An artificial exosome for regulating the fate of sepsis neutrophils to restore immune homeostasis and its preparation method.

Abstract

This invention discloses an artificial exosome for regulating neutrophil fate in sepsis to rebuild immune homeostasis and its preparation method, relating to the field of biomedical technology. The preparation method of the artificial exosome includes the following steps: extracting cell membranes from PD-1-expressing macrophages using a combination of repeated freeze-thaw cycles and physical disruption to obtain cell membrane fragments; mixing AT7519 and bovine serum albumin, followed by adding a reversible cross-linking agent to continue the reaction to obtain AT nanoparticles; and mixing the cell membrane fragments and the AT nanoparticles to obtain AT@NV-PD1 nanoparticles, which are the artificial exosomes. The artificial exosomes provided by this invention can effectively control the inflammatory cascade, alleviate immunosuppression, and ultimately promote the recovery of immune system function, providing a new therapeutic strategy for the reconstruction of immune homeostasis in sepsis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an artificial exosome that regulates the fate of sepsis neutrophils to restore immune homeostasis and its preparation method. Background Technology

[0002] Sepsis is a life-threatening systemic infectious syndrome characterized by a dual imbalance in the immune system: early excessive inflammation and later immunosuppression. This immune dysregulation makes clinical treatment extremely challenging. Initially, the body's overreaction triggers a "cytokine storm," leading to microcirculatory disturbances and multiple organ failure; later, deep immunosuppression increases the risk of secondary infections, creating a vicious cycle. Neutrophils play a crucial role in the pathology of sepsis. Under normal circumstances, after clearing pathogens, they maintain immune homeostasis through programmed apoptosis. However, the persistent inflammatory stimulation in sepsis leads to delayed neutrophil apoptosis. These "long-lived" abnormal cells not only continuously release pro-inflammatory factors and NETs but also highly express PD-L1. PD-L1 binds to PD-1 on T cells, inhibiting T cell function and exacerbating immunosuppression. This neutrophil dysfunction is a key bridge connecting sepsis's inflammation and immunosuppression. Current treatments mainly rely on antibiotics and supportive therapies, lacking specific interventions targeting the immune dysregulation. Therefore, developing therapeutic strategies that can precisely target abnormal neutrophils and repair T cell function has become the key to breaking through the bottleneck in sepsis treatment.

[0003] Given the complex immune microenvironment of sepsis, characterized by both excessive inflammation and immunosuppression, and the crucial role of neutrophil programmed cell death dysregulation in immune imbalance, there is an urgent need to develop a multidimensional synergistic therapeutic strategy that can precisely regulate neutrophil fate to improve prognosis. Summary of the Invention

[0004] The purpose of this invention is to provide an artificial exosome that regulates the fate of neutrophils in sepsis to rebuild immune homeostasis, and its preparation method, thereby addressing the problems existing in the prior art. This artificial exosome can effectively control the inflammatory cascade, alleviate immunosuppression, and ultimately promote the recovery of immune system function, providing a new therapeutic strategy for the reconstruction of immune homeostasis in sepsis.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing artificial exosomes that regulate the fate of sepsis neutrophils to reconstruct immune homeostasis, comprising the following steps: Cell membrane fragments were obtained from macrophages expressing PD-1 by repeated freeze-thaw cycles combined with physical disruption. After mixing and reacting AT7519 and bovine serum albumin, a reversible cross-linking agent was added to continue the reaction, resulting in AT nanoparticles. The cell membrane fragments and the AT nanoparticles are mixed and reacted to obtain AT@NV-PD1 nanoparticles, which are the artificial exosomes.

[0006] Furthermore, the PD-1-expressing macrophages are prepared by transfecting host macrophages with a PD-1-expressing lentivirus.

[0007] Furthermore, the reversible crosslinking agent is dialdehyde polyethylene glycol.

[0008] Furthermore, the mass ratio of the AT7519 to the bovine serum albumin is 1:100.

