A ROS-responsive hybrid vesicle based on overexpressing dual-antioxidant neutrophils and a preparation method and application thereof

Abstract

The application discloses a ROS-responsive hybrid vesicle based on neutrophil overexpressing dual antioxidant enzymes and a preparation method and application thereof, and belongs to the technical field of biological medicines. The ROS-responsive hybrid vesicle based on neutrophil overexpressing dual antioxidant enzymes provided by the application is composed of neutrophil vesicle components overexpressing SOD2 and CAT, all of which are derived from the cell itself and do not introduce exogenous toxic substances; meanwhile, the liposome is a nano delivery system that has been clinically applied, so that the hybrid vesicle has the advantages of good biocompatibility, low toxic side effects and infection site responsiveness. The hybrid vesicle has both the neutrophil's ability to tend to the microenvironment of the infected tissue and the anti-inflammatory ability, and the overexpressed SOD2 and CAT further endow the hybrid vesicle with excellent antioxidant performance, so that the hybrid vesicle can play an antioxidant role at the infection site. The hybrid vesicle is loaded with ROS-responsive liposomes, can realize the responsive and efficient release of drugs at the infection site, and exhibits a more optimal specific treatment effect on infectious diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes, its preparation method, and its application. Background Technology

[0002] Infectious diseases, particularly sepsis, are among the leading causes of death worldwide. In the infection microenvironment, excessive reactive oxygen species (ROS) not only directly damage tissues but are also key factors in inducing excessive inflammatory responses, mediating cell death (such as apoptosis, pyroptosis, and ferroptosis), and immune paralysis. Therefore, achieving precise drug delivery, effective clearance of excess ROS, and comprehensive regulation of the pathological microenvironment simultaneously has become a core issue urgently needing to be addressed in the treatment of infectious diseases.

[0003] Neutrophils, as the most abundant white blood cells in the human body (accounting for 50-70%), are the first line of defense of the body's innate immunity. They are the first to migrate to the site of infection under inflammatory signal stimulation, a characteristic that gives them a natural advantage as drug delivery carriers. However, there are significant limitations to directly using natural neutrophils: (1) Natural neutrophils are terminally differentiated cells with extremely short lifespans (blood half-life is only 6-8 hours), making it difficult to obtain large quantities and standardize their preparation; (2) In excessive inflammatory states such as sepsis, activated neutrophils release large amounts of ROS and proteases, which can exacerbate tissue damage. Therefore, simply utilizing the chemotaxis of neutrophils without controlling their harmful effects may "do more harm than good."

[0004] To address the issue of cell source, human promyelocytic leukemia HL60 cells can be efficiently differentiated into neutrophil-like cells using reagents such as dimethyl sulfoxide (DMSO), providing a scalable cell source for the preparation of cell-based drug carriers. Meanwhile, extracellular vesicles (EVs), as functional carriers after cell enucleation, retain key membrane proteins (such as adhesion molecules and chemokine receptors) while avoiding the uncontrollable risks associated with live cells. Cell-derived nanovesicles prepared using "top-down" methods such as mechanical extrusion exhibit higher yields and more uniform particle size compared to natural EVs, demonstrating significant application potential.

[0005] Nevertheless, existing delivery systems based on neutrophils or their vesicles still suffer from the following key technical defects: (1) Lack of active antioxidant capacity: Although ordinary neutrophils or their vesicles can respond to ROS, they do not have the ability to efficiently and continuously scavenge ROS. To achieve antioxidant effects, existing technologies often require additional loading of superoxide dismutase (SOD) or catalase (CAT). This exogenous loading is not only cumbersome and costly, but more seriously, it is very easy to lose enzyme activity during preparation, storage and in vivo transportation, thereby reducing the therapeutic effect; (2) Uncontrollable drug release behavior: Single cell vesicles lack intelligent responsiveness to drug loading and release. Drugs may leak prematurely in circulation or be released insufficiently or slowly at the site of infection, resulting in poor efficacy; (3) Single function: Most existing carriers only achieve a single function of "targeted delivery" or "anti-inflammatory", and it is difficult to deliver multiple molecules such as immunomodulators and anti-apoptotic drugs at the same time to regulate the complex infection microenvironment in multiple dimensions.

[0006] To address the aforementioned problems, this invention aims to provide a ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes. Summary of the Invention

[0007] The first objective of this invention is to provide a ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes. The second objective of this invention is to provide a method for preparing the aforementioned ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes, and its application.

