Preparation and application of double-membrane independently-wrapped nano drug delivery system for regulating immunity

Abstract

The invention discloses a double-membrane independently wrapped nano drug delivery system for regulating macrophages and platelets. The nano drug delivery system comprises macrophage membrane coated nanoparticles and platelet membrane coated nanoparticles, the macrophage membrane coated nanoparticles are used for adjusting macrophage immune response and loaded with a Toll-like receptor 7 (TLR7) inhibitor, the platelet membrane coated nanoparticles are used for inhibiting abnormal activation of platelets and loaded with a G protein coupled receptor P2Y12 inhibitor, the TLR7 inhibitor comprises at least one of TJ-M2010-5, hydroxychloroquine, Enpatron and IMO-8400, and the TLR7 inhibitor comprises at least one of TJ-M2010-5, hydroxychloroquine, Enpatron and IMO-8400. The P2Y12 inhibitor is prepared from at least one of ticagrelor, clopidogrel, prasugrel and cangrelor. According to the double-membrane independently-wrapped nano drug delivery system, drugs are efficiently delivered to related damaged cells of an inflammation part by utilizing related cell tropism of two cell membranes, immune disorder caused by macrophage and platelet abnormality is inhibited, diseases induced by immune imbalance are treated, an immune microenvironment is regulated and controlled, and the drug delivery effect is improved. And a new direction is provided for treatment of immune diseases.

Description

Technical Field

[0001] The present invention relates to a double-membrane independently wrapped nano drug delivery system for immune regulation, which is used to regulate the homeostasis of macrophages and platelets. The double-membrane independently wrapped nano drug delivery system includes macrophage membrane-wrapped drug-loaded nanoparticles and platelet membrane-wrapped drug-loaded nanoparticles, belonging to the field of medical technology. Background Art

[0002] Macrophages and platelets play complex and interrelated roles in immune diseases, jointly influencing the development and progression of diseases through mechanisms such as immune regulation, inflammatory responses, and tissue repair. Platelet-associated antigens released by platelets can induce macrophage polarization toward a proinflammatory phenotype. Conversely, cytokines secreted by macrophages can activate platelets, forming a vicious "macrophage-platelet" cycle. The dysregulation of these two pathways exacerbates the progression of immune-related diseases.

[0003] Toll-like receptor 7 (TLR7) is an important pattern recognition receptor in macrophages. It plays a key role in innate immune response, inflammation regulation and autoimmune diseases. It is also an important receptor for maintaining macrophage homeostasis. Its upregulation can lead to abnormal activation of autoimmune response and trigger a series of immune responses. 12 It is expressed on platelets and directly regulates platelet activation, aggregation, and immune inflammatory responses. By inhibiting these two receptors, it can alleviate the vicious cycle of "macrophage-platelet" in autoimmune reactions and repair the autoimmune response at the cellular level.

[0004] By mimicking natural cells, cell membrane-inspired nanoparticle delivery systems overcome the limitations of traditional nanocarriers, providing a more precise and safer strategy for disease treatment. Using macrophage membranes to encapsulate nanoparticles allows for precise targeting of inflammatory sites and macrophages. Similarly, platelet membranes, through their specific adhesion to inflammatory sites, can deliver drugs to the site of inflammation, achieving precise drug delivery. Summary of the Invention

[0005] In order to solve the problem of the vicious cycle of "macrophages-platelets" in autoimmune reactions, the present invention creatively uses macrophage membranes to wrap drug-loaded nanoparticles and platelet membranes to wrap drug-loaded nanoparticles to achieve precise delivery of therapeutic drugs to macrophages and platelets, inhibit the polarization of macrophages to a pro-inflammatory phenotype, reduce the secretion of pro-inflammatory cytokines by macrophages and repair their efferocytosis, while inhibiting further activation of platelets, alleviating the vicious cycle of "macrophages-platelets", and providing a new method for the treatment of related autoimmune reaction diseases such as systemic lupus erythematosus.

[0006] One of the objectives of this invention is to overcome the shortcomings of existing treatment options and provide a dual-membrane independently encapsulated nanoparticle drug delivery system for active targeted treatment of autoimmune diseases. By leveraging the inflammation-targeting and homing effects of macrophage and platelet membranes, precise drug delivery to macrophages and platelets is achieved.

[0007] Another purpose of the present invention is to treat abnormal macrophages and platelets, reduce immune response by restoring their homeostasis, repair the vicious cycle of "macrophage-platelets", and provide a reference for the treatment of autoimmune diseases.

[0008] Another purpose of the present invention is to overcome the traditional treatment of immune diseases using glucocorticoids, nonsteroidal anti-inflammatory drugs, etc., and treat related immune cells from the perspective of inhibiting receptors to achieve the purpose of precise treatment.

[0009] The present invention provides a preparation and application of a double-membrane independently coated nano drug delivery system for regulating immunity, wherein the drug delivery system comprises macrophage membrane-coated drug-loaded nanoparticles RM-NPs and platelet membrane-coated drug-loaded nanoparticles PM-NPs, wherein the drug loaded on the RM-NPs comprises a TLR7 inhibitor; the drug loaded on the PM-NPs comprises a P2Y 12 Inhibitor.