[0009] Furthermore, the physical fragmentation method employs ultrasonic processing.

[0010] Furthermore, the mass ratio of the cell membrane fragments to the AT nanoparticles is 1:1.

[0011] The present invention also provides an artificial exosome prepared according to the above-described preparation method.

[0012] The present invention also provides the use of the above-described artificial exosomes in the preparation of medicaments for treating sepsis.

[0013] The present invention also provides a medicament for treating sepsis, the active ingredient of which includes the above-mentioned artificial exosomes.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] The present invention discloses the following technical effects: This invention constructs an innovative artificial exosome nanodecoy (AT@NV-PD1) delivery system designed to target activated / delayed apoptosis neutrophils, providing an innovative approach to sepsis treatment and improving therapeutic efficacy. The nanomedicine system uses a pH-responsive bovine serum albumin nanocore loaded with AT7519 (a cyclin-dependent kinase inhibitor, CDKI; CAS: 844442-38-2) as its core component. To enhance therapeutic efficacy, this invention innovatively encapsulates it onto the membrane of macrophages expressing programmed death receptor-1 (PD-1), forming an intelligent delivery system. After intravenous administration, the PD-1 on the surface of AT@NV-PD1 specifically recognizes and binds to programmed death ligand-1 (PD-L1) expressed on activated / delayed apoptosis neutrophils, achieving precise targeting. This carefully designed nanocarrier not only precisely releases AT7519 on demand in the slightly acidic microenvironment of target cells, promoting timely apoptosis of activated neutrophils, but also effectively inhibits excessive inflammatory responses. Neutrophil membrane coating significantly enhanced the ability of the nanodecoy to neutralize bacterial toxins and inflammatory cytokines. Furthermore, the interaction between AT@NV-PD1 and PD-L1 effectively alleviated T cell exhaustion, reduced immunosuppression, and promoted the restoration of immune homeostasis. Through the synergistic effect of CDKI-mediated precise neutrophil apoptosis and the regulation of the PD-1 / PD-L1 immunosuppressive axis, this nanodecoy system demonstrated significant potential in a sepsis model: effectively controlling the inflammatory cascade, reducing immunosuppression, and ultimately promoting the recovery of immune system function, providing a novel therapeutic strategy for the re-establishment of immune homeostasis in sepsis. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram illustrating the construction of the artificial exosome AT@NV-PD1 and its regulation of sepsis immune homeostasis according to the present invention; wherein, A is a schematic diagram of the construction process of AT@NV-PD1; B is a schematic diagram of the inhibitory effect of AT@NV-PD1 on excessive inflammation in sepsis; C is a schematic diagram of the reversal effect of AT@NV-PD1 on immunosuppression in sepsis. Figure 2The images show transmission electron microscopy (TEM) images, hydration size (DLS), and zeta potential diagrams of AT@NV-PD1 and AT@NPs. Specifically, A represents the particle size of AT nanoparticles and its TEM image (scale bar: 50 nm); B represents the particle size of AT@NV-PD1 and its TEM image (scale bar: 50 nm); C shows a comparison of hydration size; and D represents the zeta potential diagram. Figure 3 The pH response validation diagram of AT@NV-PD1 is shown below; where A is the transmission electron microscopy image and hydration particle size (DLS) distribution of AT@NV-PD1 in pH 5.0 solution; B is a comparison diagram of drug release characteristics of AT@NV-PD1 in solutions with different pH values. Figure 4 The image shows the detection results of AT@NV-PD1 adsorbing inflammatory factors TNF-α (A), IL-1β (B), IL-6 (C), sPD-L1 (D), and LPS (E); Figure 