[0008] The principle of this invention is as follows: This invention constructs a ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes. First, using lentiviral genetic engineering technology, human superoxide dismutase SOD2 and catalase CAT are stably co-expressed in HL60 cells, and engineered neutrophils with inherently high and stable antioxidant activity are obtained through induced differentiation. Subsequently, nanovesicles of these engineered cells are prepared using a mechanical extrusion method, allowing them to naturally load highly active SOD2 and CAT, fundamentally avoiding the enzyme activity loss problem easily caused by traditional exogenous enzyme loading methods. Finally, the above-mentioned engineered vesicles are fused with ROS-responsive liposomes (containing thioketal TK bonds) carrying therapeutic drugs to obtain hybrid vesicles integrating multiple functions. This hybrid vesicle can achieve chemotactic targeting of inflammatory sites by leveraging the biomimetic properties of neutrophil vesicles. After reaching the high ROS microenvironment of the infection site, the liposome components respond to the rapid degradation of ROS and achieve controlled drug release. At the same time, the SOD2 / CAT dual enzyme system carried by the vesicle itself can efficiently remove excess ROS and restore the body's redox homeostasis.

[0009] Compared to single-cell vesicles, the hybrid vesicles of this invention, by introducing ROS-responsive liposomes, achieve more sensitive and precise controlled drug release at the site of infection. Compared to ordinary unmodified vesicles or exogenous enzyme loading systems, this invention, through intracellular overexpression of dual antioxidant enzymes, has significant advantages in enzyme activity preservation and ease of preparation. At the same time, this delivery platform can flexibly carry various types of drug molecules, such as those regulating cell death, anti-inflammation, and immunomodulation, providing a novel and efficient strategy for the combined treatment of infectious diseases.

[0010] The beneficial effects of this invention are as follows: 1. The ROS-responsive hybrid vesicles based on neutrophils overexpressing dioxygenase provided by this invention contain neutrophil vesicles that overexpress SOD2 and CAT, all of which are derived from the cells themselves and do not introduce exogenous toxic substances. At the same time, liposomes are a clinically used nanodelivery system. Therefore, the hybrid vesicles have advantages such as good biocompatibility, low toxicity and side effects, and responsiveness to infection sites.

[0011] 2. The hybrid vesicles of this invention possess both the neutrophil tropism for the infected tissue microenvironment and anti-inflammatory capabilities, while the overexpression of SOD2 and CAT further endows them with excellent antioxidant properties, enabling them to exert antioxidant effects at the site of infection. The hybrid vesicles of this invention encapsulate ROS-responsive liposomes, achieving responsive and efficient drug release at the site of infection. Animal experimental results show that the 7-day survival rate of septic mice treated with the hybrid vesicles of this invention increased from 20% in the model group to 60%, demonstrating superior specific therapeutic effects against infectious diseases.

[0012] 3. The hybrid vesicles of this invention, by incorporating ROS-responsive liposomes, exhibit superior drug release at the site of infection compared to single-cell vesicles. Furthermore, these hybrid vesicles are naturally rich in highly active antioxidant dual enzymes, eliminating the need for additional loading and avoiding the impact of exogenous enzyme loading on enzyme activity. They also allow for the flexible delivery of drug molecules that regulate cell death, provide antioxidant effects, and modulate immunity. Attached Figure Description

[0013] Figure 1 The diagram shows SOD2 and CAT lentiviral vectors; the top diagram shows a lentiviral vector overexpressing SOD2; the bottom diagram shows a lentiviral vector overexpressing CAT. Figure 2This document presents the preparation and characterization results of neutrophils overexpressing SOD2 and CAT. A shows the flowchart of lentiviral transfection of HL60 cells and induction of differentiation into neutrophils overexpressing SOD2 and CAT. B shows the imaging results of cells transfected with SOD2 and CAT under an inverted fluorescence microscope: from left to right and top to bottom, the images are bright field (BF) image, zsGreen fluorescence channel (labeled SOD2), mCherry fluorescence channel (labeled CAT), and bright field and dual fluorescence superimposed (Merge) image. C shows the SOD2 gene expression level in cells before and after transfection. D shows the Western spectral density of cells. Blot analysis of SOD2 and CAT protein expression bands in WT and OE group cells; E and F show the quantitative analysis results of SOD2 and CAT protein expression levels, respectively; G shows the SOD2 enzyme activity level before and after transfection; H shows the CAT enzyme activity level before and after transfection; I shows the change in absorbance of hydrogen peroxide at 240 nm at different time points under different treatments; J shows the histogram of CD11b expression on cell surface before and after DMSO treatment by flow cytometry; K shows the quantitative analysis results of CD11b mean fluorescence intensity (MFI). Figure 3 This document presents the preparation and characterization results of neutrophil vesicles overexpressing SOD2 and CAT and their ROS-responsive hybrid vesicles. A shows the flowchart for the preparation of overexpressing neutrophil vesicles; B shows a transmission electron microscope (TEM) image of the neutrophil vesicles; C shows the particle size distribution of the neutrophil vesicles; D shows the flowchart for the preparation of ROS-responsive hybrid vesicles; E shows the particle size distribution of the ROS-responsive hybrid vesicles; F shows TEM images of liposomes and ROS-responsive hybrid vesicles, from left to right: images of liposomes and ROS-responsive hybrid vesicles at different magnifications (scale bars are 200 nm and 50 nm); G shows laser confocal microscopy imaging verifying the membrane fusion of the hybrid vesicles: the left image shows DiO-labeled liposomes, the middle image shows DiO-labeled neutrophil vesicles, and the right image shows the fused hybrid vesicles; H shows the in vitro ROS-responsive drug release curve of the hybrid vesicles. Figure 4The diagram shows the cell protection effect of ROS-responsive hybrid vesicles. A represents flow cytometry analysis of apoptosis in cells after different treatments (Model: LPS treatment); B represents ROS levels in cells of different treatment groups (Model: LPS treatment); C represents mitochondrial membrane potential in cells of different treatment groups detected by the JC-1 probe (Model: LPS treatment); D represents the morphology and mitochondrial structure of cells of different treatment groups observed by transmission electron microscopy (Model: LPS + Nigericin treatment); E represents cell viability in different treatment groups detected by cck8 (Model: LPS + Nigericin treatment); (F) represents LDH release levels in cells of different treatment groups; G represents IL-18 and IL-1β release levels in cells of different treatment groups (Model: LPS + Nigericin treatment). Figure 5 The image shows the therapeutic effects of ROS-responsive hybrid vesicles in an animal model of sepsis. A represents the body weight change curves of mice in different treatment groups; B represents the survival curves of mice in different treatment groups; C represents the blood test results of mice in different treatment groups (WBC: white blood cells; Gran#: neutrophils; RBC: red blood cells); D represents serum AST, ALT, Cr, BUN, and other liver and kidney function indicators of mice in different treatment groups; and E represents H&E stained tissue sections of major organs of mice in different treatment groups. Detailed Implementation

