Application of CD177 targeted membrane modified liposome in preparation of medicine for preventing and / or treating psoriasis

Abstract

The invention discloses an application of a CD177 targeted membrane modified liposome in preparation of a medicine for preventing and / or treating psoriasis. The structure of the CD177 targeting membrane modified liposome comprises a PADI4 inhibitor loaded liposome, a targeting recognition layer formed by coupling CD177 targeting peptide on the surface of the liposome, and a bionic functional layer formed by coating a natural neutrophile granulocyte membrane on the outermost layer. Based on the core pathological mechanism of 'CD177 + neutrophil-NETs' of psoriasis, the inflammation chemotaxis / cell factor neutralization function of a neutrophil membrane, the precise targeting function of CD177 peptide and the NETs inhibition function of a PADI4 inhibitor are creatively and organically integrated, and the constructed GNLC achieves the synergistic treatment effect of 'targeting enrichment-mechanism blocking-microenvironment remodeling'; meanwhile, excellent stability and safety are achieved, and a new precise treatment strategy far better than that in the prior art is provided for psoriasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of CD177-targeted membrane-modified liposomes in the preparation of drugs for the prevention and / or treatment of psoriasis. Background Technology

[0002] Psoriasis is a chronic, relapsing inflammatory skin disease characterized by excessive epidermal proliferation and inflammatory cell infiltration in the dermis. Its pathological mechanisms are complex, involving abnormalities in the function of various immune cells such as neutrophils and T cells, as well as the continuous activation of multiple inflammatory pathways (such as the JAK-STAT and NF-κB pathways). Current clinical treatments include topical corticosteroids, vitamin D3 derivatives, oral methotrexate, and biologics. While these can control skin lesions to some extent, they generally have limitations: non-specific treatments easily cause side effects such as skin atrophy and liver and kidney damage, while some biologics face problems such as diminishing efficacy with disease progression and uneven patient response rates. Therefore, developing novel treatment strategies that combine high targeting, strong efficacy, and high safety is a core technological need to address the challenges in the clinical treatment of psoriasis.

[0003] Recent studies have confirmed that neutrophils and their released extracellular neutrophil traps (NETs) are key drivers of the pathological process of psoriasis. NETs are composed of DNA, citrullinated histones, and myeloperoxidase (MPO) released after neutrophil activation. Their accumulation in psoriatic lesions produces a dual pathogenic effect: on the one hand, as damage-associated molecular patterns (DAMPs), NETs can directly activate local innate immune cells in the skin (such as dendritic cells), inducing them to secrete pro-inflammatory cytokines such as IL-1β and IL-6, amplifying the inflammatory cascade; on the other hand, the citrullinated proteins in NETs can act as autoantigens, triggering T cell-mediated adaptive immune responses, further promoting abnormal proliferation of keratinocytes and aggravating skin lesions, forming a vicious cycle of "NETs-inflammatory factors-immune cells," leading to chronicity and recurrence of the disease.

[0004] In-depth research has revealed that the abnormal microenvironment in psoriasis patients (such as high concentrations of IL-8 and TNF-α in the skin lesions) continuously activates neutrophils, placing them in a "pre-activated" state and significantly increasing susceptibility to NETosis (the process of NET formation). More importantly, the heterogeneity of neutrophils exhibits clear functional specificity in psoriasis: CD177 positivity (CD177... + Neutrophil subsets were significantly amplified in the peripheral blood and skin lesions of patients, and their proportion was positively correlated with the psoriasis area and severity index (PASI). Furthermore, this subset showed a significantly stronger ability to produce NETs after in vitro stimulation than CD177. -Neutrophils are the main source cells of pathological NETs in psoriatic lesions. This finding indicates that CD177... + Neutrophils are a precise target for intervening in the NETs-related pathological pathways of psoriasis.

