A membrane-modified liposome drug targeting cd177-positive neutrophils and a preparation method thereof

Abstract

This invention discloses a membrane-modified liposomal drug targeting CD177-positive neutrophils and its preparation method. The membrane-modified liposomal drug has a three-layer structure, including: a liposome loaded with a PADI4 inhibitor, a targeting recognition layer formed by coupling a CD177 targeting peptide to the surface of the liposome, and an outermost biomimetic functional layer formed by coating the natural neutrophil membrane. Based on the key pathological mechanism of the "CD177-NETs axis," this invention designs a structurally sound, safe, and stable biomimetic targeted nanoformulation. It creatively combines the anti-inflammatory / chemotactic function of the neutrophil membrane, the precise targeting of the CD177 peptide to pathogenic subgroups, and the source blocking of NETs by the PADI4 inhibitor, achieving highly efficient dual-targeted enrichment and synergistic therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, specifically relating to a membrane-modified liposome drug targeting CD177-positive neutrophils and its preparation method. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a complex, chronic, systemic autoimmune disease whose pathological mechanisms involve multiple immune cell dysfunctions and abnormal activation of inflammatory pathways. Current clinical treatments, such as glucocorticoids and broad-spectrum immunosuppressants, while relieving symptoms, often lack specificity and are prone to causing serious side effects, such as increased susceptibility to infection and organ toxicity. Therefore, developing novel targeted therapies with high specificity and safety is a pressing technical challenge in this field.

[0003] Recent studies have shown that neutrophils and the extracellular traps (NETs) they release play a crucial role in the pathogenesis of SLE. NETs are mainly composed of DNA, histones, and various antimicrobial proteins. Their excessive formation exposes autoantigens (such as dsDNA) to the immune system, activating plasmacytoid dendritic cells (pDCs) to produce large amounts of type I interferon (IFN-I) and driving B cells to produce autoantibodies, thus forming a vicious cycle of "NETs-interferon-autoantibodies," which exacerbates the inflammatory response and tissue damage.

[0004] Existing research has shown that specific pathogenic factors present in SLE patients (such as circulating apoptotic microparticles) can continuously "activate" neutrophils, making them susceptible to NETosis (the process of NET formation).

[0005] In the formation of NETs, ​​peptidyl arginine deiminase 4 (PADI4) is a key rate-limiting enzyme. PADI4 catalyzes the citrullination of arginine residues in histone H3, leading to chromatin depolymerization, a necessary step for NET release. Therefore, PADI4 is considered an effective target for inhibiting pathological NETs. However, systemic administration of PADI4 inhibitors (such as GSK199) lacks cell specificity, potentially interfering with the normal physiological function of PADI4 in other tissues, leading to potential off-target effects, and making it difficult to achieve effective therapeutic concentrations in specific cells at the site of inflammation. Furthermore, studies have found that not all neutrophils participate equally in NET production. Specific subsets, such as CD177-positive (CD177+) neutrophils, have been found to be highly correlated with the activity of autoimmune diseases such as SLE and exhibit a stronger capacity for NET production. Therefore, specifically delivering PADI4 inhibitors to this highly pathogenic CD177+ neutrophil subset is crucial for improving efficacy and reducing side effects.

[0006] In the field of nanomedicine delivery, biomimetic strategies (such as cell membrane encapsulation technology) have shown great potential. For example, studies have reported the use of neutrophil membranes to encapsulate nanoparticles (without drugs), and the membrane itself has certain anti-inflammatory capabilities, such as by neutralizing inflammatory cytokines and targeting inflammatory sites.

[0007] However, existing technologies still have significant drawbacks: 1. Inaccurate drug delivery: Most existing PADI4 inhibitors are administered systemically, which cannot specifically target the CD177+ neutrophil subset that overproduces NETs, ​​resulting in limited efficacy and potential systemic toxicity.

[0008] 2. Single function: Existing drug-free neutrophil membrane biomimetic nanoparticles, although they have certain passive anti-inflammatory effects (such as adsorbing cytokines), lack the core function of actively inhibiting the formation pathway of NETs inside neutrophils (such as the PADI4 pathway).

[0009] In summary, there is currently a lack of a delivery system that can simultaneously achieve the following functions: (1) neutralizing external inflammatory factors using a biomimetic membrane; (2) actively targeting highly pathogenic CD177+ neutrophil subsets; and (3) releasing PADI4 inhibitors into target cells to block the production of NETs at the source.

[0010] Therefore, developing a biomimetic targeted nanodelivery system that integrates the above three functions is of great significance for achieving efficient and precise treatment of SLE. Summary of the Invention

[0011] The purpose of this invention is to provide a membrane-modified liposomal drug (GNLC) targeting CD177-positive neutrophils. This aims to address three core problems in the treatment of systemic lupus erythematosus (SLE) using existing technologies: 1. Existing neutrophil membrane-modified nanoparticles lack disease-specific targeting capabilities, failing to precisely accumulate at the lesion site where CD177-positive neutrophils are located, thus limiting their anti-inflammatory effects; 2. Traditional PADI4 inhibitors and other NET inhibitors have low bioavailability, are easily metabolized and cleared, and lack targeting, leading to off-target effects, making it difficult to efficiently inhibit NET production on CD177+ neutrophils; 3. Existing delivery systems do not achieve the synergistic effect of "inflammation neutralization - precise targeting - efficient drug release," failing to simultaneously address both anti-inflammatory effects and the need for NET targeted inhibition, resulting in poor efficacy in treating SLE.