[0014] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0015] Unless otherwise specified, the reagents used in the following examples can be purchased from a regular biochemical reagent store.

[0016] HL60 cells and their culture medium were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0017] Example 1: Construction and characterization of neutrophils overexpressing SOD2 and CAT 1. Screening of puromycin and hygromycin B concentrations Screening the working concentration of puromycin: HL60 cells in logarithmic growth phase were seeded into 12-well plates and cultured using standard methods. When the cell density reached approximately 70%, the medium was replaced with complete medium containing 0, 0.5, 1, 1.5, 2, 3, 4, and 5 μg / mL puromycin, respectively. The cells were cultured for another 2 days (if more than 70% of the cells died, the medium was replaced with the same concentration). The cell state was then observed and photographed under a microscope. The lowest concentration that resulted in the complete death of all cells was taken as the working concentration of puromycin.

[0018] The working concentration of hygromycin B was screened using the same method: HL60 cells in the logarithmic growth phase were seeded into 12-well plates and cultured using standard methods. When the cell density reached approximately 70%, the medium was replaced with medium containing 0, 25, 37.5, 50, 62.5, 75, 87.5, and 100 μg / mL hygromycin B, respectively, and cultured for another 7 days (if more than 70% of the cells died, the medium was replaced with the same concentration). The cell state was then observed and photographed under a microscope, and the lowest concentration group that killed all cells was taken as the working concentration of hygromycin B.

[0019] Results: The working concentrations of puromycin B at 2 μg / mL and hygromycin B at 75 μg / mL were determined to be those of puromycin B and hygromycin B, respectively.

[0020] 2. HL60 cells overexpressing SOD2 and CAT In this embodiment, the lentivirus overexpressing SOD2 and the lentivirus overexpressing CAT were purchased commercially from Hanheng Biotechnology Co., Ltd. and Yunzhou Biotechnology Co., Ltd., respectively.

[0021] The lentiviruses were removed from the -80°C freezer and dissolved in an ice bath. They were then mixed with PBS or serum-free culture medium used for HL60 cells. SOD2 was diluted to an MOI of 50 to obtain the viral stock solution. CAT was diluted to an MOI of 10 to obtain the viral stock solution.

[0022] HL60 cells in good growth condition were collected, counted, and then processed at a concentration of 1×10⁻⁶. 4Seeds were seeded at a density of 100 cells / well in 96-well plates and incubated overnight at 37°C with 5% CO2, ensuring a cell density of 30-50% for viral infection the following day. The original culture medium was aspirated, and half a volume of fresh medium was added. The SOD2 virus stock solution and polybrene at a final concentration of 4 μg / mL were mixed thoroughly and then added to the HL60 cells, gently mixing. 8-12 hours after transfection, an equal volume of culture medium was added; the cells were cultured for 2-3 days. When the infection efficiency reached approximately 80%, the infected cells were co-cultured with puromycin at a concentration of 2 μg / mL, using untransfected HL60 cells as a control group. After 2 days of selection, once all cells in the control group had died, the surviving cells in the infected group were considered successfully transfected with SOD2 (SOD2-HL60).

