous phase; S2. Add the oil phase (2 mL, 2% PLA) to the aqueous phase (total volume 10 mL; DSPE-PEG-NH2 / CTAB volume ratio 2:8). Pre-emulsify for 1 minute using a high-speed shear homogenizer (T18 digital, IKA, Germany) at 15,000 rpm, then emulsify using an ultrasonic homogenizer (950E, Scientz, China) at 300W power for 5 minutes (on / off cycle 3s / 3s) to obtain an emulsion. Disperse the obtained emulsion in a polyvinyl alcohol (PVA) solution (3 mL) and evaporate the DCM by continuous stirring in a fume hood (300 rpm, 24 h) to obtain unfunctionalized nanoparticles (unCLEAR).

[0048] S3. Unfunctionalized nanoparticles (unCLEAR) (2 mL) were mixed with MES buffer (6 mL, 100 mM, pH 6.0) containing EDC (0.7 mg), NHS (0.7 mg), a first targeting ligand (using TLR4-IN-C34-C2-COOH, 0.8 mg), and a second targeting ligand (using cGAS-IN-1, 0.7 mg). The mixture was stirred at 300 rpm for 24 h. Monomers were removed by centrifugation and ultrafiltration (100 kDa molecular weight cutoff device, 3000 × g × 30 min, 4°C), followed by washing three times with ddH2O. The purified CLEAR (2 mg / mL) was sterilized by filtration through a 0.45 μm PVDF membrane and stored at 4°C to obtain autophagy-targeted TLR4 and cGAS nanoparticles (named CLEAR).

[0049] Based on Example 1, Nile Red (1 mg / mL) was added to the oil phase to prepare CLEAR-NR.

[0050] The flowchart of the above preparation method is as follows: Figure 1 China A and Figure 2 As shown in Figure A.

[0051] Example 2: A nanoparticle for autophagy-targeted degradation of TLR4 and cGAS and its preparation method Compared with Example 1, the difference in Example 2 is that the volume ratio of DSPE-PEG-NH2 / CTAB in step S2 is 4:6, while the other steps are the same as in Example 1.

[0052] Example 3: A nanoparticle for autophagy-targeted degradation of TLR4 and cGAS and its preparation method Compared with Example 1, the difference in Example 3 is that the volume ratio of DSPE-PEG-NH2 / CTAB in step S2 is 6:4, while the other steps are the same as in Example 1.

[0053] Example 4: A nanoparticle for autophagy-targeted degradation of TLR4 and cGAS and its preparation method Compared with Example 1, the only difference in Example 4 is in step S2: the pre-emulsification speed is adjusted to 10,000 rpm and the pre-emulsification time is 5 minutes; the ultrasonic homogenization power is adjusted to 100W and the homogenization time is 1 minute. The other steps are the same as in Example 1.

[0054] Example 5: A nanoparticle for autophagy-targeted degradation of TLR4 and cGAS and its preparation method Compared with Example 1, the only difference in Example 5 is in step S3: the amount of TLR4 targeting ligand added is 1.2 mg, and the amount of cGAS targeting ligand added is 0.5 mg. The other steps are the same as in Example 1.

[0055] Example 6: A nanoparticle for autophagy-targeted degradation of TLR4 and cGAS and its preparation method Compared with Example 1, the only difference in Example 6 is the type of targeting ligand, specifically in step S3: The 0.8 mg TLR4-targeting ligand (TLR4-IN-C34-C2-COOH) and 0.7 mg cGAS-targeting ligand (cGAS-IN-1) were replaced with 0.8 mg Eritoran (TLR4-targeting ligand) and 0.7 mg RU.521 (cGAS-targeting ligand). These two small molecule ligands, via their inherent or modified carboxyl / amino groups, under EDC / NHS-mediated amidation coupling with amino groups on the surface of unfunctionalized nanoparticles (uCLEAR). All other steps were identical to those in Example 1.

[0056] Example 7: A nanoparticle for autophagy-targeted degradation of TLR4 and cGAS and its preparation method Compared with Example 1, the difference in Example 7 lies only in the type of targeting ligand and the coupling conditions, specifically in step S3: The 0.8 mg TLR4-targeting ligand (TLR4-IN-C34-C2-COOH) and 0.7 mg cGAS-targeting ligand (cGAS-IN-1) were replaced with 1.0 mg of a single-chain antibody fragment (scFv, as the TLR4-targeting ligand) that specifically binds to the extracellular region of TLR4 and 0.5 mg of a terminally carboxyl-modified A151 oligonucleotide (as the cGAS-targeting ligand). Due to the involvement of protein macromolecules in the coupling with nucleic acids, the stirring conditions in step S3 were adjusted to a gentle reaction at 4°C in the dark for 12 hours to maintain the bioactivity of the single-chain antibody, followed by purification by centrifugation and ultrafiltration (100 kDa molecular weight cutoff). Other steps were the same as in Example 1.

[0057] Among them, the single-chain antibody fragment (scFv) that specifically binds to the extracellular region of TLR4 is derived from an anti-TLR4 antagonistic monoclonal antibody. The scFv is formed by the heavy chain variable region (VH) and light chain variable region (VL) of the antibody linked together by a flexible linker. VH domain sequence (118 amino acids): QVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSDAFPYWGQGTLVTVSS (as shown in SEQ ID NO: 1).

[0058] Linker flexible linker peptide sequence (15 amino acids): GGGGSGGGGSGGGGS (as shown in SEQ ID NO: 2).

[0059] VL domain sequence (107 amino acids): EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGGGTKVEIK (as shown in SEQ ID NO: 3).

[0060] During the coupling process, the scFv, through its exposed free carboxyl group at its C-terminus or the carboxyl group on the side chain of amino acid residues on its surface, is targeted and coupled to the PEG amino terminus on the surface of the unfunctionalized nanoparticles under the action of a condensing agent.

[0061] The terminal carboxyl-modified A151 oligonucleotide has the nucleotide sequence shown in SEQ ID NO: 4 (5'-TTAGGGTTAGGGTTAGGGTTAGGG-3'), and its 5' end is modified with a free carboxyl group (e.g., 5'-Carboxyl Modifier C10) via a linker arm containing a carbon chain, so as to form a stable covalent amide bond with the amino group on the surface of the nanoparticle.

[0062] Comparative Example 1 Compared with Example 1, the difference of Comparative Example 1 is that step S2 does not contain DSPE-PEG-NH2, the total volume of CTAB is 10 mL, and the other steps are the same as in Example 1.

[0063] Comparative Example 2 Compared with Example 1, Comparative Example 2 differs in that step S2 does not contain CTAB, the total volume of DSPE-PEG-NH2 is 10 mL, and the other steps are the same as in Example 1.

[0064] Comparative Example 3 Compared with Example 1, the difference in Comparative Example 3 is that pre-emulsification is not used in step S2, and emulsification is performed directly. The other steps are the same as in Example 1.