[0012] The membrane-modified liposome drug targeting CD177-positive neutrophils provided by this invention has a three-layer structure, including: a liposome loaded with a PADI4 inhibitor as the drug core layer, a targeting recognition layer formed by coupling CD177 targeting peptides to the surface of the liposomes, and an outermost biomimetic functional layer formed by coating the natural neutrophil membrane.

[0013] In the above structure, the liposome loaded with a PADI4 inhibitor serves as the drug core layer. The liposome acts as a carrier, loading a PADI4 inhibitor (such as GSK199) to achieve controlled and sustained drug release. The target recognition layer uses PEG-modified lipid terminals with groups that react with groups in the CD177 target peptide (such as DSPE-PEG (2000)-Mal-mediated amide bonds) to couple the CD177 target peptide to the liposome surface, enabling precise recognition of CD177. + Neutrophils; the biomimetic functional layer is a coating of the natural neutrophil membrane, retaining membrane proteins such as CD62L (inflammatory chemotaxis) and TNF-αR (cytokine neutralization), endowing nanomedicines with dual properties of inflammatory homing and anti-inflammatory effects, ultimately achieving a synergistic effect of "precise targeting - sustained drug release - inflammation regulation".

[0014] In some embodiments of the present invention, the liposomes loaded with PADI4 inhibitor are prepared from raw materials including: steroids, PEG lipids, and PADI4 inhibitors; wherein the PEG end of the PEG lipid is modified with a group that can react with a group in the CD177 targeting peptide. Furthermore, the group that reacts with the group in the CD177 targeting peptide can be Mal (maleimide group), which can be coupled by Michael addition reaction with the thiol group (-SH) of cysteine ​​in the peptide; or the following groups can be selected to replace it according to the reactive groups on the peptide: NHS (N-hydroxysuccinimide), OPSS (o-pyridine dithioide), DBCO (diphenylcyclooctylene), Aldehyde (aldehyde group).

[0015] Furthermore, the steroids include, but are not limited to, cholesterol, ergosterol, lanosterol, stigmasterol, sitosterol, alfalfa, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccosterol, dehydrocholesterol, chain sterol, dihydroergocalciferol, and dihydrocholesterol. Furthermore, the PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, and PEG-modified diacylglycerol.

[0016] In some embodiments of the present invention, the PADI4 inhibitors include, but are not limited to, GSK199, GSK484, Cl-amidine, etc.

[0017] In one specific embodiment of the present invention, the steroid is cholesterol; the PEG lipid is DSPE-PEG(2000)-Mal; and the PADI4 inhibitor is GSK199.

[0018] In some embodiments of the present invention, the mass ratio of PEG lipids to steroids in the liposomes loaded with PADI4 inhibitors is 10:1.

[0019] In some embodiments of the present invention, the amino acid sequence of the CD177 targeting peptide may be CGGGTIRLNPMPKYFD.

[0020] In some embodiments of the present invention, the mass ratio of the CD177 targeting peptide to the PEG lipid is 1:(1-1.5).

[0021] In some embodiments of the present invention, the total lipids in the liposomes loaded with the PADI4 inhibitor are in a mass ratio of 11:1 to the natural neutrophil membrane.

[0022] In some embodiments of the present invention, the neutrophil membrane is prepared as follows: unactivated neutrophils are isolated from mouse bone marrow using density gradient centrifugation; neutrophil activation is induced by LPS; and the activated neutrophils are extracted by hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain the neutrophil membrane.

[0023] In some embodiments of the present invention, the average particle size of the membrane-modified liposome drug targeting CD177-positive neutrophils is 121.7-130.3 nm.

[0024] The method for preparing a membrane-modified liposome drug targeting CD177-positive neutrophils provided by this invention includes the following steps: 1) PEG lipid and CD177 targeting peptide were dissolved in HEPES buffer at a mass ratio and reacted at room temperature. After the reaction was completed, impurities were removed by dialysis and then lyophilized to obtain PEG lipid-CD177. 2) Drug-loaded targeted liposomes were prepared by thin-film hydration using PEG lipid-CD177, steroids, and PADI4 inhibitors as raw materials. 3) The drug-loaded targeted liposomes were mixed with neutrophil membranes, sonicated under ice bath conditions, and then extruded through membrane to obtain membrane-modified liposome drugs targeting CD177 positive neutrophils.

[0025] In some embodiments of the present invention, the mass ratio of the CD177 targeting peptide to the PEG lipid in step 1) is 1:(1-1.5).

[0026] In one specific embodiment of the present invention, the mass ratio of DSPE-PEG2000-Mal to CD177 targeting peptide in step 1) can be 1:1.

[0027] In some embodiments of the present invention, the HEPES buffer in step 1) is HEPES buffer (10 mM, 135 mM NaCl).

[0028] In some embodiments of the present invention, the reaction time of the room temperature reaction in step 1) can be 18-26 h.

[0029] In one specific embodiment of the present invention, the reaction time of the room temperature reaction in step 1) can be 24 hours.