[0023] SOD2-HL60 cells in good growth condition were collected, counted, and then processed at a concentration of 1×10⁻⁶. 4 Seed cells at a density of 100 cells / well in a 96-well plate and incubate overnight at 37°C with 5% CO2, ensuring a cell density of 30-50% for virus infection the following day. Aspirate the original culture medium and add 1 / 2 volume of fresh medium. Mix the CAT virus stock solution with polybrene at a final concentration of 4 μg / mL, then add this mixture to the cells and gently mix. 8-12 hours after transfection, add an equal volume of culture medium and continue culturing for 2-3 days. When the cell infection efficiency reaches approximately 80%, co-culture the infected cells with hygromycin B at a concentration of 75 μg / mL, using untransfected SOD2-HL60 cells as a control group. After 7 days of selection, once all cells in the control group have died, the surviving cells in the infected group are considered successfully transfected HL60 cells with SOD2 and CAT.

[0024] This embodiment sets up two parallel control groups: Wild-type control group (WT group): Wild-type HL60 cells that have not undergone any lentiviral infection or drug screening treatment; Overexpression group (OE group): HL60 cells successfully transfected with SOD2 and CAT constructed using the above steps.

[0025] All subsequent testing experiments were conducted in parallel in the two groups of cells mentioned above.

[0026] 2.1 Observation of transfection morphology and transfection effect Cell morphology and transfection efficiency transfected with SOD2 and CAT were observed using an inverted fluorescence microscope. The SOD2 and CAT lentiviral vectors carry zsGreen (green) and mCherry (red) fluorescent reporter genes, respectively; therefore, the transfection efficiency can be observed through fluorescence expression.

[0027] The results are as follows Figure 2As shown in Figure B, the transfected cells expressed zsGreen green fluorescence and mCherry red fluorescence, indicating that the transfection was successful.

[0028] 2.2 qPCR detection of SOD2 mRNA expression SOD2 mRNA expression levels were detected using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from cells in each group, reverse transcribed into cDNA, and amplified using the SYBR Green assay with GAPDH as an internal control. Results are shown below. Figure 2 As shown in Figure C, compared with the untransfected control group, the expression level of SOD2 gene in transfected cells was significantly upregulated (P<0.001), increasing by about 14 times.

[0029] 2.3 Western blot detection of protein expression Cell lysates were collected, and the expression of SOD2 and CAT proteins was detected by Western blot. GAPDH was used as an internal control.

[0030] The results are as follows Figure 2 As shown in the DF, compared with the wild-type control group, the protein bands of SOD2 and CAT in the overexpression group cells were significantly deepened, and the relative expression levels increased by about 1.76 times (P<0.01) and 5.69 times (P<0.001), respectively.

[0031] 2.4 SOD2 enzyme activity detection SOD2 activity in cell lysates was detected using the water-soluble tetrazolium salt (WST-1) method. After cell lysis, superoxide anions were generated using a xanthine / xanthine oxidase system, which reduced WST-1 to yellow formazan (450 nm colorimetric measurement). SOD2 in the sample inhibited this reaction; higher activity resulted in a lighter color. Since WST-1 measures total SOD activity, to determine SOD2 (Mn-SOD), a cyanide treatment group (inhibiting Cu / Zn-SOD) was simultaneously set up. The difference between the total activity and the residual activity was the SOD2-specific activity.

[0032] The results show that ( Figure 2 In the G group, the SOD2-specific activity was increased by approximately 22.95% compared with the wild-type control group (P<0.05).

[0033] 2.5 CAT enzyme activity detection CAT enzyme activity was determined using the hydrogen peroxide decomposition method (ultraviolet spectrophotometry). Cell lysate was mixed with hydrogen peroxide substrate solution, and the absorbance at 240 nm was continuously monitored over time. Kinetic curves were recorded within 0–5 min. Figure 2 (I) Enzyme activity was calculated based on the rate of decrease in absorbance, expressed as a percentage of 10... 4One unit of enzyme activity (U) is defined as the ability of one cell to catalyze the degradation of 1 nmol H2O2 per minute in the reaction system per minute. The enzyme activity change curves at different time points are plotted. Figure 2 (H). The results showed that the CAT enzyme activity in the overexpression group was 84.55% higher than that in the control group.

[0034] 3. Screening of DMSO stimulation concentration Once the cell density in the 6-well plate reached approximately 80%, the supernatant was removed by centrifugation at 800×g for 5 min. The HL60 cells were then resuspended and transferred to culture media containing 1%, 1.15%, 1.2%, 1.25%, and 1.3% DMSO, respectively. The cells were induced for 6 days, and the differentiation-inducing effect was verified using Giemsa staining. The lowest effective concentration for inducing differentiation was taken as the working concentration of DMSO.

[0035] Results: 1.2% DMSO was determined to be the optimal working concentration of DMSO.

[0036] 4. Construct neutrophils overexpressing SOD2 and CAT Take the HL60 cells overexpressing SOD2 and CAT constructed in step 2, and grow them in a 6-well plate until the cell density reaches about 80%. Centrifuge at 800×g for 5 min and remove the supernatant. After resuspending the cells, transfer them to the medium containing 1.2% DMSO as determined in step 3. Replace the medium with fresh medium containing 1.2% DMSO every 2 days and induce culture for 6 days to obtain neutrophils overexpressing SOD2 and CAT.