[0010] Nanomaterials for drug-loaded nanoparticles include polymers, phospholipids, and PEGylated lipids that can encapsulate drugs.

[0011] The polymer capable of encapsulating drugs includes at least one of polylactic acid-glycolic acid copolymer, polylactic acid, polyhydroxyalkanoate and polycaprolactone.

[0012] The phospholipids include at least one of lecithin, phosphatidylserine and choline phospholipids.

[0013] The PEGylated lipids include at least one of DSPE-PEG, DOPE-PEG, and DMPE-PEG.

[0014] The macrophage membrane includes the cell membrane of activated macrophages obtained by cytokine stimulation, and the macrophages include at least one of mononuclear macrophage leukemia cells (such as THP-1 or RAW264.7) and bone marrow-derived macrophages; the cytokines include interferon-γ (IFN-γ), tumor necrosis factor-α or interleukin-10; preferably, the cytokine is IFN-γ.

[0015] The platelet membrane is derived from at least one of resting platelets, activated platelets, and activated platelets stimulated by cytokines, wherein the cytokines include platelet-derived growth factors or chemokines; preferably, the platelet membrane is a resting platelet membrane.

[0016] In the nano drug delivery system, the mass ratio of protein to polymer in the macrophage membrane or platelet membrane is (1:0.5)-(1:10).

[0017] The TLR7 inhibitor includes at least one of TJ-M2010-5, hydroxychloroquine, Enpatoran, and IMO-8400, preferably TJ-M2010-5.

[0018] The P2Y 12 The inhibitor includes at least one of ticagrelor, clopidogrel, prasugrel, and cangrelor, preferably ticagrelor.

[0019] The autoimmune diseases include systemic lupus erythematosus.

[0020] The present invention provides a pharmaceutical composition comprising the aforementioned nano drug delivery system.

[0021] The macrophage membrane-encapsulated nanoparticles carrying the TLR7 inhibitor are prepared by the following method: (1) Treat macrophages with cytokines, collect activated macrophages after 48 hours, break the cells with a grinder, and centrifuge at ultrahigh speed to collect engineered macrophage membranes; (2) Dissolve poly(lactic-co-glycolic acid), phospholipids, pegylated lipids, and a TLR7 inhibitor (e.g., TJ-M2010-5) in dimethyl sulfoxide and dropwise add the mixture into deionized water under stirring to obtain drug-loaded nanoparticles. The drug-loaded nanoparticles are mixed with the engineered macrophage membrane prepared in (1) and ultrasonically combined to obtain engineered macrophage membrane-encapsulated TLR7 inhibitor-loaded nanoparticles RM-NPs.

[0022] The packaged P2Y 12 The platelet membrane-encapsulated nanoparticles of the inhibitor were prepared by the following method: (1) Whole blood from healthy mice was collected and gradient centrifuged to obtain resting platelets. The platelets were broken by repeated freeze-thaw method and then centrifuged at high speed to obtain resting platelet membranes. (2) Poly(lactic acid-co-glycolic acid), phospholipids, PEGylated lipids and P2Y 12 The inhibitor (e.g., ticagrelor) is dissolved in dimethyl sulfoxide and added dropwise to deionized water under stirring to obtain drug-loaded nanoparticles. The drug-loaded nanoparticles are mixed with the resting platelet membrane prepared in (1) and ultrasonicated to combine them to obtain resting platelet membrane-encapsulated P2Y nanoparticles. 12 Inhibitor nanoparticles PM-NPs.

[0023] The immune-regulating double-membrane independently wrapped nano drug delivery system can be further prepared into various forms such as injections, and drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0024] The double-membrane independently wrapped nano drug delivery system provided by the present invention accumulates at the lesion site, repairs abnormal macrophages and platelets, inhibits the transformation of macrophages to a pro-inflammatory phenotype, repairs their efferocytosis, and inhibits the abnormal activation of platelets, thereby alleviating the vicious cycle of "macrophage-platelet", regulating the immune microenvironment, and providing new ideas for the treatment of related autoimmune reaction diseases such as systemic lupus erythematosus.

[0025] Beneficial effects of the present invention: The present invention combines macrophage membrane-wrapped drug-loaded nanoparticles RM-NPs and platelet membrane-wrapped drug-loaded nanoparticles PM-NPs to regulate the circulation of macrophages and platelets, thereby achieving the purpose of regulating the inflammatory microenvironment. The relevant membrane-wrapped drug-loaded nanoparticles are used to utilize their tropism for inflammatory tissues and homing effects to achieve precise drug delivery, repair the function of macrophages in the lesion site, inhibit their polarization to the M1 proinflammatory phenotype, inhibit the secretion of proinflammatory cytokines, repair the cell efferocytosis, and at the same time inhibit the excessive activation of platelets, alleviating the vicious cycle of "macrophage-platelet". The RM-NPs and PM-NPs in the constructed double-membrane independently wrapped nano drug delivery system can synergistically treat systemic lupus erythematosus when used in combination, achieving unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Diagrams showing the preparation of dual-membrane independently encapsulated nanoparticle drug delivery systems. (A) Diagram showing the preparation of engineered macrophage membrane-encapsulated nanoparticles (IFN RM-NPs@TJ-5); (B) Diagram showing the preparation of platelet membrane-encapsulated nanoparticles (PM-NPs@TGL).

