Engineered macrophage membrane microcapsule CCR2-MM-coated PEG-PCL / DHA as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and provides an engineered macrophage membrane microcapsule CCR2-MM-coated PEG-PCL / DHA as well as a preparation method and application thereof. The engineering macrophage cell membrane microcapsule nanoparticles are prepared by transferring CCR2 overexpression plasmids into RAW 264.7 cells by using a lentiviral vector to construct RAW 264.7-CCR2 cells, then collecting cell membranes, preparing PEG-PCL / DHA nanoparticles, and mixing the cell membranes and the PEG-PCL / DHA nanoparticles according to a mass ratio of membrane protein to PEG-PCL of 1: 1. The symptoms of an MRL / LPR mouse SLE model can be effectively improved, and kidney injury is relieved. Kidney immune microenvironment is adjusted by adjusting the balance of kidney M1 / M2 macrophages, so that kidney injury of SLE mice is relieved. A new perspective is provided for treatment of SLE, and an important scientific basis is provided for drug design and clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA and a preparation method and application thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is a severe systemic autoimmune disease characterized by a variety of clinical manifestations, with a global prevalence of approximately 20 to 70 per 100,000 people [1]. Lupus nephritis (LN) is a serious complication involving renal impairment, and approximately half of SLE patients develop LN [2,3]. Current clinical treatments include glucocorticoids, immunosuppressants, antimalarials, biologics, and nonsteroidal anti-inflammatory drugs [4]. However, there is currently no cure for SLE, and long-term treatment can lead to severe organ damage, including organ failure. This highlights the urgent need for new molecular targets and therapeutic drugs for SLE. Natural products offer a promising approach to address these challenges. Dihydroartemisinin (DHA), a derivative of artemisinin, has shown potential efficacy in the treatment of autoimmune diseases such as SLE [5]. However, its poor bioavailability and significant first-pass liver effects require the identification of effective targets and strategies to improve the therapeutic efficacy of DHA.

[0003] CCL2, also known as MCP-1, is a chemokine that is primarily secreted by activated macrophages and monocytes and is produced in large quantities in a variety of cell types. Single-cell RNA sequencing of LN renal biopsies showed that CCL2 is highly expressed in infiltrating macrophages [6], and that the expression of CCL2-related genes in macrophages from SLE patients and mouse models is upregulated [7]. CCL2 levels are elevated in the early stages of disease in MRL / lpr mice. The interaction of CCL2 with its receptor CCR2 (primarily found on classical monocytes) is essential for recruiting peripheral immune cells to sites of inflammation, thereby aggravating renal damage in SLE [8,9]. In addition, CCL2 is involved in the pathogenesis of SLE by activating macrophages and promoting the release of proinflammatory cytokines [10,11]. The correlation between CCL2 levels and disease severity highlights the CCL2-CCR2 signaling axis as a promising therapeutic target for SLE.

[0004] CCR2 is a key receptor on monocytes and macrophages that regulates the directional migration of these cells to sites of inflammation through interaction with CCL2. Studies have shown that nanoparticles coated with macrophage membranes (MM) can achieve precise targeting and show good therapeutic effects in a variety of inflammatory environments. These MM nanoparticles have the combined advantages of membrane-based structures and functional cytokine receptors, which enables them to neutralize proinflammatory mediators and reduce inflammatory escalation [12-14]. In addition, MM nanoparticles can be genetically modified to enhance their function

[15] . However, despite these advances, the application of MM nanoparticles targeting the CCL2-CCR2 axis in the treatment of SLE remains unexplored. Summary of the invention

[0005] The invention provides an engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA, a preparation method and an application thereof. The microcapsule is applied to the synergistic treatment of systemic lupus erythematosus (SLE).

[0006] The microcapsules consist of an engineered macrophage membrane shell and a PEG-PCL core. The macrophage cell membrane endows the microcapsules with biomimetic properties, enhances circulation stability, and promotes the inflammatory targeting of the nanoparticles. In addition, PEG-PCL provides physical support for the cell membrane structure and is responsible for the encapsulation and controlled release of the drug. Upregulating the expression of CCR2 on the macrophage cell membrane through a genetic engineering strategy not only enhances the early mobilization and targeting ability of nanoparticles to the inflammatory area, but also acts as a "nanobait" to isolate CCL2 in the inflammatory microenvironment, targeting the CCL2-CCR2 axis, hindering the infiltration of peripheral immune cells and the activation of tissue-intrinsic immune cells, and ultimately improving kidney damage in MRL / Lpr mice.

[0007] The present invention is achieved by the following technical scheme: an engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA, a CCR2 overexpression plasmid is transferred into mouse mononuclear macrophage leukemia cells RAW 264.7 cells using a lentiviral vector to construct RAW 264.7-CCR2 cells, and then the cell membrane is collected to prepare polycaprolactone-polyethylene glycol / dihydroartemisinin, i.e., PEG-PCL / DHA nanoparticles, and then the cell membrane and PEG-PCL / DHA nanoparticles are mixed according to a membrane protein / PEG-PCL mass ratio of 1:1 to prepare CCR2-MM@PEG-PCL / DHA nanoparticles.

[0008] The method for preparing the engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA specifically comprises the following steps: (1) Obtaining RAW 264.7-CCR2 cells: The CCR2 overexpression plasmid was transferred into RAW 264.7 cells using a lentiviral vector to construct RAW 264.7-CCR2 cells; the virus was packaged into particles and their titers were determined; 1×10 6 RAW 264.7 cells were placed in a 6-well plate at a density of 1 μg / well and cultured in complete DMEM supplemented with 10% fetal bovine serum for 1 day. The cells were then infected with a lentiviral suspension containing 8 μg / mL polybrene and re-cultured with fresh culture medium 24 hours later. The fluorescence intensity of green fluorescent protein was observed under an inverted fluorescence microscope 72 to 96 hours after infection to verify the transfection efficiency. The culture was treated with puromycin at an initial concentration of 1 μg / mL and re-inoculated for amplification to screen and obtain genetically engineered RAW 264.7-CCR2 cells. (2) Isolation of RAW 264.7-CCR2 cell membranes: RAW 264.7-CCR2 cells were suspended in a homogenization buffer containing protease / phosphatase inhibitors, treated with an ultrasonic disruptor at 30W for 2 min, centrifuged at 3000 rpm for 7 min, and the supernatant was collected; the supernatant was centrifuged at 14800 rpm for 30 min) to collect the cell membranes; the cell membrane protein content was detected using a BCA kit; the cell membranes were obtained by extruding 15 times through a 400 nm polycarbonate porous membrane using an Avestin micro-extruder; the harvested cell membranes were stored in water at 4°C; (3) Preparation of PEG-PCL / DHA nanoparticles: Weigh 10 mg of DHA and 40 mg of PEG-PCL and place them in a rotary evaporator. Add 50 mL of dichloromethane to completely dissolve them. Adjust the speed to 50-120 r / min, 40°C, and perform rotary evaporation from fast to slow to form a uniform dry film. Place the rotary evaporator in a 37°C drying oven overnight to remove residual organic solvents. Add 5 mL of pure water to promote hydration of the film. Hydrate in a 40°C water bath for 60 min. Perform ultrasonic treatment at 40 Hz and 120 W for 10 min. Let stand at room temperature for 1 h. Filter 4-5 times with a 0.22 μm microporous filter membrane to remove the precipitate. This is PEG-PCL / DHA nanoparticles. (4) Preparation of engineered macrophage membrane microcapsules CCR2-MM@PEG-PCL / DHA: The macrophage vesicles obtained in step (2) and the PEG-PCL / DHA nanoparticles obtained in step (3) were mixed at a mass ratio of membrane protein / PEG-PCL / DHA of 1:1 to prepare MM@PEG-PCL / DHA nanoparticles; the mixture was then extruded 15 times through a 200 nm polycarbonate porous membrane using a mini extruder to obtain engineered macrophage membrane microcapsules CCR2-MM@PEG-PCL / DHA.