5 Targeting PD-L1 in sepsis with AT@NV-PD1 + Figures showing the results of neutrophil uptake assays; A shows a laser confocal microscope image of neutrophils stimulated with interferon-γ and lipopolysaccharide in vitro, showing up AT@NV and AT@NV-PD1. Green fluorescence indicates PD-1 expressed on the cell membrane of genetically engineered RAW264.7 cells, and red fluorescence indicates Cy5-labeled nanoparticle cores; B shows a laser confocal microscope image of inflammatory neutrophils uptake AT@NV-PD1 after 30 minutes of pre-incubation with free anti-PD-L1 antibody; C shows a laser confocal microscope image of resting neutrophils uptake AT@NV-PD1 (scale bar: 10 μm); D shows a flow cytometry analysis of PD-L1 levels on the surface of peripheral blood neutrophils from septic mice; E shows a flow cytometry image of AT@NV-PD1 uptake by blood neutrophils from healthy mice or cecal ligation and puncture model mice; F shows the quantitative analysis of E. Figure 6 Figure 1 shows the experimental results of AT@NV-PD1 improving clinical scores and reducing mortality in mice with sepsis. A is the experimental flowchart of the cecal ligation and puncture mouse model. B and D are statistical graphs of sepsis scores (B), changes in body weight (C), and survival rate (D) of mice after treatment with different preparations. Figure 7 This is a quantitative mass spectrometry imaging analysis of the distribution of AT@NV-PD1 in septic mice. Figure 8 Figure showing the experimental results of AT@NV-PD1 reducing the risk of secondary infection and mortality in septic mice. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Given the complex immune microenvironment of sepsis, characterized by both excessive inflammation and immunosuppression, and the crucial role of dysregulation of neutrophil programmed cell death in immune imbalance, there is an urgent need to develop a multidimensional synergistic therapeutic strategy that can precisely regulate neutrophil fate to improve prognosis. This strategy should integrate targeted clearance of abnormal cells, regulation of inflammatory responses, and restoration of immune balance to effectively address the complex pathophysiological processes of sepsis. Based on this concept, this invention constructs an innovative artificial exosome nanodecoy (AT@NV-PD1) delivery system (… Figure 1This nanomedicine system aims to target activated / delayed apoptosis neutrophils, providing an innovative approach to sepsis treatment and improving therapeutic efficacy. The core component of this system is a pH-responsive bovine serum albumin nanocore loaded with AT7519. To enhance therapeutic efficacy, this invention innovatively encapsulates it onto the membrane of PD-1-expressing macrophages, forming a smart delivery system. After intravenous administration, the PD-1 on the surface of AT@NV-PD1 specifically recognizes and binds to PD-L1 expressed on activated / delayed apoptosis neutrophils, achieving precise targeting. This carefully designed nanocarrier not only precisely releases AT7519 on demand in the slightly acidic microenvironment of target cells, promoting timely apoptosis of activated neutrophils and effectively inhibiting excessive inflammatory responses, but also significantly enhances the ability of the nanodecoy to neutralize bacterial toxins and inflammatory cytokines. Furthermore, the interaction between AT@NV-PD1 and PD-L1 effectively alleviates T cell exhaustion, reduces immunosuppression, and promotes the restoration of immune homeostasis. Through the synergistic effect of CDKI-mediated precise neutrophil apoptosis and PD-1 / PD-L1 immunosuppressive axis regulation, this nanodecoy system has shown significant potential in a sepsis model: effectively controlling the inflammatory cascade, alleviating immunosuppression, and ultimately promoting the recovery of immune system function, providing a new therapeutic strategy for the re-establishment of immune homeostasis in sepsis.

