Preparation and application of nanoparticles for enhancing cell burial function of macrophages

Abstract

The invention discloses preparation and application of macrophage targeting nanoparticles entrapped with a cell burial function accelerant. The medicine for enhancing the intercellular function of the macrophages is a liver X receptor stimulant, and the liver X receptor stimulant comprises at least one of T0901317, GW3965, Ouabagenin and LXR-623. The surface of the targeting nanoparticle is coated with a macrophage cell membrane, and meanwhile, a urokinase type plasminogen activator receptor (uPAR) targeting ligand is modified. The nanoparticle can be efficiently accumulated at an inflammatory focus part by utilizing a macrophage cell membrane, and the modified targeting ligand is used for targeting the macrophage with damaged cell burial function, so that the cell burial function of the macrophage is enhanced, the polarization of the macrophage to an anti-inflammatory phenotype is promoted, the release of anti-inflammatory and proinflammatory cytokines is regulated, and the anti-inflammatory effect of the nanoparticle is improved. And finally, the inflammatory microenvironment of a focus part is regulated and controlled, so that a good effect of treating autoimmune diseases is achieved.

Description

Technical Field

[0001] This invention relates to the preparation and application of actively targeted nanoparticles that enhance macrophage cell burial function. Specifically, it relates to uPAR-targeting ligand-modified macrophage cell membrane-encapsulated nanoparticles carrying a liver X receptor agonist and their applications, belonging to the field of pharmaceutical technology. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cytokine burial refers to the non-inflammatory process by which macrophages clear apoptotic cells from their environment, a process that maintains homeostasis. When macrophage burial function is impaired, apoptotic cells that are not cleared in time release substances that can trigger an autoimmune response, subsequently activating the immune response and ultimately leading to autoimmune diseases. Macrophages with impaired burial function also promote the release of various pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), thereby significantly exacerbating disease progression.

[0004] Macrophages primarily recognize apoptotic cells via the phagocytic receptor Mer proto-oncogene tyrosine kinase (Mer). Mer uses Gas6 and protein S as bridging ligands to bind to the "eat me" signal phosphatidylserine on the apoptotic cell membrane; therefore, macrophage necrolysis function depends on the Mer expressed on its surface. Furthermore, after necrolysis, macrophages further polarize to the anti-inflammatory M2 phenotype and promote the secretion of anti-inflammatory cytokines. Therefore, upregulating Mer expression to enhance necrolysis can both clear cellular debris from the lesion environment and regulate the release of inflammation-related cytokines, thereby avoiding immune responses and modulating the inflammatory microenvironment. Precise drug delivery to macrophages with impaired necrolysis is crucial for ensuring therapeutic efficacy. Macrophage cell membrane encapsulation gives nanoparticles a homing effect, allowing them to effectively accumulate at inflammatory sites rich in macrophages. Moreover, uPAR is negatively correlated with necrolysis capacity; nanoparticles modified with uPAR targeting ligands can precisely target macrophages with impaired necrolysis. Summary of the Invention

[0005] To address the current challenge of treating autoimmune diseases, this invention creatively utilizes targeted nanomaterials to encapsulate macrophage burial promoters. By enhancing the burial capacity of damaged macrophages, it clears a large number of antigen complexes and cellular debris from the lesion microenvironment, preventing the activation of the immune response. Furthermore, after burial, macrophages polarize into M2-type macrophages, inhibiting the release of pro-inflammatory cytokines. Building upon this, nanoparticles are modified with macrophage cell membranes and uPAR-targeting ligands, endowing them with the ability to target damaged macrophages, enhancing drug accumulation in the lesion, and significantly improving the therapeutic effect on autoimmune diseases.

[0006] One of the objectives of this invention is to overcome the shortcomings of existing treatment methods and provide a nanoparticle for actively targeting the treatment of autoimmune diseases. This nanoparticle can clear apoptotic cell debris that causes an immune response in the lesion environment, regulate macrophage phenotype, regulate the release of cytokines, and improve the efficacy of anti-autoimmune disease treatment.