[0009] The present invention also provides the use of the engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA or the engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA obtained by the method in the preparation of a drug for the synergistic treatment of systemic lupus erythematosus SLE.

[0010] The present invention also provides the use of the engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA in preparing a drug for alleviating kidney damage in systemic lupus erythematosus (SLE) mice.

[0011] The present invention successfully constructed a nano drug delivery system (CCR2-MM@PEG-PCL / DHA) based on engineered macrophage membranes. The expression of CCR2 on the macrophage membrane was enhanced by genetic engineering technology. The nanosystem not only improved the drug's ability to target the inflammatory area, but also served as a "nanobait" to capture CCL2 in the inflammatory microenvironment, thereby inhibiting the inflammatory response of macrophages. The results of studies in vitro and in SLE model mice showed that this biomimetic nanovesicle can be specifically recognized and internalized by inflammatory macrophages, and inhibits the inflammatory response by regulating macrophage metabolic reprogramming. In addition, SLE treatment experiments showed that DHA NPs can effectively inhibit the inflammatory progression of SLE. We finally proved that this method is convenient and safe in vivo. Therefore, the design of this biomimetic nanovesicle, which uniquely realizes the potential of macrophage membrane targeting inflammation, may provide a new solution for combining the advantages of synthetic biology and nanotechnology to meet clinical anti-inflammatory needs.

[0012] The present invention provides a new strategy for treating SLE and its complication LN. The CCR2-MM@PEG-PCL / DHA nano drug delivery system reduces the recruitment of peripheral blood macrophages to the kidneys by targeting SLE kidneys, regulates the metabolic reprogramming of tissue macrophages, and inhibits inflammatory responses, thereby improving the symptoms and kidney damage of SLE mice. This strategy not only provides a new perspective for the treatment of SLE, but also provides an important scientific basis for future drug design and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1Detailed description and analysis of the preparation and properties of CCR2-MM@PEG-PCL / DHA nanoparticles; Figure: (A) Schematic diagram of the development process of RAW 264.7-CCR2 and CCR2-MM@PPD. (B, C) Immunofluorescence microscopy (CLSM) analysis of RAW 264.7 and RAW 264.7- ccr2 cells. (D) Flow cytometry (FCM) analysis of the frequency of CCR2-positive cells in RAW 264.7 and RAW 264.7- ccr2 populations. (E) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) measurement of CCR2 mRNA levels in RAW 264.7 and RAW 264.7-CCR2 cells. (F) Transmission electron microscopy (TEM) images of macrophage membranes (MMs) and CCR2-MM@PEG-PCL / DHA nanoparticles, scale bar is 200 nm. (G) Average size and zeta potential of PEG-PCL, PPD, MM@PPD, and CCR2-MM@PPD nanoparticles. (H, I) In vitro release profiles and stability assessment of PPD, MM@PPD, and CCR2-MM@PPD formulations. (J) In vitro bioluminescence imaging of excised tissues (heart, liver, spleen, lung, and kidney) from MRL / lpr mice. (K) Quantitative data of fluorescence intensity in specific organs. Figure 2 Figure 2 shows the in vitro targeting ability of CCR2-MM@PEG-PCL / DHA; (A) Fluorescence microscopy shows that CCR2-MM@PPD (green) shows significantly higher cellular uptake in LPS-stimulated RAW 264.7 cells compared with PPD and MM@PPD; (B, C, D) Corresponding quantitative fluorescence analysis. (E) Schematic diagram of the transwell assay setup, with the lower chamber containing M1 macrophage culture medium and the upper chamber containing M0 culture medium containing nanoparticles loaded with different types of dids. (F) The DiD fluorescence intensity in the lower chamber of each formulation was measured at different time intervals. (G) Schematic diagram of the in vitro macrophage chemotaxis transwell assay, with macrophages in the upper chamber and M1 macrophage culture medium with different formulations in the lower chamber. (H, J) Transwell assay to evaluate the migration of RAW 264.7 macrophages. (I, K) Scratch assay to evaluate the migration ability of RAW 264.7 cells under different conditions. (L) ELISA was used to detect the level of CCL2 in the cell culture supernatant of each group. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 (n = 3 / group); scale bar = 10µm; Figure 3The inhibitory effect of CCR2-MM@PEG-PCL / DHA on the progression of SLE; Figure: (A) Experimental timeline for treating MRL / lpr mice. (B) Body weight of mice in the MRL / MPJ group, MRL / LPJ group, PPD group, MM@PPD group, and CCR2-MM@PPD group. (C, F) Representative images and quantitative statistics of spleen and lymph nodes. (D, E) Representative images and quantitative statistics of thymus. (I) Urine protein levels of SLE mice were monitored weekly. (H) Serum creatinine levels were measured by ELISA. (K) ANA levels were detected by ELISA. (J) Anti-double-stranded DNA antibodies were detected by ELISA. (1) IL-6 cytokine levels. (M) TNF-α cytokine levels. (N) IL-10 cytokine levels. (O) Serum C3 levels. * p<0.05; ** p<0.01; *** p<0.001; **** P<0.0001, n = 5 / group; Figure 4 Renal tissue pathology associated with SLE; in the figure: (A) he staining (first column), scale bar = 40×; Masson trichrome staining (second column), scale bar = 10μm; periodic acid Schiff (PAS) staining (third column), scale bar = 40×; anti-IgG antibody staining (fourth column), scale bar = 50μm. Extensive renal damage occurred in both the MRL-lpr group and the CsA group, while renal damage in the CCR2-MM@PEG-PCL / DHA group was alleviated compared with the MRL-lpr group. (B) Glomerulonephritis score. (C) IgG fluorescence intensity. (D) Immunohistochemical staining of CCL2 in renal tissue, 6 mice in each group, scale bar = 50 μm. Data are expressed as mean ± standard deviation (*p<0.05, ***p<0.001) (n = 3); Figure 5DHA-NPs regulate the balance of immune cells in the kidney and spleen of MRL / lpr mice; Figure: (A) CCL2 immunofluorescence staining (red), scale bar = 50 μm. (B) Immunofluorescence staining of renal monocytes with anti-CCL2 (green) and anti-Ly6C / Ly6G (red) after 8 weeks of treatment, scale bar = 50 μm. (C) Immunofluorescence staining of M1 macrophages with CD68 (green, pan-macrophage marker), iNOS (M1 marker, red), or CD206 (M2 marker, red) and nuclei (blue) with or without DHA-NPs treatment. Scale bar = 50 μm. (DK) Flow cytometry analysis of the proportions of M1 macrophages (CD86+), M2 macrophages (CD163+), Th17 cells (IL17+), and Treg cells (Foxp3+) in the spleen before and after DHA-NPs treatment. Data are expressed as mean ± SD (*p < 0.05, ***p < 0.001) (n = 3); Figure 6 The cell viability of BMDM (A) and RAW264.7 cells (B) after incubation with different concentrations of CCR2-MM@PPD for 24 hours was detected by CCK-8 method. Data are expressed as mean ± standard deviation (*p<0.05, ***p<0.001) (n = 3); Figure 7 DHA-NPs inhibit the pro-inflammatory macrophage phenotype in RAW264.7 and BMDM cells (A) LPS pretreatment of RAW264.7 and BMDM cells induced the M1 phenotype. Immunofluorescence staining of M1 phenotype macrophages with CD68 (red, pan-macrophage marker), iNOS (green, M1 marker) and nuclei (blue) was performed with or without DHA-NPs treatment. Scale bar = 100 μm. (B, C, D) Detection and quantitative analysis of iNOS and CD86 proteins, M1 macrophage markers. (E, F, G, H) Flow cytometry analysis of the proportion of M1 macrophages (CD86+) in RAW264.7 and BMDM cells before and after DHA-NPs treatment and quantitative analysis. (IN) Detection of the expression of M1 macrophage marker genes (IL-6, TNF-α, iNOS) in RAW264.7 and BMDM cells. Data are expressed as mean ± SD (*p < 0.05, ***p < 0.001) (n = 3); Figure 8DHA-NPs inhibit M1 inflammatory phenotype by downregulating glycolysis (A) Volcano plot of all genes found in this study. Red and blue dots represent significantly upregulated and downregulated proteins, respectively (p values ​​< 0.05). (B) KEGG analysis showed that pathways associated with upregulated differentially expressed genes changed significantly. DHA-NP treatment led to enrichment of genes related to the glycolysis energy metabolism pathway. (C) Heat map analysis of glycolysis-related genes. (DI) Represents the ECAR, OCR, and lactate levels of each group. ECAR, OCR, and lactate levels reflect the glycolytic capacity of cells. (JR) Presents QPCR and Western Blot detection and quantitative analysis of glycolysis-related genes. Data are presented as mean ± standard deviation (*p<0.05, ***p<0.001) (n = 3); Fig. 9 (A, B) Fluorescence images and quantitative analysis of ROS in the DHA-NPs treated and untreated groups. Data are expressed as mean ± standard deviation (*p<0.05, ***p<0.001) (n = 3); Fig.10 DHA NPs have good biosafety in vivo; HE staining of liver, spleen and lung was used to evaluate safety. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs, and the materials cited herein and those cited by them will be incorporated by reference. Equivalent technologies to the specific embodiments described that those skilled in the art recognize through routine experiments will be included in this application.