[0024] Example 1 1. Construction of a macrophage cell line expressing PD-1 Construct a stable PD-1-expressing RAW264.7 cell line (PD-1-RAW 264.7): PD-1 lentiviral expression vector (EGFP-PD-1-PLVX puro) was co-transfected with packaging plasmids pSPAX2 and pMD2.G into HEK293T cells to produce lentiviral particles. Cell culture supernatant rich in lentiviral particles was collected and used to infect RAW 264.7 cells for 72 hours. After infection, puromycin was added to the culture medium to a final concentration of 2 μg / mL for selection to obtain RAW264.7 cell lines stably expressing PD-1. Finally, successful PD-1 protein expression in the selected cells was verified by Western blotting, confocal microscopy, and flow cytometry.

[0025] Plasmids pSPAX2, pMD2.G, and pLVX-Puro were purchased from Qingke Biotechnology Co., Ltd. EGFP-PD-1-PLVX puro was constructed by Qingke Biotechnology Co., Ltd., using the following method: the PD-1 gene sequence was ligated into the linearized pLVX-Puro vector; the ligation product was transformed into competent E. coli; and the target plasmid EGFP-PD-1-PLVX puro was purified and screened.

[0026] 2. Preparation of AT nanoparticles Preparation of bovine serum albumin nanoparticles: 100 μg of AT7519 was dissolved in 100 μL of dimethyl sulfoxide (DMSO), and then slowly added dropwise to 1 mL of phosphate-buffered saline (PBS, pH 7.4) containing 10 mg of bovine serum albumin (BSA). The mixture was stirred overnight. Subsequently, 1 mg of the reversible crosslinking agent dialdehyde polyethylene glycol (CHO-PEG-CHO) (dissolved in DMSO) was added to the mixture. After reacting for 12 hours, the reaction system was diluted with 9 mL of PBS and the reaction was terminated. The solution obtained after terminating the reaction was concentrated to 10 mg / mL using an ultrafiltration device (Amicon Ultra-4, molecular weight cutoff 100 kDa).

[0027] When preparing fluorescein (Cy5) labeled bovine serum albumin nanoparticles, simply replace the unlabeled BSA in the above steps with the corresponding fluorescently labeled BSA.

[0028] Senescent neutrophils produce large amounts of lactic acid through glycolysis, creating an acidic microenvironment. Furthermore, the cytoplasm of neutrophils also becomes acidified after phagocytizing pathogens. As acid-responsive cross-linked nanoparticles, AT nanoparticles can depolymerize into ultrasmall protein clusters within acidic neutrophils, thereby promoting their diffusion within the cell and inhibiting cell cycle progression by blocking cyclin-dependent kinases in the cell nucleus.

[0029] 3. Preparation of AT-N nanoparticles: 100 μg of AT7519 was dissolved in 100 μL of dimethyl sulfoxide (DMSO), and then slowly added dropwise to 1 mL of PBS (pH 7.4) containing 10 mg of bovine serum albumin (BSA). The mixture was stirred overnight. Subsequently, 1 mg of the permanent cross-linking agent bis(sulfosuccinimidyl)suberate (dissolved in DMSO) was added to the mixture. After reacting for 12 hours, the reaction system was diluted with 9 mL of PBS to terminate the reaction. The solution obtained after terminating the reaction was then concentrated to 10 mg / mL using an ultrafiltration device (Amicon Ultra-4, molecular weight cutoff 100 kDa). To prepare the fluorescently labeled (Cy5) BSA core, the unlabeled BSA in the above steps was replaced with the corresponding fluorescently labeled BSA.

[0030] 4. Preparation of AT@NV-PD1 nanoparticles Macrophage membrane fragments were extracted using a combination of repeated freeze-thaw cycles and physical disruption: PD-1-expressing macrophages were collected and frozen three times in liquid nitrogen. The treated cells were then sonicated for 5 minutes. The sonicated cells were centrifuged at 700 g for 10 minutes (4°C), and the supernatant was collected. This supernatant was then centrifuged at 14,000 g for 30 minutes to extract cell membrane fragments. The protein content of the cell membrane fragments was quantified using the BCA method. Finally, the obtained cell membrane fragments were lyophilized and stored at -80°C for later use.