[0030] In some embodiments of the present invention, the specific method for preparing drug-loaded targeted liposomes by thin-film hydration in step 2) is as follows: PEG lipid-CD177 and steroids are dissolved in a chloroform / methanol (4:1, v / v) mixed solvent, and the mixture is rotary evaporated to form a lipid membrane; PADI4 inhibitor solution and water are added to the lipid membrane and hydrated at room temperature for 1 h; then, the mixture is sonicated for 20 min (55% power, 5 s on / 5 s off) to obtain the drug-loaded liposomes.

[0031] In some embodiments of the present invention, the mass ratio of lipids in the drug-loaded targeted liposomes to neutrophil membranes in step 3) is 11:1.

[0032] In some embodiments of the present invention, the conditions for ultrasonic treatment in step 3) are: ultrasonic treatment for 20 min (55% power, 5 s on / 5 s off).

[0033] In some embodiments of the present invention, the membrane extrusion method in step 3) is: repeated extrusion of a 200 nm polycarbonate membrane 15-20 times.

[0034] The membrane-modified liposome drug (GNLC) targeting CD177-positive neutrophils designed in this invention has the following significant advantages compared with existing technologies: 1. Elucidated the pathological basis and targets of precision treatment. The applicant first demonstrated through immunofluorescence that CD177 was present in skin lesions of lupus patients and MRL / lpr lupus model mice. + The degree of neutrophil infiltration and their NETs products (MPO) were significantly higher than those in healthy controls. Figure 1 CD177 was revealed. + Neutrophils are the main pathogenic subset for NET formation in SLE. Subsequent analysis of the SLE patient transcriptome dataset (GSE72509) confirmed that the expression levels of CD177, the key NET protein ELANE, and MPO were significantly upregulated in the blood of SLE patients. Figure 2 Furthermore, CD177 expression was significantly positively correlated with the expression of key NET enzymes PADI4 and MPO. In the subgroup of patients with high CD177 expression, the NET-related gene spectrum showed a significant upregulation trend. Figure 3 This discovery provides a basis for the present invention (targeting CD177). + This provides a solid theoretical basis for the rationality and advancement of cell-based and NET-inhibiting methods, and solves the problems of unclear pathogenic neutrophil subsets and imprecise targeting in existing technologies.

[0035] 2. The prepared GNLC nanomaterials have a reasonable and stable structure and retain key biological activities. This invention demonstrates the successful construction and stability of GNLCs through experimental data: 1) Structural characterization: TEM images show that GNLCs have a clear "core-shell" biomimetic structure; UV-Vis spectroscopy and MALDI-TOF analysis confirm that the PADI4 inhibitor GSK199 and the CD177 targeting peptide were successfully integrated. 2) Colloidal stability: GNLCs have a uniform particle size (approximately 178 nm), PDI < 0.5, and a near-neutral surface charge (-0.2 mV). This characteristic endows them with low immunogenicity, which is beneficial for prolonging in vivo circulation time. Figure 4 3) Preservation of bioactivity: Protein electrophoresis (SDS-PAGE) and Western blotting confirmed that GNLC successfully retained the inflammatory chemokine (CD62L) and cytokine neutralizing receptor (TNF-αR) of the neutrophil membrane, laying the foundation for their subsequent inflammation-targeting and anti-inflammatory functions. Simultaneously, the formulation of this invention has controllable drug sustained-release characteristics (approximately 78.64% released within 48 hours), which is beneficial for maintaining a long-term drug concentration at the lesion site. Figure 5 ).

[0036] 3. Possesses excellent drug-loading capacity and high biocompatibility. Cytotoxicity assays confirmed that GNLC showed no significant toxic side effects within the effective concentration range, and the hemolysis rate was far below the safety threshold of 5%, demonstrating high biocompatibility. Figure 6 ).

[0037] 4. Achieved dual precision enrichment through "biomimetic chemotaxis + active targeting". This invention solves the core problem of low drug delivery efficiency in existing technologies. In in vivo experiments on lupus model mice, the enrichment efficiency of fluorescently labeled GNLC in inflamed kidneys was significantly higher than that of naked liposomes, control formulations that only coated the neutrophil membrane (without targeting peptide), or those only conjugated with CD177 peptide (without membrane). Figure 7 This strongly demonstrates that the synergy of the two strategies unique to this invention, namely "neutrophil membrane biomimetic chemotaxis" (passive targeting of inflammation) and "CD177 peptide precise recognition" (active targeting of pathogenic cells), can guide drugs to the core of the lesion most efficiently.

[0038] 5. Demonstrates excellent systemic and local therapeutic effects in lupus animal models. In an imiquimod (IMQ)-induced lupus model, GNLC treatment demonstrated potent disease remission: 1) Improved systemic phenotype: significantly reduced the size of the enlarged spleen and lymph nodes in lupus mice ( Figure 8 2) Repairing kidney function: Significantly alleviates glomerulonephritis, reduces inflammatory cell infiltration in the glomeruli, and decreases IgG and complement C3 deposition. Figure 93) Significantly reduced serum levels of pro-inflammatory cytokines (such as TNF-α, IL-6, IL-1β) and the titer of anti-dsDNA antibody, a lupus marker. Figure 10 ).