[0016] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0017] 1. Materials and Methods 1. Dihydroartemisinin (DHA, CAS: 6812-81-3; catalog B20723) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). PEG-PCL was purchased from Shandong Freda Biopharmaceutical Co., Ltd. Healthy female C56, MRL / Lpr, and MRL / MPJ mice (18-22 g, 7 weeks old) were purchased from Spaifol Co., Ltd. The mice were housed in a room with controlled temperature (24 ± 2 °C) and humidity (55%) with free access to food and water. The animal experiments in this study were in accordance with the national regulations of China on experimental animals and were approved by the Ethics Committee of the Institute of Animal Protection, Shanxi Medical University. The mouse RAW264.7 cell line was purchased from the American Type Culture Collection (ATCC, USA). Dulbecco's modified Eagle's medium (DMEM) with 1% penicillin and streptomycin and 10% fetal bovine serum (GIBCO, USA) was used as the cell culture medium. The cells were incubated in a 37°C incubator containing 5% CO2.

[0018] 2. Establishment of RAW 264.7-CCR2 cells: CCR2 overexpression plasmid was transferred into RAW264.7 cells using lentiviral vector to construct RAW 264.7-CCR2 cells. Genepharma (Shanghai, China) provided plasmids containing the full-length sequence of CCR2. Virus packaging particles were prepared and their titers were determined. 1×10 6 RAW 264.7 cells were placed in a 6-well plate at a density of 1 μg / well and cultured in complete DMEM supplemented with 10% fetal bovine serum for 1 day. The cells were then infected with a lentiviral suspension containing 8 μg / mL polybrene and re-cultured with fresh medium 24 hours later. The fluorescence intensity of green fluorescent protein was observed under an inverted fluorescence microscope 72-96 hours after infection to verify the transfection efficiency. The culture was treated with puromycin at an initial concentration of 1 μg / mL and re-inoculated for amplification to screen for genetically engineered RAW 264.7-CCR2 cells with high infection efficiency and purity.

[0019] 3. Isolation of RAW 264.7 cell membranes: RAW 264.7 cells were suspended in a homogenization buffer containing protease / phosphatase inhibitors. Then, the cells were treated with an ultrasonic disruptor at 30W for 2min, centrifuged at 3000rpm for 7min, and the supernatant was collected. The cell membranes were collected by centrifugation of the supernatant (14800rpm, 30min). The cell membrane protein content was detected using a BCA kit (Beyotime, China). Macrophage vesicles were prepared by extruding 15 times through a 400 nm polycarbonate porous membrane using an Avestin microextruder. The harvested cell membranes were stored in water at 4°C.

[0020] 4. Preparation of CCR2-MM@PEG-PCL / DHA nanoparticles: Accurately weigh 10 mg of DHA and 40 mg of PEG-PCL and place them in a rotary evaporator, add 50 mL of dichloromethane to completely dissolve them, adjust the speed to 50-120 r / min, 40°C, and rotary evaporation from fast to slow to form a uniform dry film, then place the rotary evaporator in a 37°C drying oven overnight to remove residual organic solvents, add 5 mL of pure water to promote the hydration of the film, and then hydrate it in a 40°C water bath for 60 min, then ultrasonicate (frequency: 40 Hz, power: 120 W) for 10 min, place it at room temperature for 1 h, and filter it through a 0.22 μm microporous filter membrane 4-5 times to remove the precipitate.

[0021] MM@PEG-PCL nanoparticles were prepared by mixing macrophage vesicles with PEG-PCL nanoparticles at a mass ratio of 1:1. The mixture was then extruded 15 times through a 200 nm polycarbonate porous membrane using a mini extruder. The size distribution and zeta potential of the nanoparticles were determined using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). The morphology of the nanoparticles was observed using TEM (TECNAI G2, FEI, USA). The drug loading (LE) and encapsulation efficiency (EE) of DHA were determined by HPLC with a UV-vis detector at 285 nm. The serum stability of the nanoparticles was evaluated by measuring the particle size of the nanoparticles by incubating them in DMEM containing 10% FBS at room temperature for different times. CCR2-MM@MM@PEG-PCL / DHA was prepared according to the same procedure.

[0022] 5. Dynamic dialysis technique was used to determine the cumulative release of RBA: 1 mg of DHA-NPs was placed in a dialysis bag (MWCO = 7000 Da), and then dissolved in 20 mL of PBS buffer with 0.5% Tween 80. Observe for 7 days, take out 1 mL of the release solution every 12 hours, and replace it with an equal amount of fresh release solution. The concentration of released DHA was determined by UV-visible spectrophotometer.

[0023] 6. Real-time fluorescence quantitative PCR (qRT-PCR) analysis: Total RNA was extracted from cells using Trizol reagent (Takara, China), and then reverse transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (Vazyme, China). Subsequently, quantitative PCR (qPCR) was performed using AceQ qPCRSYBR Green Master Mix (Vazyme, China) in the 7500 real-time fluorescence quantitative PCR system (Applied Biosystems, Inc., USA). The reaction conditions were denaturation at 95°C for 10s, amplification at 60°C for 30s, and a total of 40 cycles. The primer sequences are shown in Table 1. The mRNA levels of the target genes were normalized to the expression of β-actin. The relative quantification of gene expression was performed using the 2^(-△△CT) method.