[0031] The extracted cell membrane fragments were mixed with AT nanoparticles at a 1:1 mass ratio and then sonicated for 30 minutes to ensure adequate binding. The mixture was then extruded 21 times using a microextruder with porous polycarbonate membranes (pore sizes of 1 μm and 400 nm) to further homogenize the nanoparticle coating. Finally, the obtained nanoparticles were collected by centrifugation and washed three times with PBS to remove unbound components.

[0032] 5. Characterization of nanoparticles (1) Morphological characteristics, hydration particle size and zeta potential of AT@NV-PD1 and AT@NPs The morphology, hydration size, and zeta potential of nanoparticles AT@NV-PD1 and AT@NPs were observed using transmission electron microscopy and a Malvern particle size analyzer. The results are shown in [Figure number missing]. Figure 2 The results showed that after being encapsulated by macrophage membranes expressing PD-1, the hydrated particle size of AT@NV-PD1 increased from approximately 110 nm to 130 nm. The increase in particle size was consistent with the cell membrane thickness. Transmission electron microscopy images revealed a distinct core-shell structure, with the membrane encapsulating the surface of the nanoparticle core. Furthermore, after cell membrane camouflage, the zeta potential of AT@NV-PD1 decreased from -17.1 mV to -24.5 mV, which may be due to the introduction of a negatively charged cell membrane onto the surface.

[0033] (2) pH response verification of AT@NV-PD1 The morphology of AT@NV-PD1 nanoparticles in pH 5.0 solution was observed using transmission electron microscopy and a Malvern particle size analyzer, and the hydrated particle size of the nanoparticles was determined. The release rate of AT7519 drug in solutions with pH values ​​of 5.0, 6.5, and 7.4 was also assessed. Drug detection was performed using mass spectrometry. Results are shown in [Figure number missing]. Figure 3 The results showed that AT@NV-PD1 releases drugs more rapidly in acidic environments.

[0034] (3) Detection of the adsorption capacity of AT@NV-PD1 for various cytokines The AT@NV-PD1 was evaluated by incubating it with a solution containing known initial concentrations of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), sPD-L1, and lipopolysaccharide (LPS). After incubation, AT@NV-PD1 was separated by centrifugation, and the remaining cytokine concentrations in the supernatant were quantitatively assessed using enzyme-linked immunosorbent assay (ELISA). The results are shown below. Figure 4 .

[0035] The results showed that AT@NV-PD1 had a significant adsorption capacity for all detected cytokines, and its scavenging efficiency was significantly better than that of bare AT nanocores without membrane encapsulation. Figure 4 The results suggest that macrophage membrane encapsulation significantly enhances the cytokine adsorption capacity of AT@NV-PD1. This may be due to the presence of a large number of cytokine receptors on the macrophage membrane surface, including tumor necrosis factor receptor, interleukin-1 receptor, and Toll-like receptor. These receptors can selectively bind to and neutralize specific pro-inflammatory cytokines.

[0036] Example 2 AT@NV-PD1 targets PD-L1 when targeting sepsis. + Detection of neutrophil capacity: 1. Experimental Methods Neutrophils were isolated from mouse bone marrow and seeded in L-polylysine-coated laser confocal microscopy culture dishes. First, neutrophils were stimulated for 2 hours with interferon-γ and lipopolysaccharide to simulate the inflammatory microenvironment of sepsis. This activation method not only delayed neutrophil apoptosis but also significantly upregulated the expression of PD-L1 on the cell surface. Then, AT@NV-PD1 nanoparticles were added to the culture medium. Senescent neutrophils treated with anti-mouse PD-L1 antibody and neutrophils not stimulated by interferon-γ and lipopolysaccharide were set up as controls. After 3 hours of incubation, the cells were washed twice with phosphate-buffered saline, and mounting media containing 4',6-diamidinyl-2-phenylindole were added to quench the fluorescence. The cells were then observed using a laser confocal scanning microscope.