[0037] Differentiation verification: Untreated and DMSO-treated cells were collected and stained with APC-labeled anti-human CD11b antibody for 30 min in the dark. After washing with PBS, the fluorescence intensity was detected by flow cytometry.

[0038] The results are as follows Figure 2 As shown in Figure J, the CD11b positivity rate of undifferentiated cells was low, while the CD11b positivity rate increased significantly after DMSO induction (P<0.01), indicating that the induced cells had a neutrophil phenotype, and that overexpression of SOD2 / CAT did not affect neutrophil differentiation.

[0039] Example 2: Preparation of ROS-responsive hybrid vesicles from neutrophils overexpressing SOD2 and CAT 1. Neutrophils overexpressing SOD2 and CAT were collected by centrifugation at 800×g for 5 min and washed three times with PBS. The collected cells were resuspended in hypotonic cell lysis buffer (0.0084 g NaHCO3 and 0.0058 g EDTA dissolved in 1 mL ultrapure water and sonicated) and a protease inhibitor, and incubated overnight at 4°C for 16 h. The cells were then centrifuged at 3200×g for 5 min, and the supernatant was collected. The supernatant was repeatedly squeezed through 0.8 μm, 0.4 μm, and 0.2 μm filters 20 times, causing cell fragmentation and enabling self-assembly of SOD2 and CAT-overexpressing neutrophil vesicles (hereinafter referred to as OE-Neu-EVs) based on the hydrophobic effect of the phospholipid bilayer. Morphology was observed using TEM. Figure 3 As shown in Figure B, OE-Neu-EVs exhibit a typical saucer-like vesicle structure, similar to naturally secreted exosomes, with a particle size of approximately 150 nm. Dynamic light scattering (DLS) was used to determine the particle size of OE-Neu-EVs. The average particle size of OE-Neu-EVs was 161.82 ± 1.71 nm, indicating good vesicle homogeneity. Figure 3 (C)

[0040] 2. Lecithin, cholesterol, DSPE-TK-PEG and the hydrophobic drug disulfiram (DSF) were dissolved in 5 mL of chloroform in a mass ratio of 10:5:1:1. The chloroform was removed by rotary evaporation, and a thin and uniform film was formed at the bottom of the flask. 2 mL of PBS was added for hydration to obtain ROS-responsive liposomes loaded with the hydrophobic drug (hereinafter referred to as DSF@Lipo).

[0041] 3. Neutrophil vesicles overexpressing SOD2 and CAT and drug-loaded ROS-responsive liposomes were mixed at a protein concentration ratio of 1:1 at room temperature using an extruder. The mixture was then repeatedly extruded through 0.8 μm, 0.4 μm, and 0.2 μm filters for homogenization, resulting in ROS-responsive hybrid vesicles (DSF@Lipo-EVs) overexpressing dual antioxidant enzymes in neutrophils. The particle size of the hybrid vesicles was detected using DLS. Figure 3 As shown in Figure E, the average particle size of the hybrid vesicles is approximately 101.04 ± 1.94 nm, indicating that they maintain a uniform dispersion after fusion. Liposomes and hybrid vesicles were observed under a negatively stained electron microscope, as shown... Figure 3 As shown in Figure F, the hybrid vesicles exhibit a spherical bilayer membrane structure with a slightly larger particle size than OE-Neu-EVs, clear boundaries, and no free liposomes were observed, indicating successful fusion.

[0042] 4. The co-localization method was used to verify the successful fusion of OE-Neu-EVs and DSF@Lipo into hybrid vesicles. OE-Neu-EVs and DSF@Lipo were labeled with their membrane lipids using fluorescent dyes DiI (red, Ex / Em=549 / 565nm) and DiO (green, Ex / Em=484 / 501nm), respectively. Equal amounts of the two labeled vesicles were mixed and processed according to the fusion method in step 3. The overlap of the dual fluorescence signals was then observed and detected using a confocal microscope.

[0043] Result: As Figure 3 As shown in G, the hybrid vesicles exhibit both red and green fluorescence, which, when superimposed, turn yellow, indicating that the two membrane components are completely fused to form hybrid vesicles.

[0044] 5. To evaluate the ROS-responsive release performance of ROS-responsive hybrid vesicles in neutrophils overexpressing dual antioxidant enzymes under oxidative stress conditions.

[0045] Take 1 mL of the DSF@Lipo-EVs prepared in step 3 and place it in a dialysis bag (molecular weight cutoff 3500 Da). After sealing, immerse it in 20 mL of PBS (pH 7.4) without H2O2 (control group) or containing 1 mM H2O2 (experimental group), and incubate in a constant temperature shaker at 37℃ and 100 rpm. At preset time points (1, 2, 4, 8, 12, 24 h), take 1 mL of the external solution and add an equal volume of fresh medium. After filtering the sample through a 0.22 μm filter membrane, determine the DSF concentration by ultraviolet spectrophotometry (detection wavelength 210 nm) and calculate the cumulative release rate.