[0024] Table 1: Primer sequences 7. Cell immunofluorescence: Cell immunofluorescence was used to detect the expression of CCR2 on the surface of RAW 264.7 cells. RAW264.7 cells or BV2 cells were seeded in 24-well plates at a density of 1×10 cells / well. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.2% Triton X-100 for 30 minutes. After incubation with 5% BSA for 1 hour, the cells were incubated at 4°C in the dark overnight and stained with CCR2 monoclonal antibody (1:200, Abcam), mouse anti-cd86 (1:200, Abcam) or rabbit anti-cd206 (1:200, Abcam). After washing with PBS, the cells were incubated with cy3-labeled (Servicebio, China) or Alexa Fluor 488 and Alexa Fluor 594-labeled secondary antibodies (1:200, Jackson) at room temperature in the dark for 2 hours, and the cell nuclei were counterstained with DAPI for 10 minutes. After washing with PBS, immunofluorescence images were captured using a confocal laser scanning microscope (CLSM, Leica, TCS SP8).

[0025] 8. Flow cytometry was used to detect the expression of CCR2 in RAW 264.7 cells, RAW 264.7-CCR2 cells, and samples. The samples were incubated with APC anti-mouse CCR2 antibody (BioLegend, USA) for 30 min at 4°C in the dark. Subsequently, the specimens were washed with PBS, collected by centrifugation (3,000 rpm, 4°C, 5 min), and examined by flow cytometry (FACSVerse 8, BD, USA).

[0026] 9. Targeting of inflammatory macrophages: RAW264.7 cells were seeded in 12-well plates at a density of 1×106 cells per well, and LPS (100 ng / mL) and IFN-γ (20 ng / mL) were added and cultured at 37°C for 24 h. DiD-encapsulated nanoparticles were incubated with cells for 0.5 h, 1, 2, and 4 h, and then washed three times with PBS. The cells were fixed, stained with DAPI, and photographed with a laser scanning confocal microscope (LSM 800, Zeiss, Germany).

[0027] 10. Biodistribution of CCR2-MM@MM@PEG-PCL / DHA: MRL / lpr mice were intravenously injected with CCR2-MM@MM@PEG-PCL / DIR (trimmnps / DiR) in vivo. The mice were killed 4 hours later, and the heart, liver, spleen, lung, and kidney were collected for fluorescence imaging using an in vivo imaging device (LB983, Berthold Technologies Gmbh&Co. KG, Bad Wildbad, Germany).

[0028] 11. Efficacy evaluation A. 8-week-old MRL / lpr mice were randomly divided into 5 groups and intravenously injected with PBS, PPD, MM@PPD, CCR2MM@PPD, and DHA (60 mg / kg) twice a week. Body weight was measured weekly. Urine protein was detected by BCA protein detection kit (Sigma-Aldrich) at 12 and 18 weeks. Spleen and axillary lymph nodes were obtained and weighed 3 days after the last administration. Kidneys were stained with HE, Masson, PAS and immunofluorescence. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory cytokines (IL-6, TNF-α, IL-10), serum creatinine and anti-double-stranded DNA antibody (PeproTech).

[0029] B. Renal histopathological evaluation: Stained sections were observed under light microscopy; glomerulonephritis was evaluated and lesions were scored by two renal pathologists in a blinded manner, ranging from 0 to 4 points: 0, normal; 1, 1-25% lesions; 2, 26-50% lesions; 3, 51-75% lesions; 4, >75% lesions. Immunohistochemistry (IHC) was used to detect immune complex deposition. Tissue sections were incubated with anti-mouse IgG overnight, followed by the addition of secondary antibodies. Sections were developed with 3,3′-diaminobenzidine (DAB). Two renal pathologists scored the IgG IHC results in a blinded manner, based on the percentage of staining area (0, <1%; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, >75%) and staining intensity (0, no reaction; 1, mild reaction; 2, moderate reaction; 3, strong reaction). At least 50 glomeruli per mouse were analyzed, and the mean of the scores of all glomeruli per mouse was recorded.

[0030] C. Immunofluorescence staining to observe the proportion of renal inflammatory macrophages: kidney tissue samples were obtained from experimental animals and fixed and sliced; then, the tissue slices were subjected to antigen repair to expose the antigen epitopes in the cells; then, specific primary antibodies were used to incubate the M1 macrophage markers iNOS and CD68; subsequently, the fluorescently labeled secondary antibody was combined with the primary antibody to make the target protein visible under a fluorescence microscope; finally, the number of M1 macrophages was observed and counted using a fluorescence microscope to obtain their proportion in the kidney tissue.

[0031] D. Analysis of the distribution of immune cells in the spleen of MRL / lpr mice by flow cytometry: After the last administration, the spleen of MRL / lpr mice was collected and lymphocyte suspension was prepared. The proportion of macrophages and Treg and Th17 immune cells was calculated based on the total number of spleen cells and flow cytometry data.

[0032] 12. Extraction and culture of primary bone marrow macrophages: Extraction and culture of primary bone marrow macrophages from C57 mice: First, euthanize healthy C57 mice, and then collect bone marrow from their femurs and tibias. After filtering the bone marrow cell suspension, count the cells and adjust the concentration to 1×10 6 , and then inoculated into a medium containing macrophage colony stimulating factor (M-CSF) and cultured in an incubator at 37°C and 5% CO2. The medium was replaced regularly during the culture process, and the cells were passaged when they grew to 80%. Macrophages were identified by flow cytometry or immunofluorescence staining using markers such as CD11b and F4 / 80. Finally, the macrophages were treated and analyzed accordingly according to the experimental design.

[0033] 13. M1 macrophage polarization: RAW264.7 and BMDM were treated with LPS (100 ng / mL) and interferon-γ (20 ng / mL) for 24 h. DHA-NPs (equivalent to 40 μM DHA) inhibited the inflammatory phenotype of M1 macrophages for 24 h. Immunofluorescence staining and flow cytometry were used to detect the proportion of M1 type. ELISA was used to detect the levels of M1 type (iNOS, TNF-α, IL-1β) and M2 type (Arg-1, IL-10) markers. The cells were blocked with blocking buffer (PBS solution containing 5% BSA), fixed with 4% paraformaldehyde, and then anti-mouse CD68 iNOS antibody was used. The cells were incubated overnight at 4°C. After DAPI counterstaining, the sections were imaged using a laser scanning confocal microscope (LSM 800, Zeiss, Germany). The cells were incubated with flow cytometry antibodies PE anti-mouse CD86; APC anti-mouseCD206; PerCP-Cy5.5 anti-mouseCD11; AF488 anti-mouse F4 / 80 for 30 minutes, and the cell ratio was detected by flow cytometry. The expression of inflammatory cytokines (IL-6, TNF-α, Inos) in the cells was detected by Q-PCR.

[0034] 14. scRNA-seq analysis: Single-cell RNA sequencing (scRNA-seq) data of PBMC samples from healthy donors and SLE patients were obtained from the GEO database (GSE135779, GSE179633). Seurat package was used for quality control, principal component analysis (PCA) dimension reduction, and t-distributed random neighbor embedding (t-SNE) visualization. The quality control criteria were set as follows: (1) exclude genes with less than 3 cells; (2) exclude cells with a total number of detected genes less than 50; (3) exclude cells with mitochondrial gene expression exceeding 5%. Macrophage subtypes were identified after unsupervised clustering. Gene set enrichment analysis (GSEA) analysis (https: / / www.gsea-msigdb.org / gsea / index.jsp, Class H) was used to analyze the enrichment of signature pathways in inflammatory macrophages.