[0065] Step S2 is as follows: the oil phase (2 mL) is directly added to the aqueous phase (10 mL), and emulsification is performed directly using an ultrasonic homogenizer at 300W for 5 minutes without using a high-speed shear homogenizer. The subsequent steps are the same as in Example 1.

[0066] Comparative Example 4 Compared with Example 1, the difference in Comparative Example 4 is that only 0.8 mg of TLR4 targeting ligand was added in step S3, and cGAS targeting ligand was not added. The other steps were the same as in Example 1.

[0067] Comparative Example 5 Compared with Example 1, the difference in Comparative Example 5 is that only 0.7 mg of cGAS-targeting ligand was added in step S3, and TLR4-targeting ligand was not added. The other steps were the same as in Example 1.

[0068] Comparative Example 6 (Target Cell Deviation) Compared with Example 1, the difference in Comparative Example 6 is that an equimolar amount of mannose was used to replace the TLR4 targeting ligand in step S3, while the other steps were the same as in Example 1.

[0069] Mannose is a specific ligand for the CD206 receptor on the surface of M2 macrophages and cannot target M1 macrophages, which are involved in the core pathological mechanism of sepsis.

[0070] Comparative Example 7 (Anti-mechanism agonist) Compared with Example 1, the difference in Comparative Example 7 is that an equimolar amount of lipopolysaccharide (LPS) was used instead of the TLR4 targeting ligand in step S3, while the other steps were the same as in Example 1.

[0071] LPS is a potent agonist of TLR4, not an antagonist or blocker.

[0072] Performance Characterization and Biological Evaluation of Experimental Example 1, Examples and Comparative Examples To verify the technical effect of the CLEAR nanoparticles of this application, the applicant conducted systematic quantitative tests on the products prepared in the above embodiments and comparative examples.

[0073] 1. Physicochemical characterization The hydrodynamic particle size and polydispersity index (PDI) of each group of nanoparticles were determined using dynamic light scattering (DLS).

[0074] Example 1: The particle size was approximately 125.4 ± 4.2 nm, and the PDI was 0.12 ± 0.03, indicating that the dual-targeted nanoparticles prepared by the emulsification method had uniform size and excellent dispersibility.

[0075] Examples 2, 3, 4, and 5: The particle size is distributed between 110 and 150 nm, and the PDI is less than 0.2, indicating good drug-like properties.

[0076] Examples 6 and 7: The nanoparticles prepared in Example 6 (Eritoran + RU.521) had a particle size of approximately 130.5 ± 5.1 nm and a PDI of 0.15. In Example 7 (scFv + A151 oligonucleotide), due to the grafting of antibody fragments and nucleic acid macromolecules, the particle size slightly increased to 145.2 ± 6.3 nm, with a PDI of 0.18. This demonstrates that even when replacing the targeting ligands with different molecular weights (small molecules, large protein molecules, nucleic acids), the preparation method of this application can still ensure the stable formation and good uniformity of dual-targeting nanoparticles.

[0077] Comparative Example 1 (without DSPE-PEG): The particle size increased to over 280 nm and was prone to aggregation after standing; Comparative Example 3 (without pre-emulsification step): The particle size was as high as 540.6±21.5 nm and the PDI soared to 0.45, indicating that the lack of a shear pre-emulsification step would lead to excessively large and non-uniform primary emulsion particles, and the ultrasonic energy could not effectively refine them.

[0078] 2. Cell-specific uptake assay (flow cytometry): The uptake of Nile Red-encapsulated nanoparticles in LPS-activated M1 macrophages (RAW264.7) was analyzed by flow cytometry.

[0079] Example 1 (CLEAR-NR): According to the gating analysis, the uptake rate of the dual-target CLEAR by M1 macrophages exceeded 80%, and the average fluorescence intensity (MFI) was extremely high.

[0080] Comparative Example 6 (targeting M2 ligand): Due to ligand mismatch, its endocytosis efficiency in M1 macrophages is extremely low, with an uptake rate of less than 20% (similar to unfunctionalized unCLEAR), thus losing its ability to target and enrich.

[0081] 3. In vitro inflammatory factor release inhibition assay: Macrophage culture supernatant after drug treatment was collected, and the secretion levels of pro-inflammatory factors TNF-α and IL-6 were detected by ELISA.

[0082] Model group (LPS stimulation): TNF-α concentration surged to 3500 pg / mL.

[0083] Comparative Example 4 (single-target TLR4) and Comparative Example 5 (single-target cGAS): The concentration of pro-inflammatory factors decreased to approximately 1800-2200 pg / mL, showing partial inhibition.

[0084] Example 1 (CLEAR): It exhibited a significant synergistic silencing effect, with TNF-α concentration sharply reduced to below 400 pg / mL and IL-6 also significantly decreased, which was superior to any single-target control group (P<0.01).

[0085] Examples 6 and 7: After treatment with LPS-stimulated models, TNF-α concentrations decreased significantly to 500-600 pg / mL, which was significantly better than the untreated model group. This confirms that the candidate pro-inflammatory transmembrane receptor ligands specified in this application and the intracytoplasmic pattern recognition receptor ligands can also achieve dual-target clearance, possessing the characteristics of a replaceable universal platform technology.

[0086] Comparative Example 7 (using LPS ligand): Inflammation was not suppressed; instead, the concentration of TNF-α increased abnormally to over 5000 pg / mL, triggering a severe cytokine storm.

[0087] 4. Animal-level pharmacodynamic evaluation (survival rate of sepsis model): Mouse cecal ligation and perforation (CLP) sepsis model: Mice treated with Example 1 (CLEAR) showed significant degradation of TLR4 and cGAS proteins in vivo, as verified by sixty-year cell biology, with an overall survival rate of approximately 80%; while the survival rate of single-target nanoparticles (Comparative Example 4, Comparative Example 5) was only 30%-40%; the Comparative Example 7 (agonist-targeted) model group developed 100% lethal shock within 24 hours.

[0088] Experimental Example 2: Nanoparticle Characterization and Biocompatibility Evaluation 1. Nanoparticle Characterization: The hydrodynamic diameter and zeta potential of unCLEAR and CLEAR were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) using a nanoparticle analyzer (Litesizer 500, Anton-Paar, Austria). The morphology was visualized by transmission electron microscopy (TEM; Tecnai G2 Spirit, FEI, USA).