[0046] Result: As Figure 3 As shown in Figure H, in PBS without H2O2, the hybrid vesicles release slowly, with a cumulative release rate of approximately 40% over 24 hours, indicating that the vesicles are stable under physiological conditions. Under H2O2 conditions, the release rate is significantly accelerated, with a release rate of approximately 90% over 24 hours. These results demonstrate that the hybrid vesicles exhibit excellent ROS-responsive release behavior, rapidly disintegrating and releasing their contents in high-concentration ROS environments, which is beneficial for targeted release of therapeutic drugs or antioxidant enzymes at sites of oxidative stress damage.

[0047] Example 3: In vitro experiment on ROS-responsive hybrid vesicles in neutrophils overexpressing dual antioxidant enzymes 1. Human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were harvested and processed at a concentration of 5 × 10⁻⁶ cells / year. 4 live cells / cm 2 The cells were seeded at a density in culture plates and cultured overnight for 16 hours. The cells were then randomly divided into the following five groups: Control group: Replace with fresh complete culture medium and incubate for 12 hours; Model group: Replace with fresh complete medium containing 1 μg / mL lipopolysaccharide (LPS) or 1 μM LPS + 5 μM Nigericin, and culture for 12 h to establish an inflammation / pyroptosis model; DSF treatment group (DSF): Replace with fresh complete culture medium containing 1 μg / mL LPS or 1 μM LPS + 5 μM Nigericin and 5 μM DSF, and incubate for 12 h; Blank ROS-responsive hybrid vesicle group (Lipo-EVs): The medium was replaced with fresh complete medium containing 1 μg / mL LPS or 1 μM LPS + 5 μM Nigericin and 50 μg / mL blank ROS-responsive hybrid vesicles, and cultured for 12 h. The preparation of blank ROS-responsive hybrid vesicles was the same as step 2 in Example 2, except that DSF was not added; ROS-responsive hybrid vesicles of neutrophils overexpressing dual antioxidant enzymes (DSF@Lipo-EVs): The culture medium was replaced with fresh complete medium containing 1 μg / mL LPS or 1 μM LPS + 5 μM Nigericin and 50 μg / mL ROS-responsive hybrid vesicles of neutrophils overexpressing dual antioxidant enzymes prepared in step 3 of Example 2, and cultured for 12 h.

[0048] 2. Apoptosis detection: Annexin V-FITC / 7-ADD double staining was performed according to the flow cytometer's instruction manual. Cells from each group were collected, resuspended in Binding Buffer, and then Annexin V-FITC and 7-ADD staining solutions were added sequentially. After incubation at room temperature in the dark for 15 minutes, the cells were analyzed by flow cytometry.

[0049] Annexin V-FITC / 7-ADD dual staining flow cytometry results show that ( Figure 4 Compared with the blank control group, the proportion of apoptosis in the LPS-treated model group was significantly increased, with the apoptosis rate rising from approximately 8.28% in the blank control group to over 18.13%. Treatment with blank ROS-responsive hybrid vesicles (Lipo-EVs) significantly reduced the apoptosis rate to 9.36%. The ROS-responsive hybrid vesicle group overexpressing dual antioxidant enzymes (DSF@Lipo-EVs) exhibited the lowest apoptosis rate (5.41%).

[0050] 3. Detection of intracellular reactive oxygen species levels After treatment, cells were collected and incubated with 10 μM DHE probe at 37 °C for 20 min. After washing with PBS, fluorescence intensity was detected by fluorescence microscopy.

[0051] The results show that ( Figure 4 In the model group (B), the intracellular fluorescence intensity was higher than that in the blank control group due to the increase in LPS treatment, indicating a large accumulation of ROS. After treatment with ROS-responsive hybrid vesicles overexpressing dual antioxidant enzymes, the fluorescence intensity of neutrophils decreased significantly. Among them, DSF@Lipo-EVs showed better ROS scavenging effect than Lipo-EVs.

[0052] 4. Mitochondrial membrane potential detection After treatment, 10 μg / mL JC-1 working solution was added and incubated at 37°C in the dark for 20 min. After washing with JC-1 buffer, the fluorescence of monomers (green) and aggregates (red) was detected by fluorescence microscopy to reflect the mitochondrial membrane potential level.

[0053] The results are as follows Figure 4 As shown in Figure C, the blank control group JC-1 exhibited red fluorescence (high membrane potential), while the red fluorescence of the LPS-treated model group was significantly reduced and the green fluorescence was enhanced. After treatment with DSF@Lipo-EVs, the red fluorescence was enhanced and the green fluorescence was reduced, indicating its protective effect on mitochondria.