[0035] 15. RNA sequencing (RNA-seq): BMDM was extracted, LPS was induced into M1 macrophages, and intervention was added for 24 hours. 40umol CCR2MM@PPD (n = 3) or DMSO (n = 3) were used for intervention, and 3 groups of paired cell samples were collected for RNASeq. Heat maps were drawn using Perseus (1.6.2.2). GO and KEGG pathway analysis was performed using GeneCodis 3.0. FDR (q value) was used to screen genes of interest. In the quantitative results, when the gene change fold was >1.5 or <0.667 and the p value was <0.05, the changed gene was considered to be differentially expressed. All relevant data can be provided upon reasonable request.

[0036] 16. Metabolic assay: The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using an XFe96 extracellular flux analyzer (Agilent Technologies). RAW264.7 cells were cultured with LPS for 24 h, and CCRMM@PPD was added or not. The glycolysis and energy phenotype of RAW264.7 cells were detected using the Agilent Seahorse XF Glycolysis Stress Test Kit and the Cell Energy Phenotype Test Kit (Agilent Technologies). Cells were counted, washed with Seahorse XF basal medium containing the required supplements, and seeded into poly-lysine pre-coated XFe96 microplates (1*10^5 cells / well for human T cells and 5*10^5 cells / well for mouse T cells). In the glycolysis stress test, 10 mM glucose, 1μM oligomycin, and 50mM 2-deoxyglucose were added in sequence to monitor the ECAR value. In the energy phenotype assay, the ECAR values ​​were monitored before and after the addition of oligomycin (1 μM) and FCCP (1 μM), an uncoupler of oxidative phosphorylation (OXPHOS) in mitochondria. Data analysis was performed using the Seahorse Wave2.6 software package (Agilent Technologies).

[0037] 17. Cellular ROS determination: A green fluorescent probe was used to detect the intracellular ROS level. RAW264.7 cell suspension was inoculated into a 12-well plate at a volume of 1 mL per well, ensuring that each well contained at least about 1 × 105 cells, and incubated for 12 h. LPS and DMSO, PPD, MM@PPD, and CCR2MM@PPD were added to each well and incubated with RAW264.7 cells for 24 h. At the same time, LPS-treated cells were used as a positive control group. DCFH-DA cells were stained with serum-free culture medium for 60 min, the cells were washed with serum-free culture medium, and fluorescence images were taken under a fluorescence microscope.

[0038] 2. Experimental Results 1. Preparation and characterization of CCR2-MM@PEG-PCL / DHA: RAW264.7-CCR2 macrophages with stable overexpression of CCR2 were established by lentiviral transduction to produce engineered macrophage membrane microvesicles ( Figure 1 A). Confocal microscopy confirmed the upregulation of CCR2 on the membrane, showing an 8.63-fold increase in fluorescence signal ( Figure 1 B, C). Flow cytometry (FCM) analysis showed that 89.89% of cells in RAW 264.7-CCR2 were CCR2+, which was 1.97-fold higher than that in RAW 264.7 cells ( Figure 1 D). RT-qPCR showed that CCR2 mRNA expression in RAW 264.7-CCR2 increased 76.3-fold compared with RAW 264.7 ( Figure 1 E). In addition, genetic engineering significantly upregulated the expression of CCR2, confirming the successful establishment of RAW 264.7-CCR2 cells.

[0039] The anti-inflammatory drug dihydroartemisinin (DHA) was encapsulated in PEG-PCL nanoparticles, with a drug loading of 9.31% and an encapsulation efficiency of 93.07%. We prepared CCR2-MM@PPD nanoparticles with a core-shell structure, which was confirmed by TEM ( Figure 1 F). Dynamic light scattering (DLS) analysis showed that the particle size was (98.1±2.8) nm, the zeta potential was (-17.6±3.6) mV, and the PDI was 0.358±0.006 ( Figure 1 G, Figure S2), good stability and uniform distribution. The DHA release rates of PPD, MM@PPD, CCR2-MM@PPD, and CCR2-MM@PPD were 80.5%, 77.6%, and 76.2%, respectively. Compared with DHA, the release curves of PPD, CCR2-MM@PPD, and CCR2-MM@PPD were slightly slower, while no significant difference was observed between CCR2-MM@PPD and MM@PPD ( Figure 1 H). CCR2-MM@PPD maintained its diameter in DMEM containing 10% FBS, indicating good colloidal stability ( Figure 1 I). This indicates that CCR2-MM@PPD has good colloidal stability. In summary, the sustained and extended release of CCR2-MM@PPD indicates its potential for sustained administration. The biodistribution in MRL / lpr mice showed that CCR2-MM@PEG-PCL / DIR nanoparticles accumulated significantly in the kidneys compared with other groups, indicating its targeting ability for inflammation ( Figure 1 J, K).

[0040] 2. Characterization of in vitro targeted delivery of CCR2-MM@PEG-PCL / NPs: In vitro experiments were performed to detect the chemotactic effect of CCR2-MM@PEG-PCL / DiD / NPs on CCL2. It has been reported that CCL2 (MCP-1) is mainly derived from renal macrophages and monocytes in lupus nephritis and plays an important role in monocyte recruitment and disease exacerbation. In order to simulate the in vivo inflammatory microenvironment of SLE macrophages, activated M1 macrophages are the main source of CCL2. We encapsulated the green fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindole dicarbocyanine (DiD) in the cores of PEG-PCL (PEG-PCL / DiD), MM@PEG-PCL (MM@PEG-PCL / DiD), and CCR2-MM@PEG-PCL (CCR2-MM@PEG-PCL / DiD), respectively. First, LPS + IFN-γ activated RAW264.7 cells were cultured in vitro, and the cell targeting of PCL-PEG / DiD, MM@PCL-PEG / DiD, and CCR2MM@PCL-PEG / DiD was detected by CLSM. The CLSM results showed that compared with PEG-PCL / DiD, the cell uptake of MM@PEG-PCL / DiD and CCR2MM@ PEG-PCL / DiD increased significantly over time, and the cell uptake of CCR2MM@ PEG-PCL / DiD increased significantly ( Figure 2 AD).

[0041] like Figure 2 E, the culture medium of M0 cells containing PEG-PCL / DiD, MM@PEG-PCL / DiD, and CCR2-MM@PEG-PCL / DiD was added to the upper chamber of the transwell system, and the culture medium of M1 cells stimulated by LPS was added to the lower chamber. The chemotaxis and fluorescence intensity of DiD passing from the upper chamber to the lower chamber were detected by fluorescence spectrophotometer to evaluate the targeting of different NPs to inflammation. Figure 2 As shown in Figure F, after 3 h, 6 h, and 12 h of treatment, the fluorescence intensity of MM@PEG-PCL / DiD and CCR2MM@PEG-PCL / DiD at each time point was significantly stronger than that of PEG-PCL / DiD. This indicates that the macrophage membrane-coated nanoparticles inherit the ability to target inflammation. It is worth noting that the overexpression of CCR2 on the macrophage membrane promoted the chemotaxis and mobilization of CCL2 by CCR2MM@PEG-PCL / DiD compared with MM@PEG-PCL / DiD. This indicates that biomimetic nanoparticles overexpressing CCR2 have better inflammation targeting and chemotaxis, which may contribute to the aggregation of NPs at the site of injury. We also examined the effect of CCR2MM@PPD on macrophage chemotaxis and inflammation.