[0089] The zeta potential (ζ) of the unfunctionalized nanoparticles (unCLEAR) prepared in Examples 1-3 was determined by electrophoretic light scattering (ELS), and the hydrodynamic diameter (Dh) and polydispersity index (PDI) of the unfunctionalized nanoparticles (unCLEAR) prepared in Examples 1-3 were evaluated by dynamic light scattering (DLS). Figure 2 In option B, select unCLEAR with a high zeta potential: In Example 1, the volume ratio of DSPE-PEG-NH2 / CTAB was 2:8 (22.78 ± 0.99 mV); in Example 2, the volume ratio was 4:6 (22.26 ± 0.71 mV); and in Example 3, the volume ratio was 6:4 (21.27 ± 0.31 mV). Simultaneous characterization showed that their Dh values ​​were 155.78 ± 2.64 nm, 196.63 ± 1.81 nm, and 203.90 ± 3.53 nm, respectively, and their PDI values ​​were 0.14 ± 0.008, 0.16 ± 0.011, and 0.18 ± 0.005, respectively. Figure 2 (C)

[0090] To maximize the preservation of the autophagy-inducing ability of the nanoparticles, CLEAR (the dual-targeting nanoparticles of Example 1), which exhibited the highest zeta potential, was selected. Specifically, DSPE-PEG-NH2:CTAB was in a ratio of 2:8, with a zeta potential of 17.89 ± 2.30 mV. Figure 2 The nanoparticle has a Dh of 162.74 ± 2.60 nm and a PDI of 0.18 ± 0.006 (in the middle D). Figure 2 (E).

[0091] In Comparative Example 1, the absence of DSPE-PEG-NH2 prevented ligand binding, resulting in no targeting or degradation activity. In Comparative Example 2, the absence of CTAB resulted in no endosome escape or autophagy induction, and the inability to degrade receptors in the cytoplasm.

[0092] Furthermore, by measuring the ζ-potential, Dh, and PDI values ​​of CLEAR (the dual-targeting nanoparticles of Example 1) during 8 weeks of storage at 4°C, it was confirmed that CLEAR has good storage stability. Figure 2 China F and Figure 8 Transmission electron microscopy (TEM) Figure 2 (G) confirmed that both unCLEAR and CLEAR (DSPE-PEG-NH2:CTAB volume ratio of 2:8) exhibited a spherical morphology. FTIR spectroscopy confirmed successful ligand coupling through characteristic amide bond signals. Figure 2 (H).

[0093] Successful nanoparticle grafting (especially amide bond formation) was confirmed using Fourier transform infrared (FTIR) spectroscopy (IRAffinity-1S, Shimadzu, Kyoto, Japan). Freeze-dried unCLEAR and CLEAR were ground, uniformly mixed with KBr, and compressed into pellets. Spectra were acquired in the 4000–400 cm⁻¹ range (20 scans per sample), confirming the covalent coupling of the ligands via amide bonds. Successful grafting of the nanoparticles with cGAS and TLR4 ligands was further confirmed using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy (Bruker Avance 600 MHz). Freeze-dried unCLEAR and CLEAR were dissolved in DMSO-d6 and analyzed in a standard 5 mm NMR tube. ¹H NMR analysis verified the effective incorporation of TLR4 and cGAS ligands. Figure 2 I and Figure 9 These results confirm the successful construction of dual-targeted nanoparticles with precisely designed positive charges.

[0094] 2. Biocompatibility evaluation: RAW264.7 cells (5 × 10⁶ cells per well) were placed in a 12-well plate. 5 Cells were incubated with CLEAR for 24 hours. Cell viability was qualitatively assessed by live / dead cell staining (fluorescence microscopy Ti2-U, Nikon, Japan Imaging), and quantitative analysis was performed using flow cytometry (CytoFLEXLX, Beckman Coulter) via calcein-AM-FITC (live cells) and propidium iodide-PE (dead cells). Simultaneously, THP-1 cells and HUVECs (96-well plates, 1 × 10⁶ cells per well) were treated with CLEAR. 4 Cell viability was detected by CCK-8 assay after 24 hours, and absorbance was measured at 450 nm using a microplate reader (Synergy H1MF, BioTek, USA).

[0095] Fresh whole blood was collected from anesthetized C57BL / 6J mice via retroorbital puncture into EDTA-2 Na tubes. Red blood cells were separated by centrifugation (2000×g, 15 min), washed, and resuspended in PBS to prepare a 4% (v / v) cell suspension. The suspension was incubated at 37°C for 2 hours with the following substances: CLEAR solution (0, 10, 20 μg / mL), PBS, 0.1% Triton X-100, or deionized water. After a second centrifugation (2000g, 15 min), the hemoglobin content of the supernatant was quantified at 540 nm using a microplate reader. Hemolysis rate (%) = (OD sample - ODPBS) / (ODH2O - ODPBS) × 100. Where, OD sample = absorbance of the CLEAR-treated sample; ODPBS = absorbance of the PBS control (0% hemolysis); ODH2O = absorbance of the water control (100% hemolysis).

[0096] Live / dead cell staining images and flow cytometry analysis showed that RAW264.7 cells maintained nearly 100% viability after treatment with 2 µg mL⁻¹CLEAR. Figure 2 China J to Figure 2 L and Figure 10 The CCK-8 assay further confirmed that human umbilical vein endothelial cells (HUVECs) are... Figure 2 (M) and THP-1 monocytes ( Figure 10 The activity of B) was not impaired.

[0097] Hemolysis assays showed negligible membrane lysis activity, with only 0.18 ± 0.02% erythrocyte damage at a concentration of 20 µg mL⁻¹, significantly below the biocompatibility threshold. Figure 2 N and Figure 10 The presence of C57BL / 6J cells indicates that the integrity of the erythrocyte membrane was well maintained. Furthermore, the system's biocompatibility was assessed by intravenous injection into C57BL / 6J mice. Histopathological examination of the major organs (heart, liver, spleen, lung, and kidney) showed intact tissue structure and no pathological damage. Figure 2 (O). Crucially, immunofluorescence analysis of lung tissue showed no significant upregulation of inducible nitric oxide synthase (iNOS), a key mediator of inflammatory response and tissue damage. Figure 2 (P). No iNOS was induced. Figure 2 The results showed that the intact lung tissue structure observed in the O cells was consistent with that observed in the O cells, further demonstrating the absence of significant inflammation or damage. In summary, the results of this application demonstrate the excellent systemic biocompatibility of CLEAR.

[0098] Experimental Example 3: In vitro evaluation of CLEAR-induced TLR4 and cGAS degradation 1. Cellular uptake of CLEAR: RAW264.7 cells were seeded in 12-well plates (5 × 10⁶ cells per well). 5(cells) were cultured for 24 hours. Subsequently, in the presence of Ec-lys or an equal volume of PBS, they were inoculated with CLEAR-NR (2 μg / mL). –1 Incubate the cells for specified times (0, 2, 4, 6, 8, and 10 hours). Discard the culture medium, wash the cells twice with pre-cooled PBS, collect the cells using a cell scraper, and detect the intracellular NR signal (APC channel) using a flow cytometer (CytoFLEX LX, Beckman Coulter).

[0099] result: The lysosomal degradation of TLR4 and cGAS proteins by CLEAR is dependent on cellular internalization. TLR4 is highly expressed in RAW264.7 mouse macrophages. Flow cytometry quantitative analysis showed that CLEAR accumulated in RAW264.7 macrophages in a time-dependent manner, with significant uptake (2 µg mL⁻¹) detectable within 2 hours of incubation. Figure 3 (A) This excellent cellular uptake capacity lays the foundation for membrane protein degradation and subsequent anti-inflammatory responses.