[0037] Simultaneously, flow cytometry was used to quantitatively assess the specific uptake capacity of neutrophils in peripheral blood of septic mice for nanoparticles: 24 hours after successfully establishing a mouse sepsis model by cecal ligation and puncture, Cy5-labeled AT@NV-PD1 nanoparticles were injected into the tail vein, with healthy mice serving as the control group; 3 hours after injection, whole blood was collected from mice via cardiac puncture using heparin anticoagulant tubes, and neutrophils in the blood were labeled with antigranulocyte receptor 1 antibody according to the reagent instructions. Subsequently, the uptake of AT@NV-PD1 nanoparticles by neutrophils was analyzed by flow cytometry.

[0038] 2. Experimental Results This invention evaluates the targeting ability of AT@NV-PD1 on apoptotic cells in vitro and in vivo. In in vitro experiments, mouse bone marrow neutrophils were isolated and stimulated with a combination of interferon-γ and lipopolysaccharide to simulate the inflammatory environment of sepsis. Previous studies have shown that this activation mode not only induces impaired neutrophil apoptosis but also significantly upregulates PD-L1 expression on their surface, exhibiting phenotypic characteristics similar to sepsis. Pretreated neutrophils were co-incubated with AT@NV-PD1 or macrophage membrane-encapsulated nanoparticles without PD-1 overexpression. For easy observation, the nanoparticles were labeled with Cy5 and imaged under a laser confocal microscope. After 4 hours of incubation, bright nanoparticle fluorescence signals were detected in senescent neutrophils co-incubated with AT@NV-PD1; while the binding ability of AT@NV nanoparticles in the control group without PD-1 expression to inflamed neutrophils was significantly reduced compared to AT@NV-PD1, suggesting that PD-1 expression enhances its binding ability to inflamed neutrophils. Figure 5 (A). To investigate the mechanism of the excellent targeting ability of AT@NV-PD1, inflammatory neutrophils were pre-incubated with free anti-PD-L1 antibody for 30 minutes before nanoparticle treatment to block the PD-L1 binding site. The results showed that the binding level of nanoparticles was significantly reduced after pre-incubation with anti-PD-L1 antibody. Figure 5 (Figure B) suggests that the targeting effect of AT@NV-PD1 on inflammatory neutrophils depends on the specific interaction between PD-1 and the neutrophil surface ligand PD-L1. Furthermore, resting neutrophils showed weak uptake of Cy5-labeled AT@NV-PD1. The fluorescence intensity of neutrophils stimulated with lipopolysaccharide + interferon-γ after co-incubation with AT@NV-PD1 was approximately three times that of the same cells co-incubated with AT nanoparticles and resting control cells co-incubated with AT@NV-PD1, confirming that AT@NV-PD1 has an enhanced targeting ability against inflammatory neutrophils. More importantly, diffuse Cy5 fluorescence signals were observed in senescent neutrophils co-incubated with AT@NV-PD1. Figure 5 In contrast, the intracellular fluorescence signal treated with the non-responsive control formulation was locally distributed, suggesting that after the nanoparticles were internalized into cells, the responsive degradation of the hydrogel core promoted the rapid release of the loaded drug.

[0039] To further elucidate the targeting ability of AT@NV-PD1 in vivo, a cecal ligation-induced sepsis mouse model was constructed. This model can realistically reproduce the pathophysiological changes of neutrophils in human sepsis and is a widely recognized animal model in sepsis research. Firstly, validation revealed that, compared with the healthy control group, the expression of PD-L1 on the surface of neutrophils isolated from cecal ligation-induced sepsis mice was significantly increased. Figure 5(D). Subsequently, the affinity of AT@NV-PD1 expressing PD-1 for damaged neutrophils in live septic mice was investigated: 24 hours after cecal ligation and puncture, Cy5-labeled AT@NV-PD1 was injected via the tail vein. Blood samples were collected 3 hours after injection. Monocytes, macrophages, T cells, and natural killer cells were identified by flow cytometry using cell-specific antibodies. The proportion of Cy5-positive cells in each subset was quantified, and the selective internalization capacity of neutrophils was assessed. The results showed that neutrophils accounted for 43.43% of AT@NV-PD1 positive cells in septic mice, while this proportion was 14.8% in sham-operated mice; monocytes accounted for 14.14% and 23.06% in septic mice and sham-operated mice, respectively; other immune cells such as B cells, T cells, and natural killer cells accounted for less than 10% of AT@NV-PD1 positive cells in both groups, with neutrophils showing the highest uptake efficiency, suggesting that AT@NV-PD1 can selectively target neutrophils that highly express PD-L1 in vivo. Figure 5 Medium EF).