[0054] 5. Observation of cell morphology and mitochondrial ultrastructure Cells from each group were collected, fixed with 2.5% glutaraldehyde, then fixed with 1% osmium tetroxide, dehydrated in a gradient manner, embedded in epoxy resin, and ultrathinly sectioned (70 nm). After double staining with uranium acetate and lead citrate, they were observed and photographed using a biological transmission electron microscope.

[0055] The ultrastructure of cells and mitochondria was observed using biological transmission electron microscopy. Figure 4 In the control group (D), cells showed intact morphology, with oval-shaped mitochondria and clear, regular cristae. Cells in the model group treated with LPS and nigericin showed numerous vacuoles, significant mitochondrial swelling, and cristae breakage. Cells treated with DSF@Lipo-EVs showed near-normal morphology, reduced mitochondrial swelling, and partial preservation of most mitochondrial cristae structures.

[0056] 6. Cell viability detection After treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 1 hour. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of blank control group - OD value of blank group) × 100%.

[0057] The results of the CCK8 experiment show that ( Figure 4In (E), the cell viability of the model group treated with LPS + Nigericin decreased to 60% of that of the blank control group (P < 0.05). After treatment with DSF@Lipo-EVs, the cell viability significantly recovered to 90% of that of the blank control group.

[0058] 7. Detection of lactate dehydrogenase release level Collect the cell culture supernatants of each group and operate according to the instructions of the lactate dehydrogenase (LDH) detection kit. By measuring the absorbance value at 490 nm, calculate the LDH release rate. LDH release rate (%) = (OD value of the treatment group - OD value of the blank control group) / (OD value of the maximum release group - OD value of the blank control group) × 100%.

[0059] The results showed that ( Figure 4 In (F), the LDH release rate of the model group treated with LPS + Nigericin was as high as 4.3 times that of the blank control group (P < 0.01), indicating that the cell membrane integrity was severely damaged. The LDH release rate of the DSF@Lipo-EVs group decreased to 1.3 times that of the blank control group.

[0060] 8. Detection of the release levels of inflammatory factors IL-18 and IL-1β Collect the cell culture supernatants of each group and use enzyme-linked immunosorbent assay (ELISA) kits to detect the concentrations of interleukin-18 (IL-18) and interleukin-1β (IL-1β) respectively. The operation steps are strictly carried out according to the kit instructions, and the absolute content (pg / mL) of cytokines in the samples is calculated through the standard curve.

[0061] ELISA detection showed that ( Figure 4 In (G), the concentrations of IL-18 and IL-1β in the cell supernatants of the model group treated with LPS + Nigericin increased to 7 times and 3 times that of the blank control group respectively (P < 0.001). After treatment with DSF@Lipo-EVs, the levels of both factors decreased significantly (P < 0.001).

[0062] Example 4: Evaluation of the therapeutic effect of ROS-responsive hybrid vesicles of neutrophils overexpressing dual antioxidant enzymes on septic mice I. Use the ROS-responsive hybrid vesicles of neutrophils overexpressing dual antioxidant enzymes prepared in Example 2 for tail vein injection to treat septic mice after cecal ligation and puncture. The C57BL / 6 mice used in this example were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., and the animal production license number is SCXK(Zhe)2024-0001. They were raised in a SPF-class animal room.

[0063] The specific experimental process is as follows: 1. Six-week-old C57BL / 6 mice were acclimatized for 7 days before modeling. After fasting for 12 hours, the mice were anesthetized by inhalation of isoflurane. The mice were placed abdomen-up, and the abdominal hair was removed with a frizz remover. The abdomen was disinfected with 75% alcohol. A 1cm incision was made in the midline of the lower abdomen to locate the cecum. The distal end of the cecum and the cecal valve were ligated using 6-0 surgical sutures. The cecum was punctured at the ligation site using a 21-gauge needle. Feces were squeezed out from the puncture site. The cecum was returned to the mouse's abdominal cavity, and the peritoneum and skin were sutured. An appropriate amount of physiological saline was administered. The mice were placed on a thermostat to maintain body temperature and prevent hypothermia-induced shock or even death.

[0064] 2. After modeling, mice were randomly divided into 5 groups of 10 mice each (for survival rate observation). After modeling, mice in each group were administered the drug via tail vein every 12 hours for a total of four administrations. The specific grouping and administration regimens are as follows: (1) Blank control group (Control group): 100 μL of normal saline was injected into the tail vein; (2) Model control group: 100 μL of normal saline was injected into the tail vein; (3) DSF administration group (DSF group): 100 μL of PBS buffer containing DSF (DSF: 30 mg / Kg) was injected via tail vein. (4) Blank ROS-responsive hybrid vesicle group (Lipo-EVs group): 100 μL of blank ROS-responsive hybrid vesicles (2.5 mg / Kg) were injected via the tail vein. (5) ROS-responsive hybrid vesicle group of neutrophils overexpressing dual antioxidant enzymes (DSF@Lipo-EVs group): 100 μL of ROS-responsive hybrid vesicles of neutrophils overexpressing dual antioxidant enzymes prepared in Example 2 were injected via tail vein (vesicles: 2.5 mg / Kg; DSF: 30 mg / Kg).