[0007] Another object of the present invention is to provide the application of nanoparticles that enhance macrophage cytotoxicity in the fight against autoimmune diseases.

[0008] Another objective of this invention is to overcome the problem of insufficient accumulation of free drugs in lesions by preparing nanoparticles that simultaneously modify macrophage cell membranes and targeting ligands, thereby achieving targeted drug delivery to damaged macrophages.

[0009] The present invention is achieved through the following technical solution: providing a nanoparticle that enhances the cytotoxicity of macrophages and its application, including a nanocarrier material, a macrophage cell membrane, a targeting ligand, and a liver X receptor agonist as an active ingredient.

[0010] The nanocarrier material is a polymer capable of encapsulating drugs, and is at least one of polylactic acid-glycolic acid copolymer, polylactic acid, and polylactic-glycolic acid; preferably, the nanocarrier material is polylactic acid-glycolic acid copolymer.

[0011] The macrophage membrane is obtained from at least one of mouse monocytic leukemia cells (RAW 264.7), bone marrow-derived macrophages (BMDM), human monocytic leukemia cells (THP-1), and activated macrophages stimulated by various cytokines; preferably, the cytokines are interleukin-4 (IL-4) and lipopolysaccharide (LPS).

[0012] The targeted ligand can bind uPAR and is at least one of AE105 (D-Cha-FsrYLWS), AE120 ((D-Cha-FsrYLWS)2-βA-K), and U11 (VSNKYFSNIHW); preferably, the targeted ligand is AE105 (D-Cha-FsrYLWS).

[0013] The macrophage burial function promoter is a liver X receptor agonist, and is at least one of T0901317, GW3965, Ouabagenin, and LXR-623, preferably T0901317.

[0014] The ligand-modified drug-loaded nanoparticles containing T0901317 are preferably prepared by the following method: (1) N Distearate phosphatidyl acetamide polyethylene glycol active ester was dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of AE105 peptide was added dropwise with stirring. After reacting at room temperature for 24 hours, unreacted AE105 peptide was removed by dialyzing with deionized water for 48 hours. The nanomaterial modified with AE105 peptide was obtained by freeze drying.

[0015] (2) Polylactic acid-glycolic acid and T0901317 were dissolved in dimethyl sulfoxide and added dropwise to deionized water under stirring to obtain drug-loaded nanomaterials. Then, the AE105 peptide-modified nanomaterials prepared in (1) were mixed with macrophage membranes and physically extruded through a 400 nm polycarbonate membrane to obtain AE105 peptide-modified cell membrane vesicles. The obtained cell vesicles and drug-loaded nanomaterials were combined under ultrasound to prepare macrophage cell membrane-encapsulated, AE105 peptide-modified T0901317-loaded nanoparticles.

[0016] The aforementioned anti-autoimmune disease drugs can be further formulated into various forms such as injections, and drugs of various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0017] The T0901319 nanoparticles, which are encapsulated in macrophage cell membranes and modified with AE105 peptide, provided by this invention accumulate in lesion skin tissue, upregulate the expression of Mer on the surface of macrophages to enhance cell burial function, and in addition, macrophages polarize to the anti-inflammatory M2 phenotype, inhibit the release of pro-inflammatory cytokines, regulate the inflammatory microenvironment of the disease, thereby treating autoimmune diseases.

[0018] The beneficial effects of this invention are: This invention provides a method for enhancing macrophage necrolysis function to treat autoimmune diseases. It involves upregulating Mer expression on the macrophage surface using a liver X receptor agonist, thereby enhancing the macrophage's ability to clear apoptotic debris and preventing inflammatory responses. The invention provides AE105 peptide and macrophage cell membrane-modified drug-loaded nanoparticles. The macrophage cell membrane encapsulation enables the nanoparticles to exhibit a homing effect, allowing them to effectively accumulate at inflammatory sites. Simultaneously, the AE105 peptide modification allows the nanoparticles to precisely target macrophages with impaired necrolysis function, improving the therapeutic effect on autoimmune diseases. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the preparation of ligand-modified drug-loaded nanoparticles.