[0027] Figure 2 Characterization diagrams of the double-membrane independently encapsulated nanoparticle drug delivery system. (A) Particle size, potential, polydispersity coefficient, encapsulation efficiency, and drug loading of the double-membrane independently encapsulated nanoparticles; (B) Transmission electron microscopy images of PLGA-NPs@TJ-5 and IFN RM-NPs@TJ-5; (B) Transmission electron microscopy images of PLGA-NPs@TGL and PM-NPs@TGL.

[0028] Figure 3 Figures show the distribution of the double-membrane independently encapsulated nanoparticle drug delivery system in lupus mice. (A) Graph showing the accumulation of PLGA-NPs@DiR and IFN RM-NPs@DiR in the spleen and kidneys of lupus mice and healthy mice; (B) Graph showing the accumulation of PLGA-NPs@DiR and PM-NPs@DiR in the spleen and kidneys of lupus mice and healthy mice.

[0029] Figure 4 Figures show the in vitro targeting ability of the double-membrane independently encapsulated nanoparticle drug delivery system for macrophages and platelets. (A) Targeting ability of IFN RM-NPs@C6 for H-RAW and I-RAW; (B) Targeting ability of PLGA-NPs@C6 and IFN RM-NPs@C6 for I-RAW; (C) Targeting ability of PLGA-NPs@C6 and PM-NPs@C6 for resting and activated platelets; (D) Targeting ability of PM-NPs@C6 for resting and activated platelets; (E) Targeting ability of PLGA-NPs@C6 for resting and activated platelets.

[0030] Figure 5 Figure 3. In vitro restoration of macrophage homeostasis by the double-membrane independently encapsulated nanoparticle delivery system. (A) IFN RM-NPs@TJ-5 restores macrophage phenotype; (B) IFN RM-NPs@TJ-5 promotes anti-inflammatory cytokine secretion in macrophages; (C) IFN RM-NPs@TJ-5 restores the expression of the macrophage efferosome recognition protein MFGE8; (D) IFN RM-NPs@TJ-5 restores the phagocytic ability of macrophages; (E) IFN RM-NPs@TJ-5 restores the acidification level of macrophage efferosomes; (F) IFN RM-NPs@TJ-5 restores the maturation level of macrophage efferosomes.

[0031] Figure 6 This is a diagram showing the effect of the double-membrane independently wrapped nanodrug delivery system in inhibiting platelet activation in vitro.

[0032] Figure 7 The therapeutic effects of the double-membrane independently encapsulated nanodrug delivery system on lupus erythematosus mice are shown. (A) Anti-dsDNA Ab levels in the treated mice were restored; (B) Spleen weight index levels in the treated mice were restored; (C) Treg levels in the thymus of the treated mice were restored; (D) IgG and C4 levels in the kidneys of the treated mice were restored.

[0033] Figure 8 Figures show the therapeutic effect of the double-membrane independently encapsulated nano-drug delivery system on the "macrophage-platelet" vicious cycle in lupus erythematosus mice. (A) Macrophage infiltration levels in the spleen of the treated mice; (B) Macrophage phenotype levels in the spleen of the treated mice; (C) Platelet levels in the spleen of the treated mice; (D) Platelet levels in the blood of the treated mice; (E) PF4 levels in the kidneys of the treated mice.

[0034] Figure 9 Schematic diagram of the preparation process of the double-membrane independently wrapped nanodrug delivery system and its in vivo circulation targeting and treatment of diseased cells. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. The present invention is exemplified below by the examples. Based on the description of the present invention, those skilled in the art may make equivalent substitutions, combinations, improvements or modifications thereto, and all such variations shall be included within the scope of protection of the present invention.

[0036] Example 1 Preparation and characterization of double-membrane independently encapsulated nanoparticles.

[0037] RAW264.7 mononuclear leukemia cells were seeded in a cell culture dish. After the cells adhered, medium containing IFN-γ at a concentration of 25 ng / mL was added. After 48 hours of induction, the cells were harvested, disrupted with a grinder, and centrifuged at 100,000 g for 1 hour. The pellet was resuspended in a storage solution containing protease inhibitors and aliquoted to obtain engineered macrophage membranes (IFN RMs). PLGA (40 mg / mL, dissolved in DMSO), DSPE-PEG2000-OME (40 mg / mL, dissolved in DMSO), soy lecithin (40 mg / mL, dissolved in MeOH), and TJ-M2010-5 (10 mg / mL, dissolved in DMSO) were prepared. The nanomaterial stock solutions were mixed at a volume ratio of 5:2:4:5 and added dropwise to stirred ultrapure water. After stirring for 5 minutes, the PLGA-NPs@TJ-5 cores were collected. An appropriate amount of engineered cell membrane stock solution was taken, and the probe was ultrasonicated in an ice bath (80 W, 3 min) to obtain engineered cell membrane vesicles. PLGA-NPs@TJ-5 core (nanomaterial PLGA: membrane protein mass ratio is 2:1) was added, and the probe was ultrasonicated in an ice bath (100 W, 5 min) to obtain engineered macrophage membrane-coated nanoparticles (IFN RM-NPs@TJ-5). The schematic diagram is shown in FIG. Figure 1 As shown in A.