[0005] In the molecular mechanism of NET formation, peptidyl arginine deiminase 4 (PADI4) is an indispensable rate-limiting enzyme. PADI4 catalyzes the citrullination of arginine residues in histone H3, disrupting chromatin structural stability and providing the necessary conditions for NET release. Therefore, inhibiting PADI4 activity has become an effective technical pathway to block pathological NETs, ​​and related inhibitors (such as GSK199) have shown significant NET inhibitory effects in in vitro experiments. However, the clinical translation of existing PADI4 inhibitors faces two major technical bottlenecks: firstly, systemic administration lacks cell specificity and cannot precisely enrich CD177. + Neutrophils not only make it difficult to achieve effective therapeutic concentrations at the site of skin lesions, but may also interfere with the physiological function of PADI4 in normal tissues (such as participating in the regulation of apoptosis), causing potential off-target toxicity; secondly, inhibitors are easily degraded or cleared by enzymes in vivo, resulting in low bioavailability and difficulty in maintaining their effects.

[0006] In the field of nanomedicine delivery, cell membrane biomimetic technology offers a new approach to solving the aforementioned problems. Previous studies have reported that blank nanoparticles encapsulated in neutrophil membranes can achieve passive targeting of inflamed sites through adhesion molecules (such as CD62L) retained on the membrane surface, and exert a certain anti-inflammatory effect by neutralizing local pro-inflammatory cytokines with proteins such as TNF-α receptors on the membrane. However, this technology still has significant functional limitations: firstly, the drug-free design prevents it from directly intervening in core pathways of NET formation (such as the PADI4 pathway), only alleviating inflammatory symptoms without blocking the root cause of the disease; secondly, it does not target CD177. + Specific modifications to neutrophils cannot achieve precise targeting of the main source cells of NETs, ​​resulting in low treatment efficiency.

[0007] In summary, the current field of psoriasis treatment still lacks a highly efficient delivery system that can integrate multiple functions, specifically in the inability to simultaneously meet the following requirements: (1) By utilizing the biological properties of biomimetic membranes, local pro-inflammatory cytokines in the skin can be neutralized, thereby alleviating immediate inflammatory responses; (2) Through specific targeted modification, it can accurately identify and enrich highly pathogenic CD177. + Neutrophils; (3) The PADI4 inhibitor is efficiently released in the target cells to block the formation of NETs at the molecular level and cut off the pathological cycle.

[0008] Therefore, developing a biomimetic targeted nanodelivery system that integrates the triple functions of "inflammation neutralization, precise targeting, and pathway inhibition" is of great technical value and clinical significance for overcoming the limitations of existing psoriasis treatments and achieving precise intervention in the disease. Summary of the Invention

[0009] The purpose of this invention is to provide pharmaceutical use of membrane-modified liposomes (GNLC) that target CD177-positive neutrophils.

[0010] The use provided by this invention is its application in the preparation of medicaments for the prevention and / or treatment of psoriasis.

[0011] Furthermore, the prevention and / or treatment of psoriasis is manifested in at least one of the following aspects: 1) Improves skin damage caused by psoriasis; 2) Repairing skin pathological damage caused by psoriasis; 3) Lower the PASI score; 4) Reduce Ly6G in skin lesions caused by psoriasis + The number of neutrophil infiltrations; 5) Inhibits MPO caused by psoriasis + The formation of NETs.

[0012] Furthermore, the skin lesions caused by psoriasis include erythema, hardening of the skin, and scaling.

[0013] Furthermore, the repair of skin pathological damage caused by psoriasis is further reflected in: the epidermal thickness returning to normal, the disappearance of parakeratosis, and a significant reduction in the number of inflammatory cells infiltrating the dermis.

[0014] The present invention also provides a medicine for the prevention and / or treatment of psoriasis.

[0015] The medicament for the prevention and / or treatment of psoriasis provided by this invention comprises a membrane-modified liposome that targets CD177-positive neutrophils as its active ingredient.

[0016] Furthermore, a pharmaceutically acceptable carrier material is also added to the drug.

[0017] The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems.

[0018] The present invention also provides methods for treating and / or preventing diseases or conditions in a subject.