[0020] Figure 2 Characterization diagram of ligand-modified drug-loaded nanoparticles.

[0021] Figure 3 Diagram illustrating the construction of an in vitro model of damaged macrophages.

[0022] Figure 4 The distribution of ligand-modified nanoparticles in psoriasis mice and their in vitro targeting ability are shown in the figure.

[0023] Figure 5 A diagram illustrating the effect of ligand-modified drug-loaded nanoparticles on enhancing the cytotoxicity of macrophages.

[0024] Figure 6 A diagram illustrating the effect of ligand-modified drug-loaded nanoparticles on the regulation of macrophage cytoplasm.

[0025] Figure 7 The therapeutic effect of ligand-modified drug-loaded nanoparticles on psoriasis mice.

[0026] Figure 8 Image showing the effect of ligand-modified drug-loaded nanoparticles on the lesion environment of psoriasis mice. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. However, those skilled in the art should understand that the implementation of the present invention is not limited to the scope of the described embodiments.

[0028] Example 1: Synthesis and characterization of ligand-modified DSPE-PEG.

[0029] Will N -Distearate phosphatidyl acetamide polyethylene glycol (DSPE-PEG) 2000-NHS) was dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of AE105 peptide was added dropwise with stirring (molar ratio of DSPE-PEG). 2000 -NHS : AE105 = 1:1). A small amount of triethylamine was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was then placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed with deionized water for 48 hours to remove unreacted peptides. Finally, lyophilization yielded DSPE-PEG. 2000 -AE105, and through 1 Its structure was characterized by H NMR and FT-IR.

[0030] The schematic diagram is shown in Figure 1A, and the result is as follows: Figure 2 As shown in A, DSPE-PEG 2000 -NHS showed a characteristic peak at 2.8 ppm; upon reaction with AE105 peptide, DSPE-PEG 2000- The disappearance of the NHS peak and the appearance of the characteristic peak of AE105 in AE105 prove that the AE105 peptide has been successfully ligated to DSPE-PEG. 2000 -NHS. (For example) Figure 2 As shown in B, in DSPE-PEG 2000 In the infrared spectrum of -AE105, 3306 cm⁻¹ -1 and 1650 cm -1 DSPE-PEG appears at wavelength 2000 The presence of a characteristic absorption peak of the AE105 peptide not found in the NHS spectrum indicates that the AE105 ligation was successful.

[0031] Example 2: Preparation and characterization of ligand-modified drug-loaded nanoparticles.

[0032] RAW 264.7 cells were cultured to a specific density, collected using a cell scraper, washed with phosphate-buffered saline (pH 7.4), and centrifuged at 1000 g. The cell pellet was then resuspended in a lysis buffer containing 20 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 7.5), 25 mM sucrose, 10 mM potassium chloride, 0.2 mM ethylenediaminetetraacetic acid (EDTA), a mixture of protease inhibitors, and a phosphatase inhibitor. The cells were homogenized 50 times using a homogenizer to disrupt the cell structure. The homogenate was then centrifuged at 3000 rpm for 10 minutes, and the supernatant was stored at 4°C. The pellet was resuspended, and the process was repeated 50 times, followed by centrifugation and collection of the supernatant. The combined supernatant was then ultracentrifuged at 100,000 g for 60 minutes. The resulting pellet was resuspended in a solution containing protease inhibitors, phosphatase inhibitors, and 0.2 mM EDTA to obtain the macrophage cell membrane stock solution, which was stored at -20°C.