[0040] Example 3 Mouse experiment showing that AT@NV-PD1 improves sepsis: 1. Experimental Methods Ten-week-old female C57BL / 6 mice provided by Beijing SPAF Biotechnology Co., Ltd. were randomly divided into different treatment groups. A mouse model of multibacterial sepsis was established by cecal ligation and puncture: After anesthetizing the mice with isoflurane, an incision was made in the midline of the abdomen to expose the cecum. The cecum was ligated with surgical sutures at the distal end of the ileocecal valve. The ligated cecum was punctured once with a 21G needle, and a small amount of fecal contents was squeezed out by gentle pressure to induce multibacterial peritonitis. Subsequently, the cecum was returned to the abdominal cavity, and the abdominal wall was sutured in layers. Different preparations were administered to the mice at 6 hours and 12 hours after surgery: phosphate buffered saline, AT nanoparticles, and AT@NV-PD1, respectively. The survival rate and body weight changes of the mice in each group were recorded daily, and Kaplan-Meier survival curves were plotted and statistically analyzed (n=15 per group).

[0041] Aerodynamic-assisted desorption electrospray ionization mass spectrometry (AFADESI-MSI) based on AT7519 was used to perform systematic analysis on mice at 6 hours and 24 hours after drug administration.

[0042] On the third day after cecal ligation and puncture, to establish a mouse model of secondary lung infection (simulating common hospital-acquired pneumonia), surviving mice were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa (ATCC 27853 strain): First, a concentration of 1×10⁻⁶ was prepared... 8A CFU / 50 μL suspension of *Pseudomonas aeruginosa* phosphate buffer was slowly injected into the trachea of ​​mice using a syringe connected to a thin polyethylene tube (PE-10) via a 45° inclined rodent tracheal intubation platform. After injection, the mice were kept supine for at least 30 seconds to allow for adequate distribution of the bacterial solution in the lungs. Finally, the mice were transferred to a heating pad and kept prone until fully recovered from anesthesia. Survival rate and body weight changes were recorded daily for each group of mice, and Kaplan-Meier survival curves were plotted and statistically analyzed (n=15 per group).

[0043] 2. Experimental Results This invention evaluated the therapeutic efficacy of AT@NV-PD1 in sepsis, according to Figure 6 The protocol described in section A established a mouse model of sepsis induced by cecal ligation and puncture. Mice were administered different formulations, including phosphate-buffered saline (PBFS), AT nanoparticles, and AT@NV-PD1, at 6 and 12 hours post-surgery. Sepsis scores and body weight were monitored daily. Results showed that, compared to the PBFS control group, administration of AT nanoparticles and AT@NV-PD1 significantly reduced sepsis scores. Figure 6 (B) alleviates sepsis-induced weight loss ( Figure 6 Among the mice, AT@NV-PD1 showed the most significant effect in preventing adverse consequences of sepsis. Furthermore, intravenous injection of AT@NV-PD1 significantly reduced the mortality rate of mice with cecal ligation-induced sepsis (C). Figure 6 (D).