[0100] After CLEAR is internalized by RAW264.7 cells, it is theoretically expected to transport TLR4 to lysosomes for degradation. To determine the subsequent fate of CLEAR, this application used immunofluorescence colocalization analysis of lysosome-associated membrane protein 1 (LAMP1) and Nile red-labeled CLEAR (CLEAR-NR). After 1 hour of incubation, partial lysosomal colocalization (2 µg mL⁻¹) was observed. Figure 3 B, Figure 3 Colocalization of lysosomes peaked at 2 hours, but by 4 hours, both fluorescence imaging and semi-quantitative analysis showed a significant decrease. These results are consistent with reports of "proton sponge effect" mediated escape from cationic nanoparticles, indicating that CLEAR has successfully achieved lysosomal escape.

[0101] This application then investigated the ability of CLEAR to induce autophagic flux. As expected, Western blot analysis showed that autophagy markers were upregulated in a dose-dependent manner in CLEAR-treated RAW264.7 cells ( Figure 3 (D). After 6 hours of incubation, the LC3B-II / I level increased to 1.4-fold and p62 decreased by 68% at a concentration of 2 μg mL⁻¹ (compared to the untreated control group), confirming the induction of anomalous autophagic flux.

[0102] 2. CLEAR's ability to degrade proteins: RAW 264.7 cells were seeded in 6-well plates (1 × 10⁶ cells per well). 6Cells were incubated with CLEAR for 6 or 24 hours with or without Ec-lys. After washing with PBS, lysis was performed on ice for 10 minutes using pre-chilled RIPA lysis buffer (100 μL, containing 1 mM PMSF). The collected lysis buffer was transferred to pre-chilled centrifuge tubes and centrifuged (12000×g, 5 min, 4°C) to collect the supernatant. Protein concentration was determined using the BCA method and normalized by dilution with lysis buffer. The sample was mixed with 5× loading buffer and denatured at 37°C with shaking (1200 rpm) for 45 min. An equal volume of protein was separated by vertical electrophoresis (Bio-Rad) on a 4-20% gradient SDS-PAGE gel (ACE) at a constant voltage of 80 V until bromophenol blue reached the bottom of the gel. Subsequently, the protein was transferred to an ethanol-activated PVDF membrane (0.45 μm, Millipore) at 4°C using a transfer module (Bio-Rad) at a constant current of 300 mA for 1.5 h. The membrane was blocked with TBST containing 5% BSA for 2 hours (room temperature, gentle shaking at 50 rpm). After washing with TBST (3 × 10 min), it was incubated overnight at 4°C with the following primary antibodies: TLR4 (1:4000), cGAS (1:1000), STING (1:100000), pSTING (1:2000), Vinculin (1:60000), and GAPDH (1:50000). After washing three times with TBST, it was incubated with HRP-labeled secondary antibody (1:2000) at room temperature for 2 hours. After a final TBST wash, the membrane was developed using ECL reagent and imaged using a chemiluminescence detection system (G:BOX Chemi XX6, Syngene). Band intensity was quantified using Fiji software (ImageJ).

[0103] result: This application used Western blotting to detect TLR4 and cGAS levels in CLEAR-treated RAW264.7 cells. Concentration-dependent TLR4 degradation was achieved within 6 hours, with a target protein degradation efficiency of 92%, significantly superior to TLR4 inhibitors (29%). Figure 3 (E); extending the action time to 24 hours, the degradation efficiency exceeds 99% ( Figure 11 (A); Parallel analyses of cGAS showed similar time- and dose-dependent degradation ( Figure 3 China F and Figure 11 Treatment with cGAS (Category B) for 6 hours resulted in 93% protein degradation, far exceeding the 36% degradation of cGAS inhibitors; extended treatment to 24 hours achieved near-complete (>99%) cGAS degradation. This dual-target efficacy demonstrates CLEAR's superior target protein degradation capabilities.

[0104] 3. The mechanism by which CLEAR degrades proteins: RAW 264.7 cells grown on the slide (1×10⁶ cells per well in a 12-well plate) 4 (cells) and CLEAR-NR (2 μg / mL) –1 Incubate for 0-8 hours. Lysosomal colocalization assessment: Wash three times with PBS, fix with 4% paraformaldehyde (PFA, prepared in PBS) (10 min, room temperature), permeabilize with 0.1% Triton X-100 (10 min, room temperature), and block with 5% donkey serum (overnight, 4°C); then incubate with eFluor™ 660-labeled mouse anti-LAMP1 antibody (1:400) at room temperature for 2 hours, wash three times with PBS, mount with DAPI-containing mounting medium, and image using confocal microscopy. Autophagosome colocalization: After the same washing / fixation / permeabilization / blocking procedure, samples were sequentially immunostained: rabbit anti-LC3B primary antibody (1:200, room temperature for 2 hours), and AF555-labeled donkey anti-rabbit secondary antibody (1:500, room temperature in the dark for 1 hour), with three PBS washes between each step. All samples were imaged using a laser confocal microscope (TCS SP8, Leica, Germany). The co-localization of CLEAR-NR / LAMP1 and CLEAR-NR / LC3B was evaluated using Fiji software.

[0105] In the study of TLR4 and cGAS degradation, RAW 264.7 cells pretreated (with or without Ec-lys) were used in 12-well plates (1 × 10⁶ cells per well). 5 Cells were divided into four groups: control group (PBS), CLEAR group (2 μg mL–1), CLEAR + chloroquine (CQ, 100 μM), and CLEAR + concanavalin A (ConA, 20 nM). After 24 hours of incubation, cells were collected, fixed / permeabilized as described above, and blocked with 5% donkey serum (room temperature, 2 hours). Intracellular staining was performed using rabbit anti-cGAS (1:400) or mouse anti-TLR4 (1:500) primary antibody, incubated overnight at 4°C, followed by incubation with AF555-labeled donkey anti-rabbit secondary antibody (1:500, room temperature, 2 hours). Flow cytometry (CytoFLEXS, Beckman) analysis was performed, and median fluorescence intensity was quantified using FlowJo software (v10.8.1). Unless otherwise specified, all washing steps involved three washes with PBS.

[0106] This application demonstrates that by inhibiting lysosomal function using chloroquine (CQ) or concanavalin A (ConA), it effectively inhibits CLEAR-mediated dual-target degradation. (Flow cytometry pseudocolor image) Figure 3 China G, Figure 3 Quantitative analysis of (I) and corresponding mean fluorescence intensity (MFI) Figure 3 H, Figure 3The results showed that the levels of TLR4 and cGAS in the inhibitor-treated group were significantly higher than those in the CLEAR-only group (p<0.05), and CQ treatment even restored TLR4 / cGAS expression to the level of untreated cells. These results demonstrate that CLEAR-mediated dual-target degradation is lysosomal mediated.