[0033] Nanoparticles loaded with T0901317 (abbreviated as T317) were prepared (PEG-NP@T317: T0901317-loaded nanoparticles without macrophage cell membrane encapsulation and AE105 peptide modification; M-NP@T317: T0901317-loaded nanoparticles only encapsulated by macrophage cell membrane; AM-NP@T317: T0901317-loaded nanoparticles encapsulated by macrophage cell membrane and modified with AE105 peptide). First, polylactic acid-glycolic acid copolymer (PLGA) loaded with T0901317 was prepared by nanoprecipitation. A dimethyl sulfoxide solution containing 2.0 mg PLGA and 0.5 mg T0901317 was slowly added dropwise to 4 mL of deionized water under stirring. Then, the organic solvent and unloaded drug were removed by centrifugation at 4500 rpm for 20 minutes using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. For AM-NP@T317, the pre-prepared membrane material was combined with DSPE-PEG. 2000 -AE105 was mixed and physically extruded through a 400 nm polycarbonate membrane to prepare AE105 peptide-modified macrophage cell membrane vesicles. The resulting mixture was then combined with PLGA loaded with T0901317 (the mass ratio of membrane protein to PLGA in the mixture was 1:4), and the mixture was then used to modify the PLGA surface using an ultrasonic probe. For M-NP@T317, DSPE-PEG was used... 2000 -AE105 is replaced with distearylphosphatidylacetamide polyethylene glycol-hydroxysuccinimide (DSPE-PEG) 2000 -OME), prepared using the same method. For PEG-NP@T317, 2.0 mg PLGA, 0.5 mg T0901317, and 0.8 mg DSPE-PEG were added under stirring. 2000 A solution of -OME and 0.2 mg phospholipid in dimethyl sulfoxide was slowly added dropwise to 4.0 mL of deionized water. Finally, the three solutions were concentrated by ultrafiltration at 4500 rpm for 20 minutes using a 30 kDa molecular weight cutoff tube. The organic solvent was removed by washing with deionized water, yielding AM-NP@T317, M-NP@T317, and PEG-NP@T317 nanoparticles, respectively. (See schematic diagram below.) Figure 1 As shown in B.

[0034] The particle size and zeta potential of AM-NP@T317 were measured using a Malvern laser particle size analyzer, and the morphology was observed using a transmission electron microscope. High-performance liquid chromatography (HPLC) was used to investigate the encapsulation efficiency and drug loading of AM-NP@T317. A C18 column was used, the mobile phase was acetonitrile-water (60:40, v / v), and the detection wavelength was 220 nm.

[0035] The results are as follows Figure 2As shown in CD, AM-NP@T317 is spherical with a core-shell structure, a particle size of about 115 nm, a potential of about -20 mV, and a particle size PDI of less than 0.3, indicating that the distribution of nanoparticles is relatively uniform.

[0036] Example 3: Construction of an in vitro model of damaged macrophages.

[0037] To construct impaired macrophages, RAW 264.7 cells were first cultured in cell culture dishes. When the cells reached a certain density, different concentrations of BMS777607 were added for treatment, with blank culture medium as a control. Cells were collected after 12 hours.

[0038] Western blotting was used to investigate Mer expression on the surface of macrophages. Collected cell samples were lysed for 10 minutes at 4°C with RIPA buffer containing a protease inhibitor. The lysate was then sonicated for 5 minutes, centrifuged at 10,000 rpm for 10 minutes, and the cell supernatant was collected. Protein quantification was performed using BCA analysis. Samples of equal protein concentration were mixed with loading buffer and heated at 95°C for 10 minutes. The prepared samples were loaded into gel wells and separated by constant voltage electrophoresis. Proteins were transferred from the gel to a polyvinylidene fluoride membrane using a constant current. The membrane was blocked with protein-free blocking buffer and incubated overnight at 4°C with anti-GAPDH and anti-Mer antibodies. The next day, the membrane was incubated with horseradish peroxidase-labeled anti-rabbit IgG. Finally, protein bands were imaged using a chemiluminescence detection system. Flow cytometry was used to detect uPAR expression on the surface of macrophages. The collected cell samples were blocked with CD16 / 32 at 4°C for 30 minutes, then centrifuged at 4500 rpm for 5 minutes to collect the cells, and then incubated with anti-uPAR antibody at 4°C for 2 hours. Finally, the cells were collected for flow cytometry analysis.