[0039] 6. The synergistic mechanism of "inhibiting NETs + reshaping the microenvironment" was verified. This invention further validated the mechanism of its therapeutic effect through histopathological examination. GNLC treatment can simultaneously achieve: 1) targeted inhibition of NETs: significantly reducing renal neutrophils (Ly6G... + ) infiltration and NETs (MPO) + ) Formation. 2) Remodeling the immune microenvironment: Significantly induces the formation of pro-inflammatory M1 type (iNOS) macrophages in the kidneys. + ) towards the anti-inflammatory and repair-promoting M2 type (CD206) + )polarization( Figure 11 ).

[0040] 7. The biosafety of GNLC nanomaterials was verified. Healthy C57BL / 6J mice were administered GNLC via tail vein injection five times over 10 days to assess its systemic toxicity. Complete blood counts showed no statistically significant differences in white blood cell (WBC), red blood cell (RBC), and hemoglobin (HGB) levels between the GNLC group and the PBS control group. Serum biochemical analysis showed that liver function indicators (ALT, AST) and kidney function indicators (CREA, UREA) in the GNLC group were within the normal range and showed no significant differences compared to the PBS control group. Histopathological examination of organ tissues showed no inflammation, necrosis, or fibrosis in H&E-stained sections of the heart, liver, spleen, lungs, and kidneys in the GNLC group; the tissue structure was consistent with the PBS control group. Figure 12 ).

[0041] In summary, this invention, based on the key pathological mechanism of the "CD177-NETs axis," designs a structurally sound, safe, and stable biomimetic targeted nanoparticle formulation. It creatively combines the anti-inflammatory / chemotactic function of neutrophil membranes, the precise targeting of pathogenic subgroups by CD177 peptides, and the source-level blocking of NETs by PADI4 inhibitors. This achieves highly efficient dual-targeted enrichment and synergistic therapy, providing a novel, highly targeted, and safer treatment strategy for autoimmune diseases such as systemic lupus erythematosus, far superior to existing technologies. Attached Figure Description

[0042] Figure 1 Immunofluorescence staining of skin tissues from SLE patients and healthy controls, as well as skin tissues from MRL lupus mice and wild-type mice, showed CD177. +Neutrophil and MPO expression were elevated in both SLE patients and MRL lupus mice.

[0043] Figure 2 The relative expression levels of CD177, ELANE, and MPO mRNA in the peripheral blood of SLE patients were significantly higher than those in healthy controls.

[0044] Figure 3 The expression levels of CD177, MPO, and PADI4 in the peripheral blood of SLE patients were positively correlated. The heatmap showed a significant upregulation trend in the NETs-related gene profile within the CD177-high expression subgroup.

[0045] Figure 4 (A) The structural formula of the PADI4 inhibitor GSK199 of this invention; (B) Transmission electron microscopy image of GNLC nanoparticles; (C) UV-Vis absorption spectra of GNLC and its components; (D) Molecular weight distribution of CD177 peptide, DSPE-PEG2000-MAL, and DSPE-PEG2000-CD177 detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS); (E) Characterization of different nanoparticle formulations by dynamic light scattering (DLS), including particle size distribution, zeta potential, and polydispersity index (PDI).

[0046] Figure 5 For (A) SDS-PAGE analysis (Coomassie Brilliant Blue staining) of proteins in LNP, neutrophils, neutrophil membrane (NM), and NM-LNP-CD177; (B) Western blot analysis of surface markers (CD62L and TNF-αR) in LNP, neutrophils, NM, and NM-LNP-CD177; (C) In vitro drug release profiles of GNLC nanoparticles; (D) Drug loading rate of GNLC. Figure 6 (A) Cell viability of neutrophils and RAW264.7 cells treated with different concentrations of GNLC was detected by CCK8 assay (n=5); (B) Hemolysis rate of different concentrations of pure liposomes (LNP) and neutrophil membrane-encapsulated liposomes (NM-LNP-CD177) was measured (n=3).

[0047] Figure 7(A) In vitro fluorescence imaging of major organs in SLE mice after treatment with DiD-labeled naked liposomes (L-DiD), targeted peptide liposomes (LC-DiD), cell membrane-encapsulated liposomes (NL-DiD), and cell membrane-encapsulated targeted peptide liposomes (NLC-DiD) to trace drug distribution; (B) Quantitative analysis of the in vitro fluorescence intensity of DiD in the kidneys of imiquimod-induced SLE mice (n=6).

[0048] Figure 8 (A) Representative appearance of spleens in imiquimod-induced SLE mice after different nanoparticle treatments (n=7); (B) Quantitative analysis of spleen weight in mice (n=7); (C) Quantitative analysis of lymph node scores in mice (n=7).

[0049] Figure 9 (A) Representative hematoxylin-eosin (H&E) staining images of mouse kidney tissue; (B) Representative periodic acid-Schiff (PAS) staining images of mouse kidney tissue; (C) Representative immunofluorescence staining images of mouse glomerular IgG (red) and C3 (yellow).

[0050] Figure 10 (A) Levels of TNF-α, IL-6 and IL-1β in mouse serum as detected by enzyme-linked immunosorbent assay (ELISA) (n=7); (B) Levels of anti-double-stranded DNA (anti-dsDNA) antibodies in mouse serum as detected by ELISA (n=7).