[0042] like Figure 2 As shown in G, macrophages were inoculated in the upper chamber and cultured, and the culture medium of M1 macrophages containing NPs was added to the lower chamber. After 24 h of co-culture, the Trans-well experiment was performed. Figure 2 As shown in H and J, M1 macrophage culture medium significantly promoted the migration of macrophages. However, MM@PPD disrupted the migration of RAW 264.7 induced by CM, and overexpression of CCR2 on the macrophage membrane enhanced this effect. No significant changes were observed after PPD treatment. This suggests that MM inhibits the inflammatory chemotaxis of macrophages, and CCR2 is one of the main receptors for this effect. A scratch test was also performed, and the results showed that compared with the PEG-PCL / DHA and MM@PEG-PCL / DHA groups, the CCR2-MM@PEG-PCL / DHA group could significantly inhibit the migration of M1 macrophages ( Figure 2 I, K).

[0043] Finally, the content of CCL2 in M1 macrophages after co-incubation with PPD, MM@PPD, and CCR2MM@PPD was detected. Figure 2 As shown in Figure 1, ELISA results showed that the content of CCL2 in the supernatant of M1 culture was significantly increased after LPS treatment (155.33±0.325pg / ml). There was no significant change in the content of CCL2 after the addition of PPD (123.67±0.154 pg / ml), while the content of CCL2 slightly decreased after the addition of PPD coated with MMs (87±0.781 pg / ml). However, the addition of PPD coated with CCR2-overexpressing MMs significantly reduced the amount of free CCL2 (75.67±0.059 pg / ml), indicating that the modified MMs-coated NPs may inhibit the chemotaxis and infiltration of macrophages to inflammation by neutralizing CCL2. This confirms our previous speculation that the overexpression of CCR2 enables MMs to act as a nanobait targeting the CCL2-CCR2 axis, capture CCL2 in the inflammatory microenvironment and exert an inhibitory effect on inflammation.

[0044] 3. The therapeutic effect of CCR2-MM@PEG-PCL / DHA on SLE: MRL / Lpr mice were intravenously injected with PBS, DHA (80 mg / kg), PEG-PCL / DHA, MM@PEG-PCL / DHA and CCR2-MM@PEG-PCL / DHA from 8 to 16 weeks ( Figure 3 A), MRL / MPJ mice were used as control mice. Figure 3 As shown in C, no significant changes in mouse body weight were observed during the treatment period, and the survival rate of CCR2-MM@PEG-PCL / DHA was much higher than that of other groups ( Figure 3B), indicating that CCR2-MM@PEG-PCL / DHA has no obvious toxic side effects. The size of spleen, axillary lymph nodes and thymus in the CCR2-MM@PEG-PCL / DHA group was significantly smaller than that in the other groups ( Figure 3 D, E and G), the body weight statistics are consistent. Urine protein and serum creatinine levels are two clinical indicators of renal function. The incidence of renal damage is high in SLE patients. The protein level of SLE mice at 12 weeks and 16 weeks was the lowest in mice treated with CCR2-MM@PEG-PCL / DHA ( Figure 3 H). Serum creatinine levels were also significantly reduced in the CCR2-MM@PEG-PCL / DHA group at week 16 ( Figure 3 I).

[0045] The serum concentration of anti-dsDHA autoantibody levels in the CCR2-MM@PEG-PCL / DHA group decreased compared with the other groups, but there was no statistical difference ( Figure 3 J), compared with other groups, the serum concentration of ANA level in the CCR2-MM@PEG-PCL / DHA group was lower than that in other groups ( Figure 3 K). Further experiments showed that CCR2-MM@PEG-PCL / DHA had the most significant inhibitory effect on inflammatory factors IL-6, TNF-α, and C3 in the serum of SLE mice, and the most significant promoting effect on the anti-inflammatory cytokine IL-10 ( Figure 3 LO). CCR2-MM@PEG-PCL / DHA has a long circulation time and can target inflammatory sites, and its therapeutic effect is better than PEG-PCL / DHA, MM@PEG-PCL / DHA or free DHA. All these data support that CCR2-MM@PEG-PCL / DHA can effectively alleviate the symptoms of SLE mice.

[0046] 4. Lupus nephritis (LN) is one of the most serious complications of SLE and a major indicator of poor prognosis. The most common symptoms of LN are glomerular and tubulointerstitial inflammation and deposition of renal immune complexes. Most SLE patients develop LN, and renal failure is one of the main causes of death in SLE patients. Therefore, the therapeutic effect of CCR2-MM@PEG-PCL / DHA on LN was further tested.

[0047] HE staining results showed that compared with free DHA, PEG-PCL / DHA and MM@PEG-PCL / DHA groups, the CCR2-MM@PEG-PCL / DHA group had reduced glomerular volume, reduced immune cell infiltration, and alleviated glomerulonephritis. The CCR2-MM@PPD group had the lowest glomerulonephritis disease score. In addition, the CCR2-MM@PPD group had significantly lower IgG deposition ( Figure 4A, B, C). Immunohistochemical staining of the kidneys of each group treated with NPs showed that CCR2-MM@PPD significantly reduced the level of CCL2 in the kidney ( Figure 4 D). In summary, CCR2-MM@PPD may inhibit the chemotaxis of monocytes to the kidney by reducing the level of CCL2 in the kidney, thereby alleviating renal damage in SLE mice. In summary, CCR2-MM@PPD may inhibit the chemotaxis of monocytes to the kidney by reducing the level of CCL2 in the kidney, thereby alleviating renal damage in SLE mice.

[0048] 5. CCR2-MM@PEG-PCL / DHA regulates the balance of macrophages and T cells in kidney and spleen: To evaluate whether CCR2-MM@PPD nanoparticles reduce monocyte recruitment to the kidneys of MRL / lpr mice by neutralizing CCL2, we performed immunofluorescence analysis of kidney tissues after 8 weeks of treatment. Figure 5 A shows that CCR2-MM@PPD treatment most significantly reduced CCL2 levels in the kidney. Figure 5 B shows that CCR2-MM@PPD treatment also significantly reduced the infiltration of CCR2-positive monocytes. Figure 5 As shown in C, staining for CD86, a marker typically associated with M1 macrophage activation, showed different intensities in the different treatment groups. The MRL / MPJ group, likely serving as a control group, showed baseline levels of CD86 expression. In contrast, kidneys from the MRL / Lpr group, which may represent an autoimmune or inflammatory condition, showed higher levels of CD86 expression, indicating increased macrophage activation. Treatment with DHA, PPD, MM@PPD, and CCR2MM@PPD appeared to reduce CD86 expression in the kidney, with the CCR2MM@PPD group showing the best treatment effect, suggesting that these treatments may have anti-inflammatory effects by inhibiting macrophage activation. CD163 is typically associated with M2 macrophage polarization, which is involved in anti-inflammatory processes and tissue repair. Figure 5 A shows that CD163 expression was lower in the MRL / Lpr group compared to the MRL / MPJ group, suggesting a possible shift toward a more pro-inflammatory M1 phenotype in the disease state. DHA, PPD, MM@PPD, and CCR2MM@PPD treatments appeared to increase CD163 expression, which could mean promotion of M2 macrophage polarization and potential resolution of inflammation.

[0049] The spleens of treated mice were analyzed by flow cytometry. Figure 5As shown in D, F, G, and H, CCR2MM@PPD can significantly inhibit the proportion of pro-inflammatory M1 macrophages in the spleen. IL-17 is a cytokine produced by Th17 cells and is associated with autoimmune and inflammatory diseases, while Foxp3 is a marker of regulatory T cells (Tregs) that helps maintain immune homeostasis. Figure 5 As shown in C, G, H, and I, the MRL / Lpr group had higher IL-17 expression and lower Foxp3 expression compared with the MRL / MPJ group, indicating that the balance between proinflammatory and regulatory immune responses was disturbed.