[0039] The safety of BMS777607 for RAW 264.7 cells was assessed using the tetrazolium blue assay. RAW 264.7 cells were seeded at a specific density in 96-well plates. After cell attachment, different concentrations of BMS777607 were added for 12 hours. After treatment, the culture medium was discarded, cells were washed with PBS, and medium containing 0.5 mg / mL tetrazolium blue was added to each well. After incubation for 4 hours, the medium was removed, and 200 μL of dimethyl sulfoxide was added to dissolve the formazan crystals. Finally, the absorbance of each well was measured at 490 nm, with untreated cells serving as a control.

[0040] Macrophage phagocytic function was assessed by flow cytometry. RAW 264.7 cells were cultured in 24-well plates and treated with 7 μg / mL BMS777607 for 12 hours, with a blank culture medium group serving as a control. Macrophages were labeled with Cell Tracker Green antibody. Human keratinocytes were incubated with 600 μM hydrogen peroxide for 12 hours as apoptotic cells and labeled with DiD. Macrophages and apoptotic cells were mixed at a 1:1 ratio and incubated at 37°C for 3 hours. The phagocytic index (%) was determined by flow cytometry using the formula: Phagocytic index (%) = (double-positive cells) / (double-positive cells + single-positive cells) × 100%. Finally, the morphology of the model cells after treatment with 7 μg / mL BMS777607 for 12 hours was observed by scanning electron microscopy.

[0041] The results are as follows Figure 3 After 12 hours of treatment with BMS777607, AC and RAW 264.7 cells showed decreased Mer expression and increased uPAR expression in a concentration-dependent manner. Cell safety assays determined that a concentration of 7 μg / mL was non-toxic to cells; therefore, macrophages treated with 7 μg / mL BMS777607 were used as a model of macrophages with impaired cell endocytosis. Figure 3 In DE, the ability of macrophages with impaired cell burial after modeling to phagocytose apoptotic cells was significantly reduced compared to normal macrophages. Simultaneously, the morphology of macrophages after modeling changed from spherical and semi-adherent to spindle-shaped.

[0042] Example 4: Distribution of ligand-modified nanoparticles in psoriasis mice and in vitro targeting ability.

[0043] Hair was removed from the back of 6-8 week old female BALB / c mice (3 cm × 5 cm area), and 62.5 g of 5% imiquimod cream was applied to the treated area to induce a psoriasis mouse model. Nanoparticle accumulation was monitored using fluorescence imaging. Mice were randomly divided into three groups (n=4) and injected with different DiR-loaded nanoparticles (PEG-NP@DiR, M-NP@DiR, and AM-NP@DiR) (DiR equivalent 1 mg / kg). Fluorescence signals on the backs of mice were captured at 2, 6, 12, and 24 hours post-injection, and semi-quantitative analysis of the fluorescence signals in each group was performed using a ROI tool.

[0044] The results are as follows Figure 4As shown in Figures AB, real-time in vivo imaging revealed that AM-NP@DiR accumulated the most in the lesioned skin at all time points. Semi-quantitative analysis confirmed that AM-NP@DiR significantly increased its accumulation in the lesioned skin compared to M-NP@DiR and PEG-NP@DiR. Furthermore, both M-NP@DiR and AM-NP@DiR are encapsulated by macrophage membranes, exhibiting a homing effect and being recruited in large quantities to macrophage-rich lesioned skin. AM-NP@DiR is modified with the AE105 peptide, thus explaining the difference in accumulation between M-NP@DiR and AM-NP@DiR at the lesion site.