[0051] Figure 11 (A) Representative immunohistochemical staining images of Ly6G and MPO expression in mouse kidney tissue; (B) Semi-quantitative analysis of Ly6G and MPO immunohistochemical staining (n=3); (C) Representative immunohistochemical staining images of iNOS and CD206 expression in mouse kidney tissue; (D) Semi-quantitative analysis of iNOS and CD206 immunohistochemical staining (n=3).

[0052] Figure 12(A) Blood routine analysis of C57BL / 6J mice after intravenous injection of PBS or GNLC (5 times in 10 days), measuring the levels of white blood cells (WBC), red blood cells (RBC), and hemoglobin (HGB) (n=8); (B) Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea (UREA) in C57BL / 6J mice injected with PBS or GNLC (n=8); (C) HE-stained sections of major organs (heart, liver, spleen, lung, and kidney) of C57BL / 6J mice injected with PBS or GNLC. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0055] Example 1: Preparation of membrane-modified liposome drug (GNLC) targeting CD177-positive neutrophils (1) Neutrophil isolation and cell membrane extraction Mouse neutrophil isolation: Bone marrow from the femur and tibia of mice was centrifuged at 300×g to obtain a single-cell suspension. After erythrocyte lysis, neutrophils were isolated using the Miltenyi Biotec Neutrophil Isolation Kit (130-097-658). The isolated neutrophils were stimulated with 50 ng / mL LPS at 37℃ for 2 h, centrifuged at 1000 rpm for 10 min, washed three times with ice-cold PBS, and resuspended in hypotonic lysis buffer (225 mM D-mannitol, 30 mM Tris-HCl pH 7.5, 75 mM sucrose, 0.2 mM EDTA, 1 mM PMSF). After incubation on ice for 20 min, the cells were sonicated on ice (160 W, 5 cycles). The cells were centrifuged at 10,000 g at 4℃ for 10 min to remove nuclear debris, and then ultracentrifuged at 100,000 g at 4℃ for 1 h to collect membrane components. The cells were washed three times with 0.2 mM EDTA and BCA. Membrane protein concentration was determined by the method and stored at -80℃. (2) Solid-phase synthesis of CD177-targeted peptides The Fmoc solid-phase synthesis method was employed: 1.0 g of Rink amide resin (0.50 mmol, Bio-Tech Pharmaceuticals) was weighed and swollen in 15 mL of dichloromethane (DCM, Beijing Chemical Plant) for 30 min; after removing DCM, 10% DBU / DMF (v / v) solution was added and reacted on a shaker for 10 min (repeated twice) to remove the Fmoc protecting group, during which time the mixture was washed twice each with DCM and DMF. Amino acids were sequentially coupled stepwise: 4 eq amino acids, 3.95 eq HBTU (Annegi Chemicals), and 6 eq DIPEA (Annegi Chemicals) were dissolved in DMF to prepare a coupling solution, which was then added to the resin and reacted at room temperature for 150 min; the deprotection step was repeated after each coupling, and the synthesis was performed stepwise in the order CGGGTIRLNPMPKYFD. After synthesis, the resin was reacted with 20 mL of TFA / TIS / H2O (95:2.5:2.5, v / v / v) mixture and 107 mg DTT for 3 h to cleave the peptide and remove side-chain protecting groups. The reaction solution was filtered, concentrated by rotary evaporation, precipitated with tert-butyl methyl ether, centrifuged 2-3 times, and vacuum dried to obtain crude product. The crude product was purified by high performance liquid chromatography (HPLC), and the product was desolvated by rotary evaporation and lyophilized to obtain purified peptide powder.

[0056] (3) Preparation of cell membrane-encapsulated targeted peptide liposome conjugates 10 mg of DSPE-PEG2000-Mal (J&K Bailingwei) and 1 mg of cholesterol (Avanti) were dissolved in 5 mL of chloroform / methanol (4:1, v / v), sonicated for 2 min, and then transferred to a 25 mL round-bottom flask. The mixture was rotary evaporated at 42 °C and 110 rpm to form a lipid membrane. 5 mL of deionized water was added for hydration at room temperature for 1 h, followed by sonication for 20 min (55% power, 5 s on / 5 s off). The membrane was then repeatedly extruded through a 200 nm polycarbonate membrane 18 times to prepare liposomes (LNPs). Further, 1 mg of neutrophil membrane (NM) was added to the preparation system, sonicated for 20 min under ice bath conditions, and repeatedly extruded through a 200 nm polycarbonate membrane 18 times to obtain membrane-fused liposomes (NM-LNPs).

[0057] To obtain targeted liposomes, DSPE-PEG2000-Mal and CD177 peptide were dissolved in HEPES buffer (10 mM, 135 mM NaCl) at a 1:1 mass ratio and reacted at room temperature for 24 h. After removing impurities by dialyzing, the mixture was lyophilized to obtain DSPE-PEG2000-CD177. Subsequently, LNP was prepared with cholesterol using the same method to obtain targeted modified liposomes (LNP-CD177). Further, 1 mg of neutrophil membrane (NM) was added to the preparation system, and the mixture was sonicated for 20 min under ice bath conditions and repeatedly extruded through a 200 nm polycarbonate membrane 18 times to obtain membrane-fused hybrid liposomes (NM-LNP-CD177).