[0038] Whole blood was collected from mice and transferred to a centrifuge tube containing sodium citrate-dextrose-sodium diphosphate anticoagulant at a volume ratio of 1:9. The whole blood was subjected to gradient centrifugation to obtain a platelet pellet containing a small number of red blood cells. This pellet was lysed with red blood cell lysis buffer at 4°C and centrifuged to obtain a resting platelet pellet. This pellet was placed in a -80°C freezer, allowed to completely freeze, and then removed from the freezer and allowed to thaw at room temperature. After five freeze-thaw cycles, the pellet was centrifuged at 10,000 rpm for 10 min to obtain platelet membranes (PMs). Stock solutions of PLGA (40 mg / mL, dissolved in DMSO), DSPE-PEG2000-OME (40 mg / mL, dissolved in DMSO), soy lecithin (40 mg / mL, dissolved in MeOH), and ticagrelor (TGL) (40 mg / mL, dissolved in DMSO) were prepared. The stock solutions of the nanomaterials were mixed at a volume ratio of 5:2:4:5 and added dropwise to stirred ultrapure water. After stirring for 5 minutes, the PLGA-NPs@TGL cores were collected. An appropriate amount of platelet membrane stock solution was taken and the probe was ultrasonicated in an ice bath (80 W, 3 min) to obtain platelet membrane vesicles. PLGA-NPs@TGL core (nanomaterial PLGA: membrane protein mass ratio is 1:1) was added and the probe was ultrasonicated in an ice bath (100 W, 5 min) to obtain platelet membrane-wrapped nanoparticles (PM-NPs@TGL). The schematic diagram is shown in FIG. Figure 1 As shown in B.

[0039] The particle size, potential, and polydispersity coefficient of the two nanoparticles were determined using a laser particle size analyzer, and their morphology was observed using transmission electron microscopy. The encapsulation efficiency and drug loading of IFN RM-NPs@TJ-5 were determined by high-performance liquid chromatography (HPLC) using a C18 column, a mobile phase of acetonitrile-water (35:65, v / v), and a detection wavelength of 264 nm. The encapsulation efficiency and drug loading of PM-NPs@TGL were determined by HPLC using a C18 column, a mobile phase of methanol-water (70:30, v / v), and a detection wavelength of 264 nm.

[0040] The results are as follows Figure 2 As shown in AC, the two membrane-wrapped nanoparticles have a membrane structure on the outside of the PLGA nanoparticles, the particle size is within 200 nm, the potential is about -20 mV, the distribution is relatively uniform, and the encapsulation efficiency and drug loading are good.

[0041] Example 2 Investigation of the distribution of double-membrane independently encapsulated nanoparticles in lupus erythematosus mice.

[0042] BALB / c mice aged 6-8 weeks were treated with 5% imiquimod cream (IMQ) (25 mg / mouse) applied to the right ear every two days for 4 weeks to induce systemic lupus erythematosus (SLE) mice. PLGA-NPs@DiR and IFN RM-NPs@DiR were injected into the tail vein. Healthy mice were also given IFN RM-NPs@DiR (ultrafiltration concentrated to a DiR equivalent of 125 μg / kg). Mice were sacrificed 4 and 12 hours after injection, and their organs were harvested. Fluorescence images of the mice's organs were captured, and fluorescence intensity was semi-quantitatively analyzed using the ROI tool.

[0043] The results are as follows Figure 3 A, In vivo imaging showed that IFN RM-NPs@DiR accumulated more strongly in the spleen and kidneys of inflammatory sites than PLGA-NPs@DiR 4 and 12 h after injection, while there was no obvious accumulation in the spleen and kidneys of healthy mice, demonstrating the accumulation ability of IFN RM-NPs@DiR in inflammatory sites.

[0044] Similarly, a lupus erythematosus mouse model was prepared according to the above method, and PLGA-NPs@DiR and PM-NPs@DiR were injected into the tail vein. At the same time, healthy mice were given PM-NPs@DiR (ultrafiltration and concentration to a DiR equivalent of 125 μg / kg). The mice were killed 12 h and 24 h after injection, respectively, and the organs were collected. The fluorescence intensity of the grind fluid of the mouse spleen and kidney tissue was detected by a microplate reader.

[0045] The results are as follows Figure 3 As shown in Figure 2, 12 h and 24 h after injection, the accumulation of PM-NPs@DiR in the spleen and kidneys of inflammatory sites was stronger than that of PLGA-NPs@DiR, while there was no obvious accumulation in the spleen and kidneys of healthy mice, demonstrating the accumulation ability of PM-NPs@DiR in inflammatory sites.