[0107] Unlike other dual-target degrading nanoparticles, the CLEAR designed in this application employs a two-phase mechanism to degrade different proteins: First, utilizing the endocytic properties of the nanoparticles, CLEAR specifically binds to TLR4 and transports it to the endosome-lysosome, degrading TLR4. The nanoparticle then escapes the lysosome through the proton sponge effect. After entering the cytoplasm, it binds to the target protein through surface ligands and induces autophagic flux through its cationic properties, ultimately achieving autophagosome-lysosomal degradation of cGAS and TLR4 in the cytoplasm. Confocal microscopy confirmed this mechanism: after 6 hours of incubation, CLEAR showed significant colocalization with lysosomes (LAMP1) and autophagosomes (LC3B). Figure 3 K Figure 3 (L). In general, CLEAR clears the membrane protein TLR4 via the endosome-lysosome pathway and degrades the cytoplasmic proteins cGAS and TLR4 via the autophagosome-lysosome pathway.

[0108] Experimental Example 4: In vitro experiment of CLEAR for the treatment of sepsis 1. Preparation of Escherichia coli lysate and construction of sepsis model: Escherichia coli (E. coli) was cultured in LB medium (37°C, 200 rpm) to mid-logarithmic growth (OD600 = 0.6). 1 mL of culture was centrifuged (6000 × g, 4°C, 10 min) to collect bacterial cells, washed twice with PBS, and resuspended in 1 mL PBS. The cells were then sonicated on ice (300 W, 5 sec on, 5 sec off, total 10 min). Debris was removed by centrifugation (4000 × g, 4°C, 15 min). The clarified lysis supernatant (Ec-lys) was aseptically filtered through a 0.22 μm PVDF membrane and stored at -20°C.

[0109] An in vitro sepsis model was established by stimulating RAW 264.7 macrophages with Ec-lys: cells were seeded into 12-well plates (5 × 10⁶ cells per well). 5 Cells) were treated with Ec-lys (0-0.6 OD) 600 Dilute to a final concentration of 30 μL / mL –1 Cells were treated for 24 hours. After staining with APC-anti-CD86, the cells were analyzed by flow cytometry, and the optimal induction concentration (OD) was confirmed by a CCK-8 viability assay (450 nm absorbance). 600 =0.4).

[0110] To overcome the limitations of LPS in simulating the in vitro state of sepsis, an E. coli lysate (Ec-lys) simulation system was established, incorporating multiple pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), flagellin, and bacterial DNA. Figure 4 As shown in Figure A, this model reproduces the characteristics of sepsis induced by multiple stimuli, and also demonstrates the mechanism by which CLEAR regulates the cGAS-STING pathway for treatment. Through exploration, this application discovered ( Figure 12 Ec-lys induced equivalent M1 macrophage polarization at OD600 values ​​of 0.4 and 0.6 (30 μL mL⁻¹, 24 h) (quantified by mean CD86+ fluorescence intensity MFI). Meanwhile, CCK-8 assays confirmed no cytotoxicity at OD600=0.4, and this concentration was consistently used in subsequent studies.

[0111] After establishing an in vitro model of sepsis, flow cytometry showed that Ec-lys stimulation significantly enhanced the uptake of CLEAR by RAW264.7 macrophages in a time-dependent manner. Figure 4 B, Figure 4 (C). Quantitative results showed that the internalization rate within 10 hours was significantly higher than that in the unstimulated control group (p<0.01), which may be due to the upregulation of TLR4 in macrophages induced by LPS, which enhances internalization by strengthening ligand-receptor binding.

[0112] Next, this application evaluates the regulatory role of the cGAS-STING pathway. Immunoblot analysis showed that 2 μg mL⁻¹ CLEAR effectively degraded cGAS (90% reduction after 6 hours) and inhibited STING phosphorylation, with pSTING levels decreasing by 88% compared to baseline. Figure 4 The D group was significantly better than the cGAS inhibitor group (which only reduced by 33%). Figure 13 ELISA analysis showed that IFNβ secretion was reduced by 26.33% compared to the control group (p<0.001). Figure 4 (E), consistent with findings in the LPS stimulation model ( Figure 4 (F). Notably, lysosomal inhibition (CQ or ConA) significantly attenuated cGAS degradation ( Figure 4 China G, Figure 4 The addition of H) increased intracellular cGAS levels to 1.52 times and 1.33 times that of the CLEAR control group alone, respectively, which is consistent with the mechanism of lysosome-dependent degradation mentioned above.

[0113] 2. Effects of CLEAR on macrophages: RAW264.7 cells were seeded into 24-well plates (1 × 10⁶ cells per well). 5 12-well plate (5 × 10 cells per well)5 (cells) and 6-well plates (1×10 cells per well) 6 Cells were cultured for 24 hours. Cells were then inoculated with Ec-lys and CLEAR (1 or 2 μg / mL). –1 The process took 24 hours.

[0114] Post-processing operations vary depending on the board type: 1) 24-well plates were washed with PBS, fixed with 4% PFA (prepared in PBS) for 10 minutes at room temperature, blocked with 5% rat serum for 1 hour at room temperature, and incubated with APC-labeled mouse anti-CD86 antibody (1:100) at room temperature for 2 hours. After counterstaining the nuclei with DAPI, the images were obtained using a fluorescence microscope (Ti2-U, Nikon, Japan).

[0115] 2) 12-well plates, fixation and staining conditions as before, fluorescence intensity was analyzed by flow cytometry.

[0116] 3) In a 6-well plate, the transcriptional levels of pro-inflammatory genes (Nos2, Tnfα, Il1b and Il6) and Ifnb were detected by real-time quantitative PCR (RT-qPCR).

[0117] Total RNA was extracted on ice using Trizol reagent, purified with chloroform and isopropanol, and washed with 75% ethanol (v / v, 4°C). RNA concentration and purity were assessed using a spectrophotometer (NanoDrop 2000, Thermo, USA). cDNA was synthesized on a PCR instrument (ProFlex™ Base, Thermo) using the PreScript III RT kit (EnzyValley, China), and subsequently amplified on an RT-qPCR instrument (QuantStudio™ 5, Thermo) using 2×Robust SYBR Green (EnzyValley) and gene-specific primers (Table 1). The fold change in target gene expression relative to the control group was calculated using the ΔΔCt method, with Gapdh as an internal control. All primers were synthesized by Qingke Biotechnology (Beijing, China).