[0045] Furthermore, we validated the in vitro targeting ability of the nanoparticles through cellular uptake experiments and explored the targeting mechanism. RAW 264.7 cells were treated with 7 μg / mL BMS777607 for 12 hours to obtain a macrophage model with impaired cell burial; untreated RAW 264.7 cells served as the control group. Coumarin 6-labeled nanoparticles AM-NP@C6 and M-NP@C6 (final C6 concentration 5 μg / mL) were prepared simultaneously. Macrophages and model macrophages were incubated with AM-NP@C6 at 37°C for 3 hours, followed by centrifugation at 3500 rpm for 15 minutes to collect cells, which were then analyzed by flow cytometry. To further explore the targeting mechanism, model macrophages were incubated with AM-NP@C6 and M-NP@C6 for 3 hours each. In addition, the model macrophages were pre-incubated with free AE105 peptide at 37°C for 30 minutes, and then incubated with AM-NP@C6 for 30 minutes. Finally, the macrophages were collected and analyzed by flow cytometry.

[0046] The results are as follows Figure 4 As shown in Figure C, compared with macrophages, model macrophages exhibited enhanced uptake of AM-NP@C6, which was due to a significant increase in uPAR expression in model macrophages. Figure 4 As shown in Figure D, AM-NP@C6 is more readily taken up by modeling macrophages than M-NP@C6. However, pretreatment with free AE105 peptide significantly inhibited the uptake of AM-NP@C6, indicating that free AE105 peptide can competitively bind to uPAR. In summary, AM-NP accumulates in psoriatic skin using cell membrane homing effects and the specific targeting ability of AE105, and is specifically recognized and taken up by cell-damaged macrophages.

[0047] Example 5: Ligand-modified drug-loaded nanoparticles enhance macrophage burial ability.

[0048] The ability of AM-NP@T317 to regulate cell necrosis was assessed by examining Mer expression on macrophages and the macrophages' ability to phagocytose apoptotic cells. Western blotting was used to examine Mer expression on the macrophage surface, and flow cytometry was used to detect the macrophages' ability to phagocytose apoptotic cells. RAW 264.7 cells were cultured in 24-well plates and treated with 7 μg / mL BMS777607 for 12 hours, with a blank medium group serving as a control. Subsequently, cells were incubated with T0901317, M-NP@T317, and AM-NP@T317 for 24 hours, respectively. Finally, cells were collected for Western blotting and flow cytometry analysis.

[0049] The results are as follows Figure 5 As shown in Figure A, Western blot analysis indicates that AM-NP@T317 can effectively enhance the expression of Mer on the surface of macrophages. Figure 5 As shown in BC, the flow cytometry results of the AM-NP@T317 treatment group showed a significantly increased proportion of double positive cells compared to the model macrophages, indicating that upregulating macrophage Mer expression can enhance the ability of macrophages to phagocytose apoptotic cells.

[0050] Example 6: Ligand-modified drug-loaded nanoparticles modulate macrophage cytotoxicity.

[0051] To assess the acidification of macrophage necrosomes, RAW 264.7 cells were seeded onto coverslips and treated with 7 μg / mL BMS777607 for 12 hours, with a blank medium treatment as a control. Cells were then treated with T0901317, M-NP@T317, and AM-NP@T317 for 24 hours, respectively. Subsequently, medium containing apoptotic cells was added to stimulate macrophage necrosome formation. This process was performed at 37°C and 60 rpm in a shaker. After 30 minutes, unphagocytosed apoptotic cells were washed away. Macrophages were then collected and incubated with 75 μM Lysotracker Red for 10 minutes, followed by fixation with 4% paraformaldehyde for 10 minutes, staining with 5 μg / mL DAPI for 5 minutes, and finally observed using a laser confocal microscope.

[0052] To assess the maturation of macrophage cytoplasm, macrophages treated in the same manner as described above were collected, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton, and then blocked with anti-mouse CD16 / 32 antibody for 30 minutes. Subsequently, the cells were incubated with APC-labeled anti-mouse CD107a antibody at 4°C for 1 hour to label lysosome-associated membrane protein 1 (LAMP-1). Finally, flow cytometry was used for analysis.