[0019] The method includes administering the membrane-modified liposomes that target CD177-positive neutrophils or the drug for the prevention and / or treatment of psoriasis to subjects in need.

[0020] The membrane-modified liposomes targeting CD177-positive neutrophils described in this invention have a three-layer structure, including: a liposome loaded with a PADI4 inhibitor as a 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.

[0021] 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 is formed by coupling the CD177 target peptide to the liposome surface with groups modified at the ends of PEG lipids that can react with groups in the CD177 target peptide (such as DSPE-PEG (2000)-Mal mediated amide bonds), 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".

[0022] Furthermore, 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. For example, 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).

[0023] 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.

[0024] Furthermore, the PADI4 inhibitors include, but are not limited to, GSK199, GSK484, Cl-amidine, etc.

[0025] 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.

[0026] 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.

[0027] Furthermore, the amino acid sequence of the CD177 targeting peptide may be CGGGTIRLNPMPKYFD.

[0028] Furthermore, the mass ratio of the CD177 targeting peptide to the PEG lipid is 1:(1-1.5).

[0029] Furthermore, 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.

[0030] Furthermore, 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 subjected to hypotonic lysis, ultrasonic disruption, and differential centrifugation to obtain the neutrophil membrane.

[0031] The membrane-modified liposomes targeting CD177-positive neutrophils described in this invention can be prepared according to the following method: 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 membrane-modified liposomes targeting CD177 positive neutrophils were obtained by membrane extrusion.

[0032] Further, the HEPES buffer mentioned in step 1) is HEPES buffer (10 mM, 135 mM NaCl).

[0033] Furthermore, the reaction time of the room temperature reaction described in step 1) can be 18-26 hours.

[0034] Further, the specific method for preparing drug-loaded targeted liposomes by the thin-film hydration method described 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.

[0035] Further, the conditions for ultrasonic treatment in step 3) are: ultrasonic treatment for 20 min (55% power, 5 s on / 5 s off).

[0036] Furthermore, the membrane extrusion method described in step 3) is as follows: repeatedly extruding a 200 nm polycarbonate membrane 15-20 times.

[0037] The drug provided by this invention solves three core problems existing in the treatment of psoriasis: 1. Existing neutrophil membrane-modified nanoparticles can only achieve non-specific enrichment through passive inflammatory homing, lacking targeting of the core pathogenic cells of psoriasis (CD177). + 1. The targeting ability of neutrophils is insufficient to precisely reach the area where these cells are located at the skin lesion, and they can only exert limited non-specific anti-inflammatory effects, making it difficult to intervene in the core pathological processes related to NETs; 2. Traditional PADI4 inhibitors and other NETs inhibitors have low bioavailability, are easily degraded by enzymes or cleared by the kidneys in vivo, and lack cell-specific targeting design, making it impossible to precisely act on CD177. + Neutrophils are difficult to reach effective therapeutic concentrations within target cells to inhibit NETs production, and may also interfere with the physiological function of PADI4 in normal tissues, posing a potential off-target toxicity. 3. Existing delivery systems have not achieved the synergistic effect of "inflammation neutralization - precise targeting - efficient drug release". They can either only neutralize some inflammatory factors through biomimetic membranes (such as drug-free neutrophil membrane nanoparticles) or only achieve single drug delivery (such as untargeted PADI4 inhibitor liposomes). They cannot simultaneously meet the dual requirements of "relieving immediate inflammation + blocking the pathological source of NETs" in psoriasis treatment, resulting in poor overall treatment efficacy.

[0038] Compared with the prior art, the present invention has the following beneficial effects: 1. Clarify the pathological basis and core targets of precision treatment for psoriasis, and resolve the issue of ambiguous targeting in existing technologies. The applicant established CD177 through dual validation using clinical samples and animal models. + Neutrophils play a central role as the primary source of pathological NETs in psoriasis. Clinical sample validation: Immunofluorescence staining of skin lesions from psoriasis patients showed that CD177... + The number of neutrophil infiltrations and the expression level of MPO, a key product of NETs, ​​were significantly higher than those in healthy controls. Figure 1 A); Mining public transcriptome datasets (GSE13355, GSE14905) revealed that the mRNA levels of CD177, NETs-related genes ELANE, and MPO were significantly upregulated in psoriatic lesions. Figure 2 Furthermore, CD177 expression is positively correlated with MPO expression. Figure 3 A), the overall NETs-related gene set in patients with high CD177 expression showed an upregulated trend ( ). Figure 3 B).