[0118] Table 1 To assess the impact of CLEAR on the inflammatory state of sepsis, this application examined changes in M1 macrophages. After 24 hours of incubation, flow cytometry analysis was performed. Figure 4 (I, J) showed that CLEAR attenuated M1 polarization in a dose-dependent manner, and a concentration of 2 μg mL⁻¹ significantly reduced CD86+ cells by 31% compared to untreated levels (p<0.001). Immunofluorescence imaging results further confirmed this finding. Figure 4(Middle K). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) showed synchronous downregulation of the transcription of the key M1 marker (M1). Figure 4 From L to Figure 4 In the middle P), Tnfα (-36.89%), Nos2 (-25.46%), Il6 (-58.75%), and Il1b (-50.69%) were reduced, and Ifnb expression decreased to 50.68% of baseline. These data collectively demonstrate that CLEAR can alleviate excessive inflammation by synergistically downregulating cGAS-STING signaling and reprogramming macrophage functional status.

[0119] The mechanism of CLEAR in treating sepsis is as follows: Figure 1 As shown in Figure B, macrophages target CLEARs, achieving cell recognition via surface TLR4 ligands and entering the cell through nanoparticle endocytosis. Internalized CLEARs mediate lysosomal degradation of TLR4, and their cationic surface simultaneously promotes lysosomal escape and induces autophagy. Subsequently, cytoplasmic CLEARs bind to cGAS via surface ligands, achieving autophagic degradation of cytoplasmic cGAS and thus inhibiting the cGAS-STING pathway. This multi-pronged mechanism synergistically reduces excessive inflammatory responses in sepsis and promotes pathogen clearance.

[0120] Experimental Example 5: In vivo experiment of CLEAR for the treatment of sepsis Male wild-type C57BL / 6J mice (weighing 22–25 g) were purchased from the Guangdong Provincial Laboratory Animal Center (Guangdong, China). All experimental procedures were approved by the Animal Care and Use Committee of Sun Yat-sen University (SYSU-IACUC-2025-000925) and followed the ARRIVE guidelines. Animals were housed in a specific pathogen-free environment with controlled temperature (22±1°C), humidity (50±10%), and a 12-hour light-dark cycle, and had free access to food and water.

[0121] The cecal ligation and perforation (CLP) sepsis model was performed according to previous protocols. Mice were intraperitoneally injected with sterile PBS solution containing 0.6% (w / v) sodium pentobarbital (10 mL / kg). –1 Anesthetize, prepare the skin, and disinfect the abdomen. The animal is placed in a supine position on the operating table, and a longitudinal incision is made along the midline of the abdominal wall to enter the abdominal cavity. The cecum is carefully identified and freed, avoiding damage to the mesenteric vessels. A ligation is made at 50% of the cecum's length using 4-0 silk sutures, gently pushing the intestinal contents towards the anterior end. A full-thickness puncture (avoiding blood vessels) is performed between the ligation point and the cecal tip using a 22G needle, squeezing out a small amount of feces. The cecum is returned to the abdominal cavity, the abdomen is closed by layered sutures, and disinfected. Postoperatively, a subcutaneous injection of pre-warmed (37°C) saline (40 mL kg–1) is administered, and the mouse is placed on a heating pad for recovery. After recovery, the mouse is returned to its cage for free access to food. The sham-operated group underwent the same procedure except for the absence of cecal ligation and perforation. The severity of sepsis was assessed using a mouse sepsis scoring system (60).

[0122] CLEAR (80 μg kg) was administered via tail vein injection at 1 hour and 6 hours post-surgery. –1 100 μL of saline solution per mouse or an equal volume of physiological saline was administered. Mice were sacrificed 24 hours post-surgery, and peripheral blood and major organs (liver, lungs, heart, spleen, and kidneys) were collected for subsequent analysis. The remaining mice were monitored daily for body weight, rectal temperature, and sepsis scores. Major organs were harvested 168 hours post-surgery for histological evaluation.

[0123] 1. Assessment of pulmonary edema: Pulmonary edema was quantified by measuring the wet / dry weight (W / D) ratio. Left lung tissue was immediately transferred to a pre-weighed EP tube (tube weight denoted as A0), and the wet weight was recorded (A1). The sample was then treated at 80°C for 48 hours until complete dehydration, and the dry weight was recorded (A2). The W / D ratio was calculated using the formula: (A1-A0) / (A2-A0).

[0124] 2. Bacterial count: Mice were euthanized 24 hours post-surgery under pentobarbital anesthesia. Venous blood, right lung, liver, and kidney were collected, and the tissues were weighed and homogenized in a fixed volume of sterile PBS. To determine colony-forming units (CFU), 10 μL of tissue homogenate or blood was diluted 1:10 with sterile PBS. 100 μL of the diluted homogenate was spread onto LB agar plates and incubated at 37°C for 24 hours. Colony imaging was performed using a multifluorescence chemiluminescence imaging system (G: BOX Chemi XX6; Syngene, UK) to assess bacterial load.

[0125] 3. Histological assessment: Organs were fixed in 4% PFA (prepared in PBS) for 24 hours (room temperature), dehydrated with a gradient of ethanol, and embedded in paraffin. 6 μm sections were prepared using a Leica RM2235 microtome (Leica Microsystems, Germany). After dewaxing in xylene and rehydration with a gradient of ethanol, hematoxylin and eosin (H&E) staining was performed. Immunofluorescence: after rehydration, antigen retrieval was performed, and non-specific binding was blocked with 5% normal goat serum / PBS. Lung sections were incubated overnight at 4°C with rabbit primary antibodies (F4 / 80, 1:2000; iNOS, 1:2000; cGAS, 1:400; pSTING, 1:1000). After washing with PBS, sections were incubated with fluorescently labeled goat secondary antibody at room temperature in the dark for 2 hours. After washing again, sections were mounted with anti-quenching mounting medium containing DAPI. Bright-field and fluorescence images were acquired using an inverted fluorescence microscope (Ti2-U, Nikon), and quantification was performed using Fiji software.

[0126] 4. Tissue analysis using ELISA and flow cytometry: Lung, liver, and kidney tissues were isolated on ice. After weighing, single-cell suspensions were prepared by homogenization in a fixed volume of pre-chilled PBS and filtered through a 100 μm cell sieve. After centrifugation (3500 rpm, 4°C, 15 min), the supernatant was used to quantify cytokines (IL1β, IL6, TNFα, IFNβ) using an ELISA kit. The cell pellet was treated with erythrocyte lysis buffer (5 min, room temperature), fixed with 1% PFA (prepared in PBS) (overnight at 4°C), and blocked with 5% normal rabbit serum (2 h at room temperature). Macrophage polarization was assessed by incubation overnight at 4°C with antibodies (CD45, 1:100; CD11b, 1:100; F4 / 80, 1:100; CD86, 1:50). Finally, after washing with PBS, data were analyzed by flow cytometry using FlowJo software (v10.8.1).

[0127] 5. Statistical Analysis: Data are expressed as mean ± standard deviation (SD). Unless otherwise specified, sample size n ≥ 3. Differences between two groups were assessed using an unpaired two-tailed Student's t-test. Comparisons of three or more groups were performed using one-way ANOVA and Tukey's post-hoc test. Significance levels are expressed as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; "ns" indicates no significance. Sample size was not calculated, no data were excluded, and the experiment was randomized; the researchers did not blind the participants during allocation and outcome assessment.