[0046] Example 3 Investigation of the in vitro targeting ability of double-membrane independently encapsulated nanoparticles.

[0047] Engineered macrophage membrane-encapsulated nanoparticles loaded with coumarin 6 (C6) (IFN RM-NPs@C6, concentrated by ultrafiltration to a PLGA equivalent of 0.5 mg / mL) were prepared. IFN RM-NPs@C6 were incubated with healthy RAW264.7 cells (H-RAW) and RAW264.7 cells with impaired efferocytosis (I-RAW). After 4 hours, the cells were harvested and washed three times with PBS. Flow cytometry was used to analyze the uptake of nanoparticles by macrophages. Figure 4 As shown in A, I-RAW has a higher uptake of IFN RM-NPs@C6, which is 1.2 times higher than that of H-RAW, proving that IFN RM-NPs@C6 has a stronger targeting effect on I-RAW cells with damaged efferocytosis than H-RAW.

[0048] Next, we investigated the targeting ability of the two nanoparticles on I-RAW cells damaged by cell burial. RAW264.7 cells were seeded at an appropriate density in a confocal microplate to construct damaged I-RAW cells. They were incubated with culture medium containing PLGA-NPs@C6 and IFN RM-NPs@C6 (ultrafiltration and concentration to a PLGA equivalent of 0.5 mg / mL), respectively. After 4 hours, the nanoparticles were removed, washed three times with PBS, and fixed with 4% paraformaldehyde. The intracellular fluorescence intensity was observed using a laser confocal microscope to investigate the targeting ability of the two nanoparticles on I-RAW cells. The results are shown in Figure 2. Figure 4 As shown in Figure 2B, the targeting ability of IFN RM-NPs@C6 to I-RAW is significantly stronger than that of PLGA-NPs@C6, reflecting its strong targeting and homing ability to damaged macrophages.

[0049] Furthermore, we investigated the targeting of PM-NPs@C6 to platelets. Fresh mouse platelets were extracted, and adenosine diphosphate was added to a final concentration of 50 μmol / L. Platelet activation was induced by shaking on a shaker for 15 minutes. PLGA-NPs@C6 and PM-NPs@C6 were added to both activated and unactivated platelets. After incubation for 30 minutes, the targeting of nanoparticles to platelets was detected by flow cytometry. The results are shown in Figure 2. Figure 4 As shown in CE, PM-NPs@C6 has a stronger targeting ability to activated platelets (act.) than PLGA-NPs@C6 ( Figure 4 C). It is worth noting that PM-NPs@C6 has a stronger targeting ability to activated platelets than to resting platelets (res. Figure 4 D), while there was no significant difference in the targeting ability of PLGA-NPs@C6 to resting and activated platelets ( Figure 4 E), demonstrating that PM-NPs@C6 possess optimal targeting ability for activated platelets. This dual-membrane independently encapsulated nanoparticle drug delivery system targets damaged macrophages and activated platelets, achieving precise and effective drug delivery.

[0050] Example 4 Engineered macrophage membrane-coated nanoparticles repair macrophage homeostasis in vitro.

[0051] First, the polarization of macrophages and the secretion of inflammatory factors were investigated. Macrophage RAW264.7 cells were seeded into 24-well plates. After the cells adhered to the wall, a medium containing 100 ng / mL lipopolysaccharide was added to stimulate the macrophages. After 12 hours, the medium was removed and a medium containing PLGA-NPs@TJ-5 and IFN RM-NPs@TJ-5 (containing 5 μg / mL TJ-5) was added. After 12 hours, the cells were collected, washed with PBS, and blocked with goat serum for 30 minutes. Then, they were incubated with CD206 antibody (1:200 dilution) for 1.5 hours at 4°C. After washing with PBS, the fluorescence intensity was detected by flow cytometry. The results are shown in Figure 2. Figure 5 As shown in A, the proportion of M2 anti-inflammatory phenotype of macrophages after treatment increased by nearly 3 times compared with the control group, showing a good anti-inflammatory effect overall.

[0052] Then, the secretion of inflammatory factors was investigated. Macrophages were seeded in 24-well plates. After the cells adhered to the wall, a medium containing 100 ng / mL LPS was added to stimulate the macrophages. After 12 hours, the medium was removed and a medium containing PLGA-NPs@TJ-5 and IFN RM-NPs@TJ-5 (containing 5 μg / mL TJ-5) was added. After 12 hours, the medium was collected and the content of the anti-inflammatory cytokine IL-10 in the medium was detected using an ELISA kit. The results are shown in Figure 2. Figure 5 As shown in Figure 2, the anti-inflammatory cytokine IL-10 was significantly upregulated in the culture medium, among which IFN RM-NPs@TJ-5 had the strongest upregulation ability, indicating that the use of engineered macrophage membrane-encapsulated drug-loaded nanoparticles can upregulate the level of anti-inflammatory cytokines by inhibiting the signal transduction of TLR7 to downstream, thereby inhibiting the inflammatory response involving macrophages.