[0053] The results are as follows Figure 6As shown in Figure A, flow cytometry analysis revealed that LAMP-1 expression was significantly downregulated in model macrophages compared to macrophages, while AM-NP@T317 significantly upregulated LAMP-1 expression in model macrophages. Figure 6 As shown in BC, in confocal imaging and semi-quantitative analysis of cytoplasmic acidification, the fluorescence intensity of the AM-NP@T317 group was significantly increased compared with that of the model macrophage group, indicating that AM-NP@T317 enhanced the maturation of cytoplasmic cells and upregulated the acidification level of cytoplasmic cells.

[0054] Example 7: The therapeutic effect of ligand-modified drug-loaded nanoparticles on psoriasis mice.

[0055] Female BALB / c mice aged 6-8 weeks were randomly divided into groups. Hair was removed from a 3 cm × 5 cm area of ​​skin on the back, recorded as day 0. From day 1 to day 7, healthy control mice received 62.5 mg of petrolatum cream daily at the hair removal site, while psoriasis model mice received 62.5 mg of 5% imiquimod cream daily at the hair removal site. On days 3, 5, and 7, the following treatments were administered: healthy mice received no other treatments, while psoriasis mice in each group received a tail vein injection of the following drugs: saline, PEG-NP@T317, M-NP@T317, or AM-NP@T317. Mouse body weight and Psoriasis Area and Severity (PASI) scores were recorded daily. On day 8, mice were sacrificed, and the spleen was removed to calculate the spleen weight index (spleen / body weight ratio).

[0056] The results are as follows Figure 7 As shown in AB, compared with the saline group, the psoriatic mice in the M-NP@T317 and AM-NP@T317 groups showed only mild desquamation and mild erythema on the back of their skin on day 8. Figure 7 As shown in Figure C, the mice in the saline group showed a significant decrease in body weight, while the mice in the nanoparticle treatment group did not show a significant decrease in body weight. Figure 7 As shown in Figure D, compared to the saline group, the PASI scores of psoriatic mice in the AM-NP@T317 group were significantly lower. Figure 7 As shown in Figure E, mice in the saline group exhibited splenomegaly due to macrophage activation, while mice in the AM-NP@T317 group showed reduced spleen size and a significantly decreased spleen weight index, suggesting that inflammation was suppressed. Based on these results, AM-NP@T317 can alleviate psoriasis symptoms.

[0057] Example 8: Effect of ligand-modified drug-loaded nanoparticles on the lesion environment of psoriatic mice.

[0058] To further investigate the effect of ligand-modified drug-loaded nanoparticles on regulating the lesion microenvironment, skin lesions on the backs of psoriatic mice were collected and stained with hematoxylin-eosin (HE), Ki67, and Mer. In addition, the back skin was digested with collagenase and ground into a single-cell suspension to detect macrophage infiltration and macrophage phenotype. Interleukin-17A (IL-17A) is widely believed to play an important role in the pathogenesis of psoriasis; therefore, enzyme-linked immunosorbent assay (ELISA) was used to examine the level of IL-17A in the skin lesions of psoriatic mice. Simultaneously, the levels of the anti-inflammatory factor transforming growth factor-β (TGF-β) and the pro-inflammatory factor tumor necrosis factor-α (TNF-α) were also examined.