[0065] 3. Ten mice were used in each group to observe survival rate. Mice were considered dead when their body weight fell below 80% of their initial body weight. Weight changes in mice were recorded and scored during the experiment. Mice were sacrificed after the septic mice began to recover, and their organs and blood were collected for analysis.

[0066] 4. The collected mouse organ samples were embedded in paraffin. After embedding, tissue sections were prepared. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (HE). The stained sections were then used for subsequent histopathological observation.

[0067] II. Results Analysis like Figure 5As shown in Figure A, after CLP-induced sepsis, the body weight of mice in the model group decreased significantly starting on day 1. The body weight of mice in the DSF@Lipo-EVs group decreased less and gradually recovered from day 2 onwards. Meanwhile, the 7-day survival rate of mice in the model group was only 20%, while the survival rate of mice in the DSF@Lipo-EVs group significantly increased to 60%. Figure 5 (For Class B), the increase is as high as 40%.

[0068] like Figure 5 As shown in Figure C, the white blood cell count and neutrophil ratio were significantly increased in the model group mice, while the red blood cell count was decreased. In the DSF@Lipo-EVs group, the white blood cell and neutrophil ratios decreased significantly, and the red blood cell count returned to near normal levels. In other words, DSF@Lipo-EVs can inhibit excessive systemic inflammatory response and alleviate sepsis-induced coagulation abnormalities and immune disorders.

[0069] The liver and kidney function indicators (AST, ALT, Cr, BUN) were tested, such as... Figure 5 As shown in Figure D, compared with the blank control group, AST, ALT, Cr, and BUN were significantly elevated in the model group, indicating severe liver and kidney dysfunction. The DSF@Lipo-EVs group showed significantly lower levels of these indicators compared to the model group, and the improvement in liver and kidney function directly demonstrates that hybrid vesicles have a protective effect on distal organs.

[0070] The histopathological results of H&E directly confirmed ( Figure 5 Compared to the model group, the DSF group and Lipo-EVs group only showed partial improvement in organ damage, while DSF@Lipo-EVs treatment almost completely reversed myocardial arrangement disorder, hepatocyte junction disruption, and renal tubular structure damage, indicating that DSF@Lipo-EVs can alleviate sepsis-induced multi-organ damage such as heart, liver, and kidney, and has a broad-spectrum tissue protective effect.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes, characterized in that, The hybrid vesicles are formed by fusing neutrophil-derived vesicles overexpressing SOD2 and CAT with drug-loaded ROS-responsive liposomes.

2. The ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes according to claim 1, characterized in that, The drug is at least one of the drugs that regulate cell death, antioxidant response, or autoimmune response.

3. The ROS-responsive hybrid vesicle based on neutrophils overexpressing dual antioxidant enzymes according to claim 2, characterized in that, The drug in question is disulfiram.

4. A method for preparing ROS-responsive hybrid vesicles based on neutrophils overexpressing dual antioxidant enzymes as described in claim 1, characterized in that, Follow these steps to achieve the following: 1) HL60 cells were simultaneously infected with lentiviruses that overexpress SOD2 and lentiviruses that overexpress CAT, and then screened with puromycin and hygromycin B antibiotics to obtain HL60 cells that stably co-express SOD2 and CAT. Neutrophils overexpressing SOD2 and CAT were obtained by DMSO induction differentiation. 2) The neutrophils obtained in step 1) were treated with hypotonic cell lysis buffer and protease inhibitor, and then processed by mechanical extrusion to self-assemble into neutrophil vesicles overexpressing SOD2 and CAT. 3) Lecithin, cholesterol, and thioketal bond-containing lipid material DSPE-TK-PEG were mixed with the drug and hydrated using a thin-film dispersion method to obtain drug-loaded ROS-responsive liposomes; 4) The neutrophil vesicles obtained in step 2) and the drug-loaded ROS-responsive liposomes obtained in step 3) are repeatedly squeezed by extrusion to fuse their membranes, resulting in ROS-responsive hybrid vesicles of neutrophils overexpressing dual antioxidant enzymes.

5. The method for preparing ROS-responsive hybrid vesicles based on overexpressed dual antioxidant enzyme neutrophils according to claim 4, characterized in that, In step 1), the working concentration of DMSO for inducing differentiation is 1.2%.

6. The method for preparing ROS-responsive hybrid vesicles based on neutrophils overexpressing dual antioxidant enzymes according to claim 4, characterized in that, In step 2), the mechanical extrusion method involves repeatedly extruding the material through polycarbonate films of 0.8 μm, 0.4 μm, and 0.2 μm in sequence.

7. The use of the hybrid vesicles according to any one of claims 1-3 as a drug delivery system.

8. The use of the hybrid vesicles according to any one of claims 1-3 in the preparation of a medicament for treating infectious diseases, characterized in that, The infectious disease is sepsis, peritonitis, or lung infection.

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