[0053] Finally, the recovery of macrophage efferocytosis was investigated. Macrophage efferocytosis is mainly divided into three processes, namely the recognition, phagocytosis and processing of apoptotic cells by macrophages. Therefore, the level of efferocytosis recognition protein MFGE8 was first investigated. I-RAW was given a culture medium containing PLGA-NPs@TJ-5 and IFN RM-NPs@TJ-5 (containing 5 μg / mL TJ-5). The drug was withdrawn after 12 hours, and apoptotic thymocytes were added and incubated for 30 minutes to stimulate the occurrence of I-RAW efferocytosis. The cell culture medium was collected, centrifuged (3500rpm, 5 min) to remove the cells, and the content of efferocytosis-related protein MFGE8 in the culture medium was detected using an ELISA kit. The results are shown in Figure 2. Figure 5As shown in Figure C, the secretion of MFGE8 in the untreated I-RAW group (control group) was significantly lower than that in the healthy group. After administration of the two nanoparticles, the secretion of MFGE8 in both the I-RAW group and the healthy group was significantly increased. Due to the strong targeting of the engineered macrophage membrane-wrapped nanoparticles, the IFN RM-NPs@TJ-5 group had the strongest ability to increase the level of MFGE8, which was significantly higher than PLGA-NPs@TJ-5, demonstrating its strongest ability to repair macrophages from recognizing apoptotic cells.

[0054] Then, the phagocytic ability of macrophages for apoptotic cells was investigated. I-RAW was given a culture medium containing PLGA-NPs@TJ-5 and IFN RM-NPs@TJ-5 (containing 5 μg / mL TJ-5). The drug was withdrawn after 12 hours. The macrophages were stained with the live cell dye FITC-CFMDA (2μM) and then apoptotic thymocytes (DiD 5 μg / mL, staining for 30 min) were added and incubated for 30 minutes to stimulate the occurrence of I-RAW efferocytosis. The cells were collected and the phagocytosis of apoptotic cells by macrophages was detected by flow cytometry. The results are shown in the figure. Figure 5 As shown in Figure 3, the phagocytic ability of macrophages in the healthy group was the strongest, which was nearly 2.3 times higher than that in the control group. After administration of the two nanoparticles, the phagocytic ratio of macrophages increased, among which the IFN RM-NPs@TJ-5 group had the highest phagocytic ratio, which was significantly stronger than that of the PLGA-NPs@TJ-5 group, showing the strongest effect of improving the phagocytic ability of macrophages.

[0055] Finally, we investigated the acidification and maturation of ectopic bodies. Coverslips were placed on the bottom of 12-well plates, and I-RAW cells were seeded on the coverslips. Culture media containing PLGA-NPs@TJ-5 and IFN RM-NPs@TJ-5 (5 μg / mL TJ-5) were then administered. After 12 hours, the drugs were withdrawn, and apoptotic thymocytes were added for 30 minutes to stimulate I-RAW ectopic body formation and ectopic body formation. Apoptotic cells were then removed, washed three times with PBS, and incubated in serum-free DMEM for 10 minutes. The cells were then stained with the lysosomal probe LysoTracker Red (DND-99) for 10 minutes. Following paraformaldehyde fixation, nuclei were stained with 5 μg / mL DAPI solution. After washing with PBS and mounting, the slides were mounted and fluorescence intensity was observed under a confocal microscope to assess the degree of acidification of I-RAW ectopic bodies. Next, I-RAW was given a culture medium containing PLGA-NPs@TJ-5 and IFN RM-NPs@TJ-5 (containing 5 μg / mL TJ-5). The drug was withdrawn after 12 hours, and apoptotic thymocytes were added for 30 minutes to stimulate the occurrence of I-RAW burial and the formation of burial bodies. Apoptotic cells were removed, washed three times with PBS, fixed with 4% paraformaldehyde for 10 minutes, washed with PBS, permeabilized with 0.1% Triton X-100 for 10 minutes, blocked with goat serum for 30 minutes, and then APC-anti-mouse CD107a antibody (1:200 dilution) was added. The cells were incubated at 4°C for 1.5 hours, washed three times with PBS, and the fluorescence intensity was detected by flow cytometry to examine the maturity of burial bodies. The results are shown in Figure 2. Figure 5 As shown in E, the acidification degree of I-RAW corpora in the untreated group was the lowest, and the acidification degree increased significantly after the drug administration. Among them, the IFN RM-NPs@TJ-5 group had the highest acidification degree, which was significantly stronger than the PLGA-NPs@TJ-5 group. The expression level of LAMP-1 can determine the degree of maturation of macrophage corpora. The results show that the maturation of I-RAW corpora was inhibited after modeling ( Figure 5 F), there was a significant recovery after administration, among which the IFN RM-NPs@TJ-5 group had the strongest ability to relieve the inhibition of efferosome maturation, which was stronger than the PLGA-NPs@TJ-5 group, repairing the damaged effect of macrophage efferosome from the perspective of macrophages' ability to process apoptotic cells.

[0056] Example 5: Resting platelet membrane-coated nanoparticles inhibit platelet activation and aggregation in vitro.