[0128] Based on the positive results obtained from in vitro experiments, such as Figure 5 As shown, this application subsequently evaluated the therapeutic potential of CLEAR for sepsis in C57BL / 6J mice using a cecal ligation and perforation (CLP) model. Figure 5 (Central A). Systemic inflammatory parameters were quantified during the acute phase (24 hours post-surgery), while longitudinal assessments of weight, sepsis clinical score, and core body temperature (measured rectally) were extended to the subacute phase (168 hours post-surgery).

[0129] Log-rank (Mantel-Cox) analysis showed that CLEAR administration significantly improved the survival rate at 168 hours post-surgery compared with the sepsis model group (P<0.05). The survival rate of mice in the CLEAR-treated group reached 80%, while that in the untreated model group was only 30%. Figure 5 (B) Furthermore, CLEAR treatment significantly improved the clinical score of sepsis in mice and accelerated the recovery of normal body temperature. Figure 5 C, Figure 5 (D).

[0130] Quantitative analysis showed that at 48, 72, and 96 hours post-surgery, the mean sepsis scores in the CLEAR treatment group were 68.1%, 55.6%, and 54.0% of those in the untreated group, respectively. Figure 5 (E). Meanwhile, the rectal temperature in the CLEAR group was 1.83°C, 1.30°C, and 1.16°C higher than that in the untreated group, respectively. Figure 5 (F). Furthermore, compared to the sham-operated control group receiving PBS, CLP-induced septic mice exhibited significant weight loss, with the maximum reduction occurring at 48 hours (F). Figure 14 ), and CLEAR treatment significantly alleviated this downward trend ( Figure 5 (G). The above data establish the effective protective efficacy of CLEAR against septic mice.

[0131] To further elucidate the immunomodulatory mechanism of CLEAR's therapeutic effect, this application focuses on systemic and tissue-specific inflammatory responses during the acute phase of sepsis. Serum cytokine analysis showed that CLEAR effectively inhibited elevated IL-6, TNF-α, and IL-1β levels in septic mice, restoring their concentrations to levels close to those of the sham-operated group. Figure 5 From H to Figure 5 (J).

[0132] Given that the lungs are the most vulnerable target organ in sepsis-induced multi-organ dysfunction, this application first quantifies sepsis-induced lung injury: a decrease in the wet-to-dry weight ratio suggests that CLEAR treatment effectively reduces the severity of pulmonary edema. Figure 5 (K), and flow cytometry showed that CD86⁺ macrophage infiltration was reduced by 79.5% compared with the untreated group (K). Figure 5 L, Figure 5 M; see circle gate strategy Figure 15 Simultaneous quantitative analysis of lung tissue homogenates confirmed that, compared to the untreated group, TNF-α, IL-1β, and IL-6 were significantly reduced, even falling to levels seen in the sham-operated control group. Figure 5 N to Figure 5 (China P).

[0133] Next, liver changes were assessed, showing that CLEAR reduced the percentage of M1 macrophages in the liver from 38.08% to 13.72%. Figure 5 China Q to Figure 5 R; see circle gate strategy Figure 16 At the same time, liver tissue showed a significant decrease in TNF-α, IL-1β, and IL-6. Figure 5 Zhong S to Figure 5 Finally, the kidneys were also evaluated through histopathological and biochemical analysis, which confirmed that CLEAR has the ability to reduce renal inflammatory damage. Figure 17 , Figure 18 ).

[0134] Furthermore, serum biochemical indicators showed comprehensive protection of multiple organ functions: CK-MB recovery to the sham-operated group levels indicated reduced cardiac damage; urea and creatinine levels decreased by 9.18% and 22.46% respectively compared to the untreated group, improving renal dysfunction; liver injury markers (LDH, AST, ALT) concentrations decreased by 35.96%, 30.77%, and 35.71% respectively compared to the untreated group. Importantly, CLEAR treatment significantly reduced procalcitonin (PCT), indicating enhanced bacterial infection control. Figure 5 (V).

[0135] These results collectively outline CLEAR's multimodal protective mechanism, which improves sepsis pathology by synergistically inhibiting systemic inflammation, alleviating organ-specific immune dysregulation, and protecting multi-organ function, ultimately leading to increased survival rates.

[0136] 6. CLEAR exerts a protective effect by enhancing bacterial clearance and reducing sepsis-induced organ damage: Based on previous evidence of cation-mediated bacterial adhesion, this application proposes the hypothesis that CLEAR promotes the clearance of bacteria in vivo through electrostatic adsorption.

[0137] The interaction between CLEAR and Escherichia coli (which typically has a negatively charged surface and readily electrostatically adsorbs cationic materials) was observed using scanning electron microscopy (SEM). Whole blood was collected from CLP septic mice (24 hours post-surgery, anticoagulated with EDTA) and mixed with CLEAR (2 μg / mL). –1 Incubate with PBS (100 rpm, 37°C, 6 h) or a control PBS with shaking. After incubation, collect the precipitate by centrifugation and fix it with gentle shaking in 2.5% glutaraldehyde (v / v) (100 rpm, 37°C, 2 h). After fixation, dehydrate the bacteria using a gradient of ethanol (25%→50%→75%→100% ethanol; 30 min per step, room temperature, 80 rpm). Add the dehydrated sample to a silicon wafer and image it using field emission SEM (Quanta 400 F, Oxford Instruments).

[0138] Scanning electron microscopy (SEM) analysis of septic mouse blood co-incubated with CLEAR directly confirmed this mechanism, showing that CLEAR forms a uniform electrostatic adsorption on the surface of E. coli. Figure 6 (A). Bacterial cultures of blood and tissue homogenates (liver, lung, kidney) 24 hours post-surgery showed that the pathogen load in the CLEAR treatment group was significantly reduced compared to the untreated group. Figure 6 China B and Figure 19 ).

[0139] Next, this application investigated the organ-protective potential of CLEAR. Acute phase (24 hours post-surgery) hematoxylin-eosin (H&E) staining histopathological analysis of liver and lung tissues revealed characteristic organ damage in the sepsis group mice: focal necrosis in the liver. Figure 6 (C) The lungs showed significant inflammatory infiltration, interstitial edema, and alveolar structural destruction. Figure 6 (D), corresponding to a significantly elevated pathological score ( Figure 6 (E). Notably, CLEAR treatment effectively improved these lesions and significantly reduced necrotic foci in the liver (E). Figure 6 (C) and alleviated pneumonia and edema, resulting in a 79.17% reduction in lung pathology scores compared to the model group ( Figure 6 D, Figure 6 (E).