[0044] Aerodynamic-assisted desorption / electrospray ionization mass spectrometry (AFADESI-MSI) based on the AT7519 was used to systematically analyze mice at 6 and 24 hours post-drug administration. Results ( Figure 7The study showed that AT@NV-PD1 was widely distributed in multiple vital organs of mice in both the sepsis model group and the sham-operated control group, including the heart, liver, spleen, lungs, kidneys, skeletal muscle, intestines, and stomach. Notably, AT@NV-PD1 exhibited significant retention in lung tissue at 6 hours post-administration. This distribution pattern may be related to two key pathological changes caused by sepsis: increased permeability due to vascular endothelial cell damage and the large accumulation of inflammatory-activated neutrophils in the lungs. In kidney tissue, AT@NV-PD1 exhibited a unique distribution pattern, primarily enriched in the renal medulla and renal pelvis. Interestingly, the distribution intensity of AT@NV-PD1 was significantly higher in the CLP group than in the sham-operated group. This suggests that AT@NV-PD1 may be primarily cleared via the kidneys, and that sepsis-induced renal impairment may reduce its clearance efficiency. Another important finding was the preferential accumulation of AT@NV-PD1 in organs such as the liver, lungs, and kidneys. Given that these organs are the most common target organs of sepsis, this distribution pattern of AT@NV-PD1 may have potential therapeutic significance in alleviating disease-related organ damage. This finding provides important experimental evidence for further optimizing the application of AT@NV-PD1 in the treatment of sepsis.

[0045] After the acute inflammatory phase, sepsis patients often face a high risk of secondary infections due to immunosuppression, which significantly increases the probability of hospital-acquired infections and multiple organ dysfunction, leading to poor prognosis. Clinical data shows that approximately one-third of sepsis survivors die from various complications within one year of discharge. Therefore, preventing and treating immunosuppression-related secondary infections is crucial for improving the long-term survival rate of sepsis patients. To simulate the clinical scenario of secondary infections in sepsis patients, this invention constructs a two-hit model, inducing bacterial pneumonia by intratracheal administration of *Pseudomonas aeruginosa* seven days after cecal ligation and puncture. In a 14-day survival study, *P. aeruginosa* infection showed a relatively low mortality rate in healthy mice, but significantly increased mortality in mice with sepsis induced by cecal ligation and puncture. The results showed that the cumulative mortality rate of septic mice treated with phosphate buffer after challenge with Pseudomonas aeruginosa was 57.1%, while the survival rate of mice treated with AT nanoparticles or AT@NV-PD1 nanomedicine was significantly improved, with the AT@NV-PD1 treatment group showing the most significant effect, with a mortality rate of only 16.6%. Figure 8 ).

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method of preparing artificial exosomes to modulate neutrophil fate in sepsis to reestablish immunological homeostasis, characterized in that, Includes the following steps: Cell membrane fragments were obtained from macrophages expressing PD-1 by repeated freeze-thaw cycles combined with physical disruption. After mixing and reacting AT7519 and bovine serum albumin, a reversible cross-linking agent was added to continue the reaction, resulting in AT nanoparticles. The cell membrane fragments and the AT nanoparticles are mixed and reacted to obtain AT@NV-PD1 nanoparticles, which are the artificial exosomes.

2. The production method according to claim 1, characterized by, The PD-1-expressing macrophages were prepared by transfecting host macrophages with a PD-1-expressing lentivirus.

3. The production method according to claim 1, characterized by, The reversible crosslinking agent is dialdehyde polyethylene glycol.

4. The method of claim 1, wherein, The mass ratio of AT7519 to bovine serum albumin is 1:

100.

5. The preparation method according to claim 1, characterized in that, The physical fragmentation method employs ultrasonic processing.

6. The method of claim 1, wherein, The mass ratio of the cell membrane fragments to the AT nanoparticles is 1:

1.

7. An artificial exosome prepared by the preparation method according to any one of claims 1-6.

8. The use of an artificial exosome as described in claim 7 in the preparation of a medicament for treating sepsis.

9. A medicament for treating sepsis, characterized by, The active ingredient includes the artificial exosomes as described in claim 7.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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