[0039] Animal model validation: In an imiquimod (IMQ)-induced mouse model of psoriasis, CD177 was detected in the skin lesions. + Neutrophil infiltration and MPO deposition were significantly higher in wild-type control mice than in wild-type control mice. Figure 1 B).

[0040] The above results clarify that "CD177" + Neutrophil-NETs are a key pathogenic pathway in psoriasis, and this invention targets CD177. + The design of neutrophils provides a solid theoretical basis and solves the core problems of unclear identification of pathogenic neutrophil subsets in psoriasis and lack of specificity in targeting in existing technologies.

[0041] 2. The prepared GNLCs are structurally stable and retain key biological activities, providing a material basis for therapeutic effects. Through systematic physicochemical characterization and functional verification, it was confirmed that GNLC possesses a rational structure and stable performance, while retaining the core functions of biomimetic membranes and drugs. Structure and composition verification: Transmission electron microscopy (TEM) images showed that GNLCs were uniformly spherical with a clear "neutrophil membrane shell-liposome core" nucleoshell structure. Figure 4 B); The UV-Vis absorption spectrum contains characteristic absorption peaks of GSK199 (PADI4 inhibitor) and the CD177 targeting peptide, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) confirms the successful coupling of DSPE-PEG2000 with the CD177 peptide. Figure 4(C, D) proves that the functional components are effectively integrated.

[0042] Colloidal stability: Dynamic light scattering (DLS) analysis showed that the GNLC particle size was approximately 121.7-130.3 nm, with a polydispersity index (PDI) <0.5 and a surface zeta potential close to neutral (-0.2 mV). Figure 4 E); This property can reduce the recognition and clearance of immune cells in the blood, prolong the circulation time in the body, and create conditions for drug delivery to the skin lesion site.

[0043] Bioactivity retention: SDS-PAGE protein electrophoresis showed that the protein profile of NLCG was highly consistent with that of the native neutrophil membrane. Figure 5 A); Western blotting confirmed that NLCG successfully retained the inflammatory chemokine CD62L (mediating homing to inflammatory sites) and the cytokine neutralizing receptor TNF-αR (directly neutralizing pro-inflammatory factors) on the neutrophil membrane. Figure 5 B), ensuring its biomimetic anti-inflammatory function; in vitro drug release experiments showed that NLCG released approximately 78.64% of GSK199 within 48 hours, exhibiting sustained-release characteristics that are continuously controllable. Figure 5 (C) can maintain a long-lasting effective drug concentration at the site of skin lesions, avoiding insufficient efficacy caused by the rapid metabolism and clearance of the drug.

[0044] 3. GNLC exhibits excellent biocompatibility, addressing the potential toxicity issues of existing drug delivery systems. Cytotoxicity, hemolysis, and in vivo safety assessments confirmed that GNLC had no significant toxic side effects. Cytotoxicity validation: The cytotoxicity of different concentrations of GNLC on neutrophils and RAW264.7 macrophages was detected using the CCK-8 assay and Calcein AM / PI double staining method. The results showed that within the effective therapeutic concentration range (≤100 μg / mL), the cell viability was >90%, and there was no significant difference compared with the blank control group. Figure 6 (A) This demonstrates that GNLC does not cause significant damage to target cells or normal immune cells.

[0045] Blood compatibility verification: Different concentrations (10~500 μg / mL) of GNLC were co-incubated with 4% mouse erythrocyte suspension. The results showed that its hemolysis rate was consistently <5%, a safe threshold, and significantly lower than that of naked liposomes without neutrophil membrane coating. Figure 6 (B) meets the blood safety requirements for intravenous administration.