[0058] (4) Preparation of cell membrane-encapsulated drug-targeting liposomes (GSK199@NM-LNP-CD177) During the preparation of LNP-CD177, 50 μL of 10 mM GSK199 (MCE) was added, and the product was subjected to rotary evaporation, hydration, sonication, and extrusion as described above. The product was centrifuged at 2000 rpm for 6 min to remove unencapsulated free drug, and the supernatant was collected as the drug-loaded membrane-fused hybrid liposome (GSK199@NM-LNP-CD177). GSK199@LNP, GSK199@NM-LNP, and GSK199@LNP-CD177 can be obtained by referring to the above method.

[0059] Example 2: Characterization and efficacy evaluation of membrane-modified liposomal drugs targeting CD177-positive neutrophils I. Experimental Methods 1. Nanoparticle characterization methods 1.1 Morphological and Physicochemical Property Testing 1) Morphology of liposomes: Observed by transmission electron microscopy (TEM, Hitachi HT7800, Japan), with negative staining of 1% phosphotungstic acid; 2) Particle size and zeta potential: Measured using a particle size and zeta potential analyzer (Zetasizer Nano ZS90, Malvern, UK); 3) Identification of lipopeptide conjugates: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF, Ultraflextreme, Bruker).

[0060] 1.2 Drug-loaded sustained-release detection To determine the drug loading (DLC%), lipid nanoparticles were disrupted with ethanol. Drug content was quantified using UV-Vis spectrophotometry. The efficiency value was calculated using the following formula: DLC (wt.%) = Drug loading / Total mass of drug loading and nanoparticles × 100%. To detect release kinetics, GNLC nanoparticles dissolved in PBS buffer (1 mL) were placed in a dialysis bag (molecular weight cutoff of 1000 Da) and immersed in 2 mL of dissolution medium. The entire release unit was placed in an incubator at 37 °C with shaking. At set time intervals, 0.5 mL of dialysis buffer was removed and an equal volume of fresh dialysis medium was added. The drug released from the dialysis buffer was quantified using UV-Vis spectrophotometry.

[0061] 1.3 Membrane protein detection (Coomassie blue staining + Western blot) Samples were lysed in RIPA buffer containing protease inhibitors at 4 °C for 30 min, followed by sonication (5 cycles), centrifuged at 13,000 g for 10 min, and the supernatant was collected. Protein concentration was quantified using the BCA method. An equal volume of protein sample was mixed with loading buffer and heated at 100 °C for 10 min.

[0062] After protein separation by SDS-PAGE electrophoresis, a portion of the gel was stained with Coomassie Brilliant Blue to visualize the bands; the other portion was transferred to a polyvinylidene fluoride (PVDF) membrane by constant current and blocked with 5% skim milk powder for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies (anti-CD62L and anti-TNF-α, both rabbit-derived antibodies). The next day, the membrane was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit IgG secondary antibody and developed using a chemiluminescence detection system.

[0063] 2. Animal model construction (IMQ-induced SLE model) 1) Animals and modeling: 8-10 week old C57BL / 6J mice were treated with 5% imiquimod (IMQ) cream on the inner side of both ears three times a week for six consecutive weeks; 2) Grouping and administration: On day 14 of modeling, the participants were randomly divided into 6 groups (n=8 / group) and injected with different components of nanomaterials (3mg / kg, once every 3 days) via tail vein. Normal control (NC, no modeling) and model control (MC, modeling only) were set up and injected with equal volume of physiological saline. 3) Sample collection: Mice were sacrificed in week 6 of the experiment, and spleens, lymph nodes (weighed and scored), and kidneys were collected.

[0064] 3. In vivo and in vitro functional testing methods 3.1 In vitro targeting studies 1) Fluorescent labeling: DiD labeling of four nanomaterials (LNP: liposomes; LNP-CD177: targeted peptide modified liposomes; NM-LNP: cell membrane-coated liposomes; NM-LNP-CD177: cell membrane-encapsulated targeted peptide liposomes). 2) Detection: The model mice were divided into 4 groups (n=6 / group) and injected with 3mg / kg fluorescent particles via the tail vein. Whole-body fluorescence (excitation 644nm, emission 665nm) was collected at 1, 3, 6, 12 and 24h using an IVIS imaging system (PerkinElmer, USA). After dissection, the mice were imaged in vitro and the tissue fluorescence intensity was quantified.

[0065] 3.2 Histology and Immunostaining 1) Histological analysis: Heart, liver, spleen, lung, kidney and target organs were harvested; kidney of SLE model was stained with HE and PAS. 2) Immunofluorescence staining: Paraffin sections were dewaxed and hydrated → microwave antigen retrieval → permeabilized with 0.2% Triton X-100 for 5 min → blocked with 1% donkey serum + 5% BSA for 1 h; incubated overnight at 4℃ with primary antibody (anti-mouse C3, FITC-IgG, MPO, Ly6G, F4 / 80, iNOS, CD206); incubated at room temperature with Alexa Fluor-conjugated secondary antibody for 45 min, counterstained with Hoechst 33342, and observed under a confocal microscope (ZEISS); 3) Immunohistochemical staining: Dewaxing and hydration of paraffin sections → antigen retrieval (thermal retrieval / enzymatic digestion) → quenching of peroxidase with 3% hydrogen peroxide → serum blocking; incubation with primary antibody at 4℃ overnight, HRP secondary antibody binding at room temperature; DAB staining, hematoxylin counterstaining, gradient dehydration, xylene clearing, mounting with neutral resin, and microscopic observation.