[0057] Platelets were resuspended in PBS containing the fluorescent dye DiI (5 μg / mL) and incubated at 37°C for 25 minutes. After washing three times with PBS, DiI-labeled platelets were obtained. Labeled platelets were incubated with a PBS solution containing PLGA-NPs@TGL and PM-NPs@TGL (containing 100 μg / mL ticagrelor) at 37°C for 30 minutes. Adenosine diphosphate was added to a final concentration of 50 μmol / L, and the platelets were shaken on a shaker for 15 minutes to induce platelet activation and aggregation. The sample was then dripped onto a glass slide, sealed, and platelet aggregation observed under a confocal microscope.

[0058] The results are as follows Figure 6 As shown in the results, PM-NPs@TGL has the strongest inhibitory ability on activated platelet aggregation, which is significantly stronger than the PLGA-NPs@TGL group, and exhibits a good anti-platelet activation and aggregation effect in vitro.

[0059] Example 6 Double-membrane independently encapsulated nano drug delivery system alleviates systemic lupus erythematosus in mice.

[0060] A systemic lupus erythematosus (SLE) mouse model was established by smearing the right ear of mice with IMQ. Starting from the 4th week, normal saline (normal saline group), IFN RM-NPs@TJ-5 (TJ-5 equivalent to 5 mg / kg), PM-NPs@TGL (ticagrelor equivalent to 10 mg / kg), and IFN RM-NPs@TJ-5+PM-NPs@TGL (double-membrane independently encapsulated nanodrug delivery system group, i.e., combination group, TJ-5 equivalent to 5 mg / kg, ticagrelor equivalent to 10 mg / kg) were injected into the tail vein. The drugs were administered twice a week for a total of 4 weeks. After the urine protein was detected, the mice were killed, and the kidneys, spleen, thymus, and plasma of the mice were collected for the detection of subsequent indicators.

[0061] After whole blood was centrifuged (3500 rpm, 15 min), plasma was collected and the anti-dsDNA Ab content of lupus erythematosus related indicators in mouse plasma was detected. Figure 7 As shown in A, the anti-dsDNA Ab content in the plasma of mice in the normal saline group increased significantly, and the level decreased after drug administration. The combination group showed the best therapeutic effect, which was better than the single drug group. The spleen and thymus of the mice were taken, the spleen was weighed, and the spleen weight index (%) of the mice was calculated. The results are shown in Figure 7As shown in B, the spleen of the modeling mice was obviously enlarged, and the spleen weight index of the saline group was nearly 6 times higher than that of the healthy group. After administration, the spleen weight index decreased significantly, and the combination group decreased the most, proving that the combination group had the best therapeutic effect on reducing the enlargement of the spleen. Then, the thymus of the mice was ground to obtain a thymus single-cell suspension. After lysis by red blood cell lysis solution for 10 min and blocking with goat serum for 30 min, the thymus single-cell suspension was incubated with PBS containing FITC anti-mouse CD3 antibody (1:200 dilution) and PC5.5 anti-mouse CD4 antibody (1:200 dilution) for 1.5 h. After removing the antibodies, the thymus single-cell suspension was washed with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and washed with PBS three times. Then, the thymus single-cell suspension was blocked with goat serum for 30 min, and then incubated with PBS containing PE anti-mouse Foxp3 antibody (1:200 dilution) for 1.5 h. After washing, the Treg level in the thymus of the mice was detected by flow cytometry to determine the activation state of the immune system. The results are shown in Figure 7 As shown in C, the Treg cell level of the saline group was generally low, and the level increased after administration. The Treg level recovered to a level similar to that of the healthy group in the IFN RM-NPs@TJ-5 group, which was stronger than that in the PM-NPs@TGL group. The Treg cell level in the combination group was higher than that in the healthy group, which showed that the combination group had a stronger ability to suppress the activation of the immune system.

[0062] Finally, the kidney damage of the lupus mice was investigated. The kidneys of the mice were subjected to immunofluorescence staining to investigate the deposition of IgG antibodies and C3 complements in the kidneys. The results are shown in Figure 7 As shown in D, the fluorescence semi-quantitative results showed that almost no deposition of IgG and C3 was found in the kidneys of the healthy mice, and the deposition in the kidneys of the lupus mice increased significantly after modeling. After administration, the deposition decreased, and the deposition of immune complexes in the combination group was the lowest compared with the saline group and the single-drug group, which was significantly better than that in the single-drug group, proving that the combination strategy inhibited the deposition of immune complexes and achieved a therapeutic effect on the kidney damage of the lupus mice.

[0063] Example 7 Treatment of the “macrophage-platelet” vicious cycle of lupus mice by the double-membrane independent nano drug delivery system

[0064] After the experiment, the spleen of the mice was removed and ground to obtain a spleen single-cell suspension. The cells were lysed with red blood cell lysis buffer for 10 min and blocked with goat serum for 30 min. A portion of the cells was incubated with PBS containing FITC anti-mouse F4 / 80 antibody (1:200 dilution), PE anti-mouse CD11b antibody (1:200 dilution), and APC anti-mouse CD206 antibody (1:200 dilution) for 1.5 h to label the macrophages in the spleen; another portion of the cells was incubated with PBS containing FITC anti-mouse CD41 antibody (1:200 dilution) for 1.5 h to label the platelets in the spleen. After removing the antibodies and washing with PBS, the macrophage infiltration and platelet levels in the mouse spleen were detected by flow cytometry.