[0046] The above results indicate that GNLC has excellent biocompatibility, which solves the problem that some existing nanodelivery systems are limited in clinical application due to insufficient biocompatibility.

[0047] 4. Achieve dual enrichment through "biomimetic chemotaxis + active targeting," significantly improving drug delivery efficiency. In an IMQ-induced mouse model of psoriasis, the targeted delivery capability of GNLC was validated using fluorescence imaging: In vitro imaging validation: In vitro organ imaging was performed on mice intravenously injected with different formulations of DiD fluorescently labeled (naked liposome L-DiD, LC-DiD conjugated only with CD177 peptide, NL-DiD covering only the neutrophil membrane, and NLC-DiD corresponding to GNLC). The results showed that the fluorescence intensity of NLC-DiD in the skin lesions was significantly higher than that in other control groups. Figure 7 (A, B) proves that it has the best enrichment efficiency at the lesion site.

[0048] In vivo imaging verification: Dynamic in vivo imaging showed that NLC-DiD began to accumulate at the lesion site 3-6 hours after injection, reaching a fluorescence peak at 12 hours, and the fluorescence signal highly overlapped with the lesion boundary, lasting for more than 24 hours. Figure 7 C).

[0049] This result confirms that NLCG achieves drug delivery to CD177 in psoriatic lesions through a synergistic effect of "neutrophil membrane-mediated passive chemotaxis" and "CD177 peptide-mediated active recognition of pathogenic cells." + Precise delivery of neutrophils solves the problems of existing technologies, such as the difficulty in targeting and enriching drugs at the lesion site and low delivery efficiency.

[0050] 5. Demonstrates remarkable therapeutic efficacy in psoriasis models, simultaneously improving systemic symptoms and local pathological lesions. In an IMQ-induced mouse model of psoriasis, GNLC treatment was significantly superior to existing control formulations (naked GSK199, drug-free NLC, and GNL without a target peptide): Macroscopic phenotypic improvement: Daily observation and recording of erythema, scaling, and thickening of the dorsal skin of mice were performed. Assessment based on the Psoriasis Area and Severity Index (PASI) score (0–12 points) showed that the model control group (MC) mice exhibited typical psoriasis-like changes on their dorsal skin, including severe erythema, sclerosis, and scaling. The GNLC treatment group showed rapid and significant improvement in skin lesions; by day 5, the skin had essentially returned to smoothness. Its total PASI score and scores for sclerosis, scaling, and erythema were the lowest, significantly superior to the bare GSK group, the GNL group without a targeted peptide (GSK199@NM-LNP), and the unloaded NLC group (NM-LNP-CD177). Figure 8 B); Macroscopic photographs visually demonstrate the significant regression of skin lesions after GNLC treatment ( Figure 8 A).

[0051] Histopathological repair: H&E staining of the skin showed typical pathological changes in the skin of mice in the model control group, including significant epidermal thickening (acanthosis), parakeratosis, and a large number of inflammatory cell infiltrations in the dermis; the GNLC treatment group effectively reversed the above pathological changes, the epidermal thickness returned to normal, the parakeratosis disappeared, the number of inflammatory cell infiltrations in the dermis was significantly reduced, and the repair effect was better than that of other control groups. Figure 9 ).

[0052] 6. Achieve a synergistic effect of "inhibiting NETs + reshaping the immune microenvironment" to block the pathological cycle. Targeted inhibition of NETs: The formation of NETs and the "Munro microabscesses" formed by neutrophil aggregation in psoriatic lesions are classic pathological features and key drivers of persistent inflammation. Immunofluorescence staining of skin lesions showed that a large amount of Ly6G was present in the skin lesions of the model control mice. + Neutrophil infiltration and MPO + NET formation; GNLC treatment group significantly reduced intralesional Ly6G. + The number of neutrophil infiltrations, while fundamentally inhibiting MPO. + The formation of NETs was significantly reduced, and the inhibitory effect was significantly better than that of the bare GSK group and the GNL group without the target peptide. Figure 10 A) demonstrates that it can specifically act on CD177. + Neutrophils, blocking NETs-related pathological processes.