[0066] 3.3 Serum marker detection (ELISA) The levels of TNF-α, IL-6, IL-1β and anti-dsDNA antibody in the serum of lupus mice were detected using the Biolegend ELISA kit.

[0067] 4. Biosafety assessment methods 4.1 Cytotoxicity Detection 1) CCK8 method: RAW264.7 cells / neutrophils were co-incubated with different concentrations of nanomaterials for 24 h, then incubated with 10% CCK8 medium for 1 h, and the OD value at 450 nm was measured by microplate reader; 2) Calcein AM / PI staining: After co-incubation, discard the culture medium, add the detection working solution and incubate for 30 min, then observe under a fluorescence microscope.

[0068] 4.2 Hemolysis rate detection A 4% erythrocyte suspension was prepared from mouse peripheral blood and incubated with 10-500 μg / mL LNP / NLC solution (PBS negative control, H2O positive control); after incubation at 37℃ for 4 h, the suspension was centrifuged at 3000 rpm for 20 min, and 100 μL of the supernatant was taken to measure the absorbance at 542 nm. The hemolysis rate was calculated (>5% indicates potential hemolysis).

[0069] 4.3 In vivo safety evaluation To evaluate the in vivo toxicity of this material, C57BL / 6J mice were divided into a control group and an experimental group. The control group was injected with PBS via the tail vein, while the experimental group was injected with GNLC (6 mg / kg) via the tail vein, both administered five times over 10 days. After the experiment, blood and major organs such as the heart, liver, spleen, lungs, and kidneys were collected from the mice. The organ tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to observe pathological changes. Red blood cell count, white blood cell count, and hemoglobin content were measured using a small animal blood routine instrument. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (BUN) were also measured.

[0070] II. Experimental Results 1. Nanoparticle characterization results This invention demonstrates the successful construction and stability of GNLC through experimental data: 1) Structural characterization: TEM images showed that GNLC had a clear "core-shell" biomimetic structure; UV-Vis spectroscopy and MALDI-TOF analysis confirmed that the PADI4 inhibitor GSK199 and the CD177 targeting peptide were successfully integrated. Figure 4 The drug loading rate of GSK199 in GNLC was 16.97%.

[0071] 2) Colloidal stability: GNLC particles have uniform size (approximately 121.7-130.3 nm), PDI < 0.5, and a near-neutral surface charge (-0.2 mV). This characteristic endows them with low immunogenicity, which is beneficial for prolonging in vivo circulation time. Figure 4 ).

[0072] 3) Preservation of bioactivity: Protein electrophoresis (SDS-PAGE) and Western blotting confirmed that GNLC successfully retained the inflammatory chemokine (CD62L) and cytokine neutralizing receptor (TNF-αR) of the neutrophil membrane, laying the foundation for their subsequent inflammation-targeting and anti-inflammatory functions. Simultaneously, the formulation of this invention has controllable drug sustained-release characteristics (approximately 78.64% released within 48 hours), which is beneficial for maintaining a long-term drug concentration at the lesion site. Figure 5).

[0073] 2. Biosafety evaluation results Both cytotoxicity and hemolysis assays confirmed that GNLC had no significant toxic side effects within the effective concentration range, and the hemolysis rate was far below the safety threshold of 5%, demonstrating high biocompatibility. Figure 6 ).

[0074] 3. Results of in vivo and in vitro functional tests of IMQ-induced SLE model mice In in vivo experiments on SLE model mice, the enrichment efficiency of fluorescently labeled GNLC in inflamed kidneys was significantly higher than that of naked liposomes, control formulations coated only with neutrophil membranes (without targeting peptides), or formulations conjugated only with CD177 peptides (without membranes). Figure 7 This strongly demonstrates that the synergy of the two strategies unique to this invention, namely "neutrophil membrane biomimetic chemotaxis" (passive targeting of inflammation) and "CD177 peptide precise recognition" (active targeting of pathogenic cells), can guide drugs to the core of the lesion most efficiently.

[0075] In an imiquimod (IMQ)-induced lupus model, GNLC treatment demonstrated potent disease remission: 1) Improved systemic phenotype: significantly reduced the size of the enlarged spleen and lymph nodes in lupus mice ( Figure 8 2) Repairing kidney function: Significantly alleviates glomerulonephritis, reduces inflammatory cell infiltration in the glomeruli, and decreases IgG and complement C3 deposition. Figure 9 3) Significantly reduced serum levels of pro-inflammatory cytokines (such as TNF-α, IL-6, IL-1β) and the titer of anti-dsDNA antibody, a lupus marker. Figure 10 ).

[0076] GNLC treatment can simultaneously achieve: 1) Targeted inhibition of NETs: significantly reducing renal neutrophils (Ly6G) + ) infiltration and NETs (MPO) + ) Formation. 2) Remodeling the immune microenvironment: Significantly induces the formation of pro-inflammatory M1 type (iNOS) macrophages in the kidneys. + ) towards the anti-inflammatory and repair-promoting M2 type (CD206) + )polarization( Figure 11 ).