[0059] The results are as follows Figure 8 AC and HE images showed that AM-NP@T317 significantly reduced the thickening of the stratum corneum and stratum spinosum, alleviating lesions in the epidermis and dermis. The decreased Ki67 expression indicated that AM-NP@T317 could inhibit excessive proliferation of keratinocytes. The expression of Mer on macrophages in the lesion tissue showed that, compared with the saline group, macrophages in AM-NP@T317-treated mouse skin expressed higher levels of the cytotoxic receptor Mer. Figure 8 As shown in Figure D, flow cytometry analysis indicated that macrophage infiltration in the saline group was approximately 2.3 times that of the healthy group. AM-NP@T317 significantly reduced macrophage infiltration, achieving a level comparable to that of healthy individuals. Figure 8 As shown in Figure E, macrophage phenotypes were detected and the M2 / M1 ratio was calculated, i.e., (F4 / 80 positive CD206 positive) / (F4 / 80 positive CD86 positive), to evaluate the suppression of skin inflammation. The M2 / M1 ratio in the AM-NP@T317 group was significantly higher than that in the saline group, but not significantly different from that in the healthy group, indicating a reduction in psoriatic inflammation in mice. Figure 8 As shown in Figure FG, AM-NP@T317 treatment significantly reduced IL-17A and TNF-α levels in the lesion tissue, while significantly increasing TGF-β levels to near healthy levels. In summary, macrophages are highly infiltrated in psoriatic lesions, with most macrophages exhibiting a decreased M2 / M1 ratio and displaying a pro-inflammatory phenotype. AM-NP@T317 treatment reduced macrophage infiltration, increased their M2 / M1 ratio, and increased expression of cell burial-related receptors. Furthermore, AM-NP@T317 regulates cytokine secretion, further improving the inflammatory microenvironment of the disease and effectively alleviating psoriasis.

Claims

1. A nanoparticle that enhances the cytotoxic function of macrophages, characterized in that: This includes nanocarrier materials, macrophage cell membranes, targeting ligands, and liver X receptor agonists as active ingredients.

2. The nanoparticles for enhancing macrophage cytotoxicity according to claim 1, characterized in that: The nanocarrier material is a polymer capable of encapsulating drugs, and is at least one of polylactic acid-glycolic acid copolymer, polylactic acid, and polylactic-glycolic acid; preferably, the nanocarrier material is polylactic acid-glycolic acid copolymer.

3. The nanoparticles for enhancing macrophage cytotoxicity according to claim 1, characterized in that: The macrophage cell membrane is obtained from at least one of mouse monocytic macrophage leukemia cells (RAW 264.7), bone marrow-derived macrophages (BMDM), human monocytic leukemia cells (THP-1), and activated macrophages stimulated by various cytokines; preferably, the cytokines are interleukin-4 (IL-4) and lipopolysaccharide (LPS).

4. The macrophage cell membrane according to claim 3, characterized in that: The mass ratio of protein to nanoparticles in the macrophage cell membrane is (1:1) to (1:8).

5. The nanoparticles for enhancing macrophage cytotoxicity according to claim 1, characterized in that: The macrophage cell membrane encapsulated on the surface of the nanoparticles retains macrophage membrane surface proteins.

6. The nanoparticles for enhancing macrophage cytotoxicity according to claim 1, characterized in that: The targeting ligand is capable of binding to urokinase-type plasminogen activator receptor (uPAR) and is at least one of AE105 (D-Cha-FsrYLWS), AE120 ((D-Cha-FsrYLWS)2-βA-K), and U11 (VSNKYFSNIHW); preferably, the targeting ligand is AE105 (D-Cha-FsrYLWS).

7. The targeted ligand according to claim 6, characterized in that: The mass ratio of the targeting ligand to the nanocarrier material is (1:100) to (1:5).

8. The nanoparticles for enhancing macrophage cytotoxicity according to claim 1, characterized in that: The active ingredient is a liver X receptor agonist, and is at least one of T0901317, GW3965, Ouabagenin, and LXR-623; preferably, the liver X receptor agonist is T0901317.

9. The liver X receptor agonist according to claim 8, characterized in that: The liver X receptor agonist is the active ingredient, and the mass of the active ingredient accounts for 0.1% to 90% of the total weight of the nanoparticles.

10. The application of the nanoparticles that enhance macrophage burial function according to claim 1 in the preparation of drugs for treating autoimmune diseases.

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