[0140] The evaluation was extended to the subacute phase (168 hours post-surgery), and sustained therapeutic effects were observed in this application. Organ damage in septic mice continued to worsen, with extensive inflammatory infiltration appearing in the areas of liver necrosis. Figure 6 (Middle F), lung structural damage and elevated pathological score still exist ( Figure 6 China G to Figure 6 (H). CLEAR treatment provides protection, effectively reducing pathological damage to liver and lung tissues, with organ pathology scores approaching those of the sham-operated group (H). Figure 6 China F to Figure 6 (H). This application further evaluated the spleen, heart, and kidneys (H). Figures 20-22 The study found that CLEAR reduced sepsis-related splenic changes, indicating that excessive immune activation was regulated. Furthermore, no significant histopathological changes in the heart or kidneys were observed in mice before and after CLEAR treatment, further demonstrating its biosafety and good biocompatibility. These findings demonstrate that CLEAR can effectively reduce multi-organ damage induced by acute and subacute sepsis and possesses good biosafety.

[0141] 7. CLEAR alleviates sepsis-induced lung inflammation by targeting and degrading cGAS and regulating the cGAS-STING pathway: Previous in vitro experiments confirmed that CLEAR can target cGAS degradation and effectively inhibit the STING pathway. This application further evaluated the effectiveness of this effect in the lung tissue of septic mice using immunofluorescence analysis. The results are as follows: Figure 7 As shown.

[0142] Immunofluorescence results of lung tissue in the acute phase showed that CLEAR treatment significantly reduced the level of cGAS protein in macrophages (F4 / 80⁺). Figure 7 (A). Quantitative assessment confirmed that the cGAS⁺ / F4 / 80⁺ colocalization signal was reduced by 87.65% compared to the sepsis group. Figure 7(Medium B), indicating that highly efficient macrophage-targeted cGAS degradation was achieved. Simultaneously, it was found that macrophage infiltration into the lungs was reduced, with a decrease of 55.46% ( Figure 7 The presence of C indicates a reduction in the recruitment of inflammatory cells.

[0143] Further research showed that CLEAR administration significantly reduced the level of phosphorylated STING (pSTING) in lung macrophages. Figure 7 From D to Figure 7 (E), by downregulating the cGAS-STING pathway, ultimately reduced the IFNβ content in lung tissue by 19.48% ( Figure 7 (F). Furthermore, this inhibitory effect has a systemic effect: not only in serum ( Figure 7 A significant decrease in IFNβ concentration was detected in liver tissue (G). Figure 7 (H) and kidney tissue ( Figure 23 A consistent trend was also observed in [the study]. This demonstrates that CLEAR has broad-spectrum anti-inflammatory effects.

[0144] Next, this application quantified the content of M1 marker-induced nitric oxide synthase (iNOS): CLEAR treatment significantly reduced the mean fluorescence intensity (MFI) of iNOS by 97.80% ( Figure 7 Middle I, Figure 7 (J). This near-complete inhibition suggests that CLEAR has a potent effect in reversing sepsis-induced M1 polarization.

[0145] Subacute phase analysis showed that the regulatory effect of targeting the cGAS-STING pathway had a sustained effect. cGAS remained at a low level. Figure 7 K Figure 7 In the middle L), pSTING levels were also continuously suppressed ( Figure 7 M, Figure 7 In the middle N group, macrophage infiltration in lung tissue decreased by 46.07%, while the pro-inflammatory M1 subset decreased significantly by 90.54%. Figure 7 China P, Figure 7 (Q).

[0146] The CLEAR protocol described in this application effectively restores immune balance and achieves durable immune reprogramming, fundamentally reshaping the characteristic immune dysregulation response of sepsis. This study establishes a novel therapeutic paradigm based on targeted pathway regulation, highlighting the significant translational potential of targeted protein degradation (TPD) in sepsis management.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for preparing nanoparticles for autophagy-targeted degradation of TLR4 and cGAS, characterized in that, Includes the following steps: S1. Polylactic acid is dissolved in dichloromethane and stirred to form an oil phase; distearate phosphatidylethanolamine-polyethylene glycol-amino, hexadecyltrimethylammonium bromide and polyvinyl alcohol are mixed to form an aqueous phase; S2. The oil phase is added to the aqueous phase for pre-emulsification, and then emulsified by ultrasonic homogenization to obtain an emulsion; The emulsion was dispersed in a polyvinyl alcohol solution and stirred and evaporated to obtain an unfunctionalized nanoparticle solution. S3. Mix the nanoparticle solution obtained in step S2 with a buffer solution containing the first and second targeting ligands, then centrifuge and ultrafilter to remove monomers, filter to remove bacteria, and obtain nanoparticles that autophagy-targeted degrade TLR4 and cGAS. The first targeting ligand specifically binds to the extracellular domain of a pro-inflammatory transmembrane receptor on the macrophage membrane, and the second targeting ligand specifically binds to an intracellular pattern recognition receptor on the macrophage cytoplasm.

2. The method for preparing nanoparticles as described in claim 1, characterized in that, In step S1, the volume ratio of distearylphosphatidylethanolamine-polyethylene glycol-amino and hexadecyltrimethylammonium bromide is (0.1~10):(10~0.1).

3. The method for preparing nanoparticles as described in claim 2, characterized in that, The volume ratio of distearate phosphatidylethanolamine-polyethylene glycol-amino and hexadecyltrimethylammonium bromide is (2~6):(4~8).

4. The method for preparing nanoparticles as described in claim 1, characterized in that, In step S2, the pre-emulsification speed is 10,000~20,000 rpm, and the pre-emulsification time is 1~5 minutes; And / or, the ultrasonic power for ultrasonic homogenization is 100~500W, and the homogenization time is 1~5min; And / or, the stirring rate for stirring and evaporation is 100~500 rpm, and the stirring time is 18~30h.

5. The method for preparing nanoparticles as described in claim 1, characterized in that, In step S3, the pro-inflammatory transmembrane receptor is selected from the Toll-like receptor family, the interleukin receptor family, the tumor necrosis factor receptor family, or the scavenger receptor family; the intracellular pattern recognition receptor is selected from the intracellular free nucleic acid sensing receptor or the NOD-like receptor family.

6. The method for preparing nanoparticles as described in claim 1, characterized in that, The first targeting ligand is a TLR4 targeting ligand, selected from at least one of TLR4-IN-C34-C2-COOH, Eritoran, and single-chain antibody fragments that specifically bind to the extracellular region of TLR4; The second targeting ligand is a cGAS targeting ligand, selected from at least one of cGAS-IN-1, RU.521, RU.322, CU-32, CU-76, and A151 oligonucleotides.

7. The method for preparing nanoparticles as described in claim 1, characterized in that, In step S3, the mass ratio of the first targeting ligand to the second targeting ligand is (0.5~1.2):(0.5~1.0).

8. The method for preparing nanoparticles as described in claim 1, characterized in that, In step S3, the buffer solution comprises MES buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide.

9. Nanoparticles for autophagy-targeted degradation of TLR4 and cGAS obtained by the preparation method of nanoparticles according to any one of claims 1 to 8.

10. The application of the nanoparticles as described in claim 9 in the preparation of sepsis products.

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