[0077] 4. In vivo safety assessment results Healthy C57BL / 6J mice were repeatedly injected via tail vein with GNLC over 10 days, at a total of 5 high doses (6 mg / kg), to assess its systemic toxicity. Complete blood counts showed no statistically significant differences in white blood cell (WBC), red blood cell (RBC), and hemoglobin (HGB) levels between the GNLC group and the PBS control group. Serum biochemical analysis showed that liver function indicators (ALT, AST) and kidney function indicators (CREA, UREA) in the GNLC group were within the normal range and showed no significant differences compared to the PBS control group. Histopathological examination of organ tissues showed no inflammation, necrosis, or fibrosis in H&E-stained sections of the heart, liver, spleen, lungs, and kidneys in the GNLC group; the tissue structure was consistent with the PBS control group. Figure 12 ).

[0078] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A membrane-modified liposomal drug targeting CD177-positive neutrophils, comprising a three-layer structure, including: The drug consists of a liposome loaded with a PADI4 inhibitor as the drug core layer, a target recognition layer formed by coupling a CD177 targeting peptide to the surface of the liposome, and a biomimetic functional layer formed by coating the outermost layer with a natural neutrophil membrane.

2. The membrane-modified liposome drug targeting CD177-positive neutrophils according to claim 1, characterized in that: The liposomes loaded with the PADI4 inhibitor are prepared from the following raw materials: steroids, PEG lipids, and PADI4 inhibitors; wherein the PEG end of the PEG lipid is modified with a group that can react with the group in the CD177 targeting peptide. Furthermore, the PADI4 inhibitors include, but are not limited to, GSK199, GSK484, and Cl-amidine; Furthermore, the steroids include, but are not limited to, cholesterol, ergosterol, lanosterol, stigmasterol, sitosterol, alfalfa, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccosterol, dehydrocholesterol, chain sterol, dihydroergocalciferol, and dihydrocholesterol. Furthermore, the PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, and PEG-modified diacylglycerol; Furthermore, the mass ratio of the PEG lipid to the steroid is 10:

1.

3. The membrane-modified liposome drug targeting CD177-positive neutrophils according to claim 1 or 2, characterized in that: The amino acid sequence of the CD177 targeting peptide is CGGGTIRLNPMPKYFD.

4. The membrane-modified liposomal drug targeting CD177-positive neutrophils according to any one of claims 1-3, characterized in that: The mass ratio of the CD177 targeting peptide to the PEG lipid is 1:(1-1.5).

5. The membrane-modified liposomal drug targeting CD177-positive neutrophils according to any one of claims 1-4, characterized in that: The total lipid content in the liposomes loaded with the PADI4 inhibitor is in a mass ratio of 11:1 to the mass of the native neutrophil membrane.

6. The membrane-modified liposomal drug targeting CD177-positive neutrophils according to any one of claims 1-5, characterized in that: The method for preparing the neutrophil membrane is as follows: Unactivated neutrophils are isolated from mouse bone marrow using density gradient centrifugation; neutrophil activation is induced by LPS; and the activated neutrophils are extracted by hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain the neutrophil membrane.

7. A method for preparing the membrane-modified liposome drug targeting CD177-positive neutrophils according to any one of claims 1-6, comprising the following steps: 1) PEG lipid and CD177 targeting peptide were dissolved in HEPES buffer at a mass ratio and reacted at room temperature. After the reaction was completed, impurities were removed by dialysis and then lyophilized to obtain PEG lipid-CD177. 2) Drug-loaded targeted liposomes were prepared by thin-film hydration using PEG lipid-CD177, steroids, and PADI4 inhibitors as raw materials. 3) The drug-loaded targeted liposomes were mixed with neutrophil membranes, sonicated under ice bath conditions, and then extruded through membrane to obtain membrane-modified liposome drugs targeting CD177 positive neutrophils.

8. The preparation method according to claim 7, characterized in that: In step 1), the mass ratio of the CD177 targeting peptide to the PEG lipid is 1:(1-1.5). And / or, the reaction time of the room temperature reaction in step 1) is 18-26 h.

9. The preparation method according to claim 7 or 8, characterized in that: The specific method for preparing drug-loaded targeted liposomes using the thin-film hydration method in step 2) is as follows: PEG lipid-CD177 and steroids are dissolved in a chloroform / methanol (4:1, v / v) mixed solvent, and the mixture is rotary evaporated to form a lipid membrane; PADI4 inhibitor solution and water are added to the lipid membrane, and the mixture is hydrated at room temperature for 1 h; then, the mixture is sonicated for 20 min (55% power, 5 s on / 5 s off) to obtain the drug-loaded liposomes.

10. The preparation method according to any one of claims 7-9, characterized in that: In step 3), the mass ratio of lipids to neutrophil membranes in the drug-loaded targeted liposomes is 11:

1. And / or, the conditions for ultrasonic treatment in step 3) are: ultrasonic treatment for 20 min (55% power, 5 s on / 5 s off); And / or, the membrane extrusion method described in step 3) is: repeated extrusion 15-20 times through a 200 nm polycarbonate membrane.

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