[0050] These results suggest that CCR2-MM@PPD effectively reduces the migration of monocytes to the kidneys and regulates the balance of macrophages and T cells in the inflammatory sites of the kidneys, alleviating chronic renal inflammation. The efficacy of this therapy is better than that of PPD, MM@PPD or free DHA. 5. CCR2-MM@PEG-PCL / DHA regulates M1 / M2 balance: It has been reported that balancing the M1 / M2 ratio in SLE kidneys can alleviate lupus nephritis because M1 appears to drive the pathological process of SLE. Since CCR2-MM@PEG-PCL / DHA can be effectively internalized by M1 macrophages and significantly inhibits inflammation and tissue damage, it is necessary to explore its role in M1 / M2 regulation.

[0051] To test this possibility, C57 mouse bone marrow macrophages BMDM and macrophage cell line RAW264.7 were used. These cells were pretreated with LPS to induce M1 phenotype polarization. RAW264.7 cells were then analyzed by immunofluorescence staining for CD68 (a marker for all macrophages) and iNOS (a marker for M1).

[0052] First, the CCK8 assay was used to screen the safe doses of NPs for BMDM and RAW264.7, and the results were 40µM and 80µM, respectively ( Figure 6 AB). RAW264.7 cells were immunofluorescently stained for CD68 (pan-macrophage marker) and iNOS (M1-type marker). As expected, iNOS expression was significantly increased in RAW264.7 and BMDM cells pretreated with LPS. After treatment with CCR2-MM@PPD, the iNOS fluorescence intensity decreased, indicating that CCR2-MM@PPD can inhibit the inflammatory phenotype of macrophages ( Figure 7 A). In addition, the expression of M1 marker proteins CD86 and iNOS in RAW264.7 cells was significantly reduced after CCR2-MM@PPD treatment ( Figure 7BD). Flow cytometric analysis of RAW264.7 and BMDM cells further supported these findings, showing that after CCR2-MM@PPD treatment, the proportion of M1 macrophages decreased from 74.3% and 86.1% to 48.2% and 58.3%, respectively ( Figure 6 EH). Finally, CCR2-MM@PPD significantly inhibited the expression of M1 markers such as iNOS, TNF-α, and IL-6 in BMDM and RAW264.7 cells ( Figure 7 IN). These results collectively indicate that CCR2-MM@PPD effectively alleviates the inflammatory phenotype of macrophages.

[0053] 6. CCR2-MM@PEG-PCL / DHA inhibits macrophage inflammatory phenotype by downregulating glycolysis level: RNA-seq was used to study the mechanism of CCR2-MM@DHA reprogramming BMDM macrophages. Compared with the LPS group, a total of 5,414 differentially expressed genes were identified, of which 1,821 genes were upregulated and 3,593 genes were downregulated ( Figure 8 A). Functional pathway analysis showed that these genes were mainly involved in metabolic processes, immune system function, innate and adaptive immune responses, inflammatory responses, and catalytic activity. KEGG enrichment analysis further highlighted that these genes were involved in glycolysis, FAO, and OXPHOS pathways, among which glycolysis was identified as the main metabolic pathway ( Figure 8 B). Heat map analysis of glycolysis-related genes showed that CCR2-MM@PPD inhibited the expression of these genes compared with the LPS group ( Figure 8 C). To evaluate the effect on metabolic energy regulation, we assessed the glycolytic potential of M1 macrophages by measuring the extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR), markers of glycolytic flux. The ECAR of M1 macrophages was 2-fold higher and the OCR value was 1.85-fold lower than that of control cells. After treatment with 80 μM CCR2-MM@PPD, the ECAR of M1 macrophages approached that of normal cells ( Figure 8 DH). In addition, CCR2-MM@PPD reduced the production of lactate, a key byproduct of glycolysis ( Figure 8 I). QPCR analysis confirmed that CCR2-MM@PPD significantly inhibited the expression of glycolysis-related genes such as LDHA, Eno1, PKM2, Glut1 and aldolase ( Figure 8 JN). Western blotting further showed that CCR2-MM@PEG-PCL / DHA inhibited the expression of LDHA, Eno1, and PKM2 proteins ( Figure 8In addition, CCR2-MM@PPD effectively reduced LPS-induced ROS generation in macrophages, further demonstrating its role in downregulating glycolysis and inhibiting the M1 inflammatory phenotype ( Fig. 9 A, B).

[0054] 7. Biosafety evaluation: After treatment, the liver, spleen, lung and other major organs of mice in different groups were taken for histological analysis to evaluate biosafety. Fig.10 As shown in A, no obvious damage or lesions were found under an optical microscope. In summary, the present invention reveals the good biocompatibility of the functionalized platform, making it possible for potential biomedical applications.

[0055] The present invention successfully developed a nano drug delivery system (CCR2-MM@PEG-PCL / DHA) based on engineered macrophage membranes and demonstrated its therapeutic potential for SLE. The results showed that by genetically enhancing the expression of CCR2 on macrophage membranes, the nanosystem not only enhanced the targeting of drugs to inflammatory sites, but also acted as a "nanobait" to capture CCL2 in the inflammatory microenvironment. This reduced the recruitment of SLE peripheral blood monocytes to the kidney and inhibited macrophage-driven inflammation. By targeting this axis, the nanosystem effectively alleviated the symptoms of the MRL / lpr mouse SLE model and reduced kidney damage. The key role of monocytes and macrophages in SLE requires not only the inhibition of their infiltration into inflamed tissues, but also the inhibition of their proinflammatory phenotypes to reduce disease activity or restore tissue tolerance.

[0056] RNA-seq analysis showed that the CCR2-MM@PPD nanosystem regulated macrophage metabolic reprogramming, especially by downregulating glycolysis to inhibit the inflammatory M1 macrophage phenotype. In vitro experiments and studies in SLE model mice were conducted to explore how CCR2-MM@PPD improves SLE symptoms by inhibiting macrophage glycolysis. The results showed that this biomimetic nanovesicle was specifically recognized and internalized by inflammatory macrophages, reprogramming M1 macrophages into M2 macrophages, inhibiting the expression of inflammatory cytokines and promoting tissue repair. This approach showed an effective anti-SLE therapeutic effect.

[0057] The present invention successfully constructed a nano drug delivery system (CCR2-MM@PEG-PCL / DHA) based on engineered macrophage membranes. The expression of CCR2 on the macrophage membrane was enhanced by genetic engineering technology. The nanosystem not only improved the drug's ability to target the inflammatory area, but also served as a "nano bait" to capture CCL2 in the inflammatory microenvironment, thereby inhibiting the inflammatory response of macrophages. The results of studies in vitro and in SLE model mice showed that this biomimetic nanovesicle can be specifically recognized and internalized by inflammatory macrophages, and inhibits the inflammatory response by regulating macrophage metabolic reprogramming. In addition, SLE treatment experiments showed that DHA NPs can effectively inhibit the inflammatory progression of SLE. It was finally proved that this method is convenient and safe in vivo. Therefore, the design of this biomimetic nanovesicle uniquely realizes the potential of macrophage membrane targeting inflammation, which may provide a new solution for combining the advantages of synthetic biology and nanotechnology to meet clinical anti-inflammatory needs.