[0053] Immune microenvironment remodeling: In chronic inflammation of autoimmune diseases, macrophage phenotypic balance is crucial. Pro-inflammatory M1 macrophages drive tissue damage, while anti-inflammatory M2 macrophages promote inflammation resolution and tissue repair. Immunofluorescence staining directly confirmed that after GNLC treatment, the expression of the M1 marker iNOS in skin lesions was decreased, while the expression of the M2 marker CD206 was significantly increased. Figure 10 B).

[0054] These results confirm that GNLC not only blocks NET formation at its source but also actively guides the immune response towards anti-inflammatory and repair-promoting directions, overcoming the limitation of existing technologies that can only focus on anti-inflammation and cannot block the pathological cycle.

[0055] 7. Long-term in vivo safety validation provides a guarantee for clinical translation. Systemic safety was assessed by administering high-dose repeated administration (5 tail vein injections over 10 days, at twice the treatment dose) to healthy C57BL / 6J mice. Blood routine and biochemical indicators: After the administration, there were no significant differences in the levels of white blood cells (WBC), red blood cells (RBC), and hemoglobin (HGB) in the GNLC group mice compared with the PBS control group. Figure 11 A); Serum liver function indicators ALT, AST, kidney function indicators, and UREA were all within the normal physiological range, and there was no statistically significant difference compared with the PBS control group. Figure 11 B).

[0056] Organ pathological examination: H&E staining of the heart, liver, spleen, lungs, and kidneys showed that the tissue structure of each organ in the GNLC group was intact, without inflammatory infiltration, cell necrosis, fibrosis, or other pathological changes, consistent with the PBS control group. Figure 11 C).

[0057] In summary, this invention is based on psoriasis "CD177". + The core pathological mechanism of neutrophils-NETs creatively integrates the inflammatory chemotaxis / cytokine neutralization function of neutrophil membrane, the precise targeting function of CD177 peptide, and the NETs inhibition function of PADI4 inhibitor. The constructed GNLC achieves a synergistic therapeutic effect of "targeted enrichment-mechanism blockade-microenvironment remodeling", while also possessing excellent stability and safety, providing a new precision treatment strategy for psoriasis that is far superior to existing technologies. Attached Figure Description

[0058] Figure 1 Immunofluorescence staining of skin tissues from psoriasis patients, healthy controls, imiquimod-induced psoriasis mice, and wild-type mice showed CD177. + Neutrophil and MPO expression were elevated in both psoriasis patients and psoriasis mice.

[0059] Figure 2 The relative expression levels of CD177, ELANE, and MPO mRNA in the skin tissue of psoriasis patients were significantly higher than those in healthy controls.

[0060] Figure 3 The expression levels of CD177 and MPO in the skin tissue of psoriasis patients were positively correlated. The heatmap showed that in the subgroup of patients with high CD177 expression, the spectrum of NETs-related genes exhibited a significant upregulation trend.

[0061] 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).

[0062] 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) of LNP, neutrophils, NM, and NM-LNP-CD177; (C) In vitro drug release curves of GNLC nanoparticles; (D) Drug loading of GNLC.

[0063] 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).

[0064] Figure 7 (A) In vitro fluorescence imaging of major organs in psoriatic 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 skin tissue of imiquimod-induced psoriatic mice (n=4); (C) Representative in vivo imaging of nanoparticles in mice at different time points after different treatments.

[0065] Figure 8(A) Phenotypic changes of dorsal skin in a mouse model of psoriasis on days 1–5 (left: PASI quantitative heatmap; right: corresponding skin phenotype map) (n=8); (B) Trends in the scores of dorsal skin severity index (PASI), duration, desquamation, and erythema in a mouse model of psoriasis over time (n=8).

[0066] Figure 9 Representative H&E staining images of skin tissue from a mouse model of psoriasis.