[0065] The results are as follows Figure 8 As shown in A, the level of macrophage infiltration in the saline group was nearly 2 times higher than that in the healthy group. After administration, the infiltration level decreased. The combined group recovered to a level similar to that of the healthy group, and the effect was stronger than that of the single drug group. Afterwards, we examined the phenotype of macrophages infiltrating into the spleen, using CD86 antibodies to label M1 macrophages and flow cytometry to detect macrophage phenotypes. The results are shown in Figure 8 As shown in B, the macrophages infiltrating into the spleen showed an increase in the proportion of M1 pro-inflammatory phenotype, which was nearly 4 times higher than that of the healthy group. After administration, the proportion of M1 decreased. The combination group repaired the platelet homeostasis while repairing the macrophages, showing an anti-inflammatory effect better than the single-drug group. Next, we examined the platelet level in the spleen of mice, and the results were as follows Figure 8 As shown in CD, compared to the healthy group, mice in the saline group showed significant thrombocytopenia. Platelet levels recovered significantly after drug administration, with the combination group showing the strongest repair ability, significantly better than the single-drug group. Similarly, platelet levels in the mouse blood also recovered significantly, with the combination group demonstrating superior platelet repair capabilities to the single-drug group.

[0066] Then, immunofluorescence staining was performed on the kidneys of mice to investigate the deposition of platelet-associated antigen PF4 in the kidneys of lupus mice. Figure 8 As shown in E, almost no PF4 deposition was found in the kidneys of healthy mice. After modeling, the amount of PF4 deposition in the kidneys of lupus mice increased significantly. After drug administration, the deposition amount decreased. The fluorescence semi-quantitative results showed that the combination group had the lowest deposition compared with the normal saline group and the single-drug group, which was significantly better than the single-drug group.

[0067] In summary, the double-membrane independently encapsulated nanodrug delivery system effectively alleviates the disease progression of lupus erythematosus in mice by repairing macrophage and platelet homeostasis, and its therapeutic effect is better than that of single-membrane encapsulated nanoparticles.

Claims

1. A double-membrane independently encapsulated nano drug delivery system for regulating immunity, characterized in that: The double-membrane independently wrapped nano drug delivery system includes macrophage membrane wrapped drug-loaded nanoparticles RM-NPs and platelet membrane wrapped drug-loaded nanoparticles PM-NPs; the macrophage membrane is the cell membrane of activated macrophages obtained by cytokine stimulation, and the macrophages include at least one of mononuclear macrophage leukemia cells and bone marrow-derived macrophages; the drug loaded on the RM-NPs includes a TLR7 inhibitor; the drug loaded on the PM-NPs includes a P2Y 12 Inhibitor.

2. The nano drug delivery system according to claim 1, characterized in that The mononuclear macrophage leukemia cells include THP-1 or RAW264.7, and the cytokines include interferon-γ (IFN-γ), tumor necrosis factor-α or interleukin-10; preferably, the cytokine is IFN-γ.

3. The nano drug delivery system according to claim 1 or 2, characterized in that: The platelet membrane is derived from at least one of resting platelets, activated platelets, and activated platelets stimulated by cytokines, wherein the cytokines include platelet-derived growth factors or chemokines; preferably, the platelet membrane is a resting platelet membrane; and the drug-loaded nanoparticles include nanomaterials.

4. The nano drug delivery system according to claim 3, characterized in that The nanomaterials include polymers, phospholipids and PEGylated lipids that can encapsulate drugs.

5. The nano drug delivery system according to claim 4, characterized in that The mass ratio of the protein to the polymer in the macrophage membrane or platelet membrane is (1:0.5)-(1:10).

6. The nano drug delivery system according to claim 4, characterized in that The polymer is at least one of polylactic acid-co-glycolic acid, polylactic acid, polyhydroxyalkanoate and polycaprolactone; the phospholipid includes at least one of lecithin, phosphatidylserine and choline phospholipid; the PEGylated lipid includes at least one of DSPE-PEG, DOPE-PEG and DMPE-PEG.

7. The nano drug delivery system according to claim 1, characterized in that The TLR7 inhibitor includes at least one of TJ-M2010-5, hydroxychloroquine, Enpatoran, and IMO-8400; preferably, the TLR7 inhibitor is TJ-M2010-5.

8. The nano drug delivery system according to claim 1, characterized in that The P2Y 12 The inhibitor includes at least one of ticagrelor, clopidogrel, prasugrel, and cangrelor; preferably, the P2Y 12 The inhibitor is ticagrelor.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nano drug delivery system according to any one of claims 1 to 8.

10. Use of a double-membrane independently encapsulated nano drug delivery system for regulating immunity according to any one of claims 1 to 8 in the preparation of drugs for treating autoimmune diseases, characterized in that: The autoimmune disease is systemic lupus erythematosus.

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