[0058] CCR2-MM@PEG-PCL / DHA is an innovative therapeutic strategy that is rationally designed and effectively applied to SLE. This platform has several significant advantages. First, unlike the unilateral inflammation inhibition by targeting specific cytokines, CCR2-MM@PEG-PCL / DHA proposes a unique and indispensable approach by focusing on the CCL2-CCR2 axis. This strategy effectively reduces the overall production of inflammatory factors by inhibiting macrophage infiltration and pro-inflammatory polarization of macrophages in LN kidneys, thereby significantly alleviating the secondary inflammatory cascade. In addition, the genetically engineered macrophage membrane structure can be used as a nanocarrier and nanobait to modulate inflammation while delivering therapeutic drugs. More importantly, compared with the cell membrane-coated nanoparticles without any engineering modification, the macrophage membrane-coated nanoparticles with enhanced CCR2 expression have higher drug delivery efficiency, significant in vivo anti-inflammatory effects, and demonstrate their practical value and prospects in the treatment of SLE.

[0059] References: [1] Pons-Estel GJ, Alarcón GS, Scofield L, Reinlib L, Cooper GS. Understanding the epidemiology and progression of systemic lupuserythematosus. Semin Arthritis Rheum . 2010;39(4):257-268. doi:10.1016 / j.semarthrit.2008.10.007. [2]Bajema IM, Wilhelmus S, Alpers CE, et al. Revision of theInternational Society of Nephrology / Renal Pathology Society classificationfor lupus nephritis: clarification of definitions, and modified NationalInstitutes of Health activity and chronicity indices. Kidney Int . 2018;93(4):789-796. doi:10.1016 / j.kint.2017.11.023. [3]Fanouriakis A, Tziolos N, Bertsias G, Boumpas DT. Update οn thediagnosis and management of systemic lupus erythematosus. Ann Rheum Dis . 2021;80(1):14-25. doi:10.1136 / annrheumdis-2020-218272. [4]Fava A, Petri M. Systemic lupus erythematosus: Diagnosis andclinical management. J Autoimmun . 2019;96:1-13. doi:10.1016 / j.jaut.2018.11.001. [5]Shi X, Liao T, Chen Y, et al. Dihydroartemisinin inhibitsfollicular helper T and B cells: implications for systemic lupuserythematosus treatment. Arch Pharm Res . 2024;47(7):632-644.doi:10.1007 / s12272-024-01505-1. [6]Kulkarni O, Pawar RD, Purschke W, et al. Spiegelmer inhibition ofCCL2 / MCP-1 ameliorates lupus nephritis in MRL-(Fas)lpr mice. J Am Soc Nephrol JASN . 2007;18(8):2350-2358. doi:10.1681 / ASN.2006121348. [7]Shu B, Fang Y, He W, Yang J, Dai C. Identification of macrophage-related candidate genes in lupus nephritis using bioinformatics analysis. Cell Signal . 2018;46:43-51. doi:10.1016 / j.cellsig.2018.02.006. [8]Lema GPD, Maier H, Nieto E, et al. Chemokine expression precedesinflammatory cell infiltration and chemokine receptor and cytokine expressionduring the initiation of murine lupus nephritis. J Am Soc Nephrol JASN . 2001;12(7):1369-1382. doi:10.1681 / ASN.V1271369. [9]Charo IF, Myers SJ, Herman A, Franci C, Connolly AJ, Coughlin SR.Molecular cloning and functional expression of two monocyte chemoattractantprotein 1 receptors reveals alternative splicing of the carboxyl-terminaltails. Proc Natl Acad Sci USA. 1994;91(7):2752-2756. doi:10.1073 / pnas.91.7.2752.

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Claims

1. An engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA, characterized by: The CCR2 overexpression plasmid was transferred into mouse mononuclear macrophage leukemia cells RAW 264.7 cells using a lentiviral vector to construct RAW 264.7-CCR2 cells. The cell membranes were then collected to prepare polycaprolactone-polyethylene glycol / dihydroartemisinin (PEG-PCL / DHA) nanoparticles. The cell membranes were then mixed with PEG-PCL / DHA nanoparticles at a membrane protein / PEG-PCL mass ratio of 1:1 to prepare CCR2-MM@PEG-PCL / DHA nanoparticles.

2. A method for preparing the engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA according to claim 1, characterized in that: The specific steps include: (1) Obtaining RAW 264.7-CCR2 cells: The CCR2 overexpression plasmid was transferred into RAW 264.7 cells using a lentiviral vector to construct RAW 264.7-CCR2 cells; the virus was packaged into particles and their titers were determined; 1×10 6 RAW 264.7 cells were placed in a 6-well plate at a density of 1 μg / well and cultured in complete DMEM supplemented with 10% fetal bovine serum for 1 day. The cells were then infected with a lentiviral suspension containing 8 μg / mL polybrene and re-cultured with fresh culture medium 24 hours later. The fluorescence intensity of green fluorescent protein was observed under an inverted fluorescence microscope 72 to 96 hours after infection to verify the transfection efficiency. The culture was treated with puromycin at an initial concentration of 1 μg / mL and re-inoculated for amplification to screen and obtain genetically engineered RAW 264.7-CCR2 cells. (2) Isolation of RAW 264.7-CCR2 cell membranes: RAW 264.7-CCR2 cells were suspended in a homogenization buffer containing protease / phosphatase inhibitors, treated with an ultrasonic disruptor at 30W for 2 min, centrifuged at 3000 rpm for 7 min, and the supernatant was collected; the supernatant was centrifuged at 14800 rpm for 30 min) to collect the cell membranes; the cell membrane protein content was detected using a BCA kit; the cell membranes were obtained by extruding 15 times through a 400 nm polycarbonate porous membrane using an Avestin micro-extruder; the harvested cell membranes were stored in water at 4°C; (3) Preparation of PEG-PCL / DHA nanoparticles: Weigh 10 mg of DHA and 40 mg of polycaprolactone-polyethylene glycol PEG-PCL and place them in a rotary evaporator. Add 50 mL of dichloromethane to completely dissolve them. Adjust the speed to 50-120 r / min, 40°C, and evaporate from fast to slow to form a uniform dry film. Then place the rotary evaporator in a 37°C drying oven overnight to remove residual organic solvents. Add 5 mL of pure water to promote hydration of the film. Hydrate in a 40°C water bath for 60 min, perform ultrasound at 40 Hz and 120 W for 10 min, let stand at room temperature for 1 h, and filter 4-5 times with a 0.22 μm microporous filter membrane to remove the precipitate, which is the PEG-PCL / DHA nanoparticles. (4) Preparation of engineered macrophage membrane microcapsules CCR2-MM@PEG-PCL / DHA: The macrophage vesicles obtained in step (2) and the PEG-PCL / DHA nanoparticles obtained in step (3) were mixed at a mass ratio of membrane protein / PEG-PCL / DHA of 1:1 to prepare MM@PEG-PCL / DHA nanoparticles; the mixture was then extruded 15 times through a 200 nm polycarbonate porous membrane using a mini extruder to obtain engineered macrophage membrane microcapsules CCR2-MM@PEG-PCL / DHA.

3. Use of the engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA described in claim 1 or the engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA obtained by the method described in claim 2 in the preparation of a drug for the synergistic treatment of systemic lupus erythematosus (SLE).

4. The use according to claim 3, characterized in that: The engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA is used in preparing a drug for targeted inhibition of inflammation of SLE.

5. The use according to claim 3, characterized in that: The engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA is used in preparing a drug for alleviating kidney damage in systemic lupus erythematosus (SLE) mice.

6. The use according to claim 5, characterized in that: The engineered macrophage membrane microcapsule CCR2-MM@PEG-PCL / DHA is used in preparing a drug for inhibiting the inflammatory response of inflammatory macrophages.

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