[0067] Figure 10 (A) Representative immunofluorescence staining images of Ly6G (red) and MPO (green) expression in skin tissue of a mouse model of psoriasis. Cell nuclei were counterstained with Hoechst (blue); (B) Representative immunofluorescence staining images of F4 / 80 (green), iNOS (red), and CD206 (yellow) expression in skin tissue of a mouse model of psoriasis. Cell nuclei were counterstained with Hoechst (blue).

[0068] Figure 11 (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

[0069] 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.

[0070] 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.

[0071] The preparation method of the membrane-modified liposomes (GNLC) targeting CD177-positive neutrophils used in the following examples is as follows: (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.

[0072] (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).

[0073] 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).

[0074] (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.

[0075] Characterization of membrane-modified liposomes (GNLC) targeting CD177-positive neutrophils: I. Nanoparticle Characterization Methods 1. Morphology 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).

[0076] 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.

[0077] 3. Membrane protein detection (Coomassie blue staining + Western blotting) 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.

[0078] 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.

[0079] II. Nanoparticle Characterization Results This invention demonstrates the successful construction and stability of GNLC through experimental data: 1) Structural characterization: TEM images show that GNLCs are uniformly spherical with a clear "neutrophil membrane shell-liposome core" nucleoshell structure. Figure 4 B); The UV-Vis absorption spectrum contains characteristic absorption peaks of GSK199 (PADI4 inhibitor) and the CD177 targeting peptide, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) confirms the successful coupling of DSPE-PEG2000 with the CD177 peptide. Figure 4 C, D).

[0080] 2) Colloidal stability: GNLC particles have uniform size (approximately 121.7-130.3 nm), PDI < 0.5, and surface charge is close to neutral (-0.2 mV). Figure 4 E); This property can reduce the recognition and clearance of immune cells in the blood, prolong the circulation time in the body, and create conditions for drug delivery to the skin lesion site.

[0081] 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 ).

[0082] 4) Drug loading rate: The drug loading rate of GSK199 in GNLC was 16.97%.

[0083] Example 1: The pathological basis and core target of the drug of the present invention for achieving precision treatment of psoriasis The specific experimental method is as follows: Immunofluorescence staining: Immunofluorescence staining was used to detect target protein expression and immune complex deposition. Paraffin-embedded sections were baked, dewaxed, and rehydrated, then placed in antigen retrieval solution and microwaved for antigen retrieval. The sections were permeabilized with 0.2% Triton X-100 for 5 min, followed by blocking with blocking buffer (PBS containing 1% donkey serum and 5% bovine serum albumin) at room temperature for 1 h. The sections were incubated overnight at 4°C with MPO and CD177 primary antibodies, washed three times with PBS, and then incubated with the corresponding Alexa Fluor 488 / 555 / 647 labeled secondary antibodies at room temperature for 45 min. The nuclei were stained with Hoechst 33342, mounted with ProLong mounting medium, and finally images were acquired using a ZEISS A1 confocal fluorescence microscope.

[0084] Bioinformatics analysis.

[0085] Gene expression datasets (GSE13355, GSE14905) were obtained from the Gene Expression Comprehensive Database (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ). High-throughput sequencing data were screened for differentially expressed genes (DEGs) using the DESeq2 software package (v.1.48.1), and microarray data were standardized using the limma software package (v.3.64.1). The Mann-Whitney U test was used to compare the expression levels of individual genes between the two groups. In addition, gene set enrichment analysis (GSEA) was performed using GSEA software (v1.70.0) in conjunction with the MSigDB gene set, and genes were sorted by log2 fold change (log2 FoldChange).

[0086] This invention establishes CD177 through dual validation using clinical samples and animal models. + Neutrophils play a central role as the primary source of pathological NETs in psoriasis. Clinical sample validation: Immunofluorescence staining of skin lesions from psoriasis patients showed that CD177... + The number of neutrophil infiltrations and the expression level of MPO, a key product of NETs, ​​were significantly higher than those in healthy controls. Figure 1 A); Mining public transcriptome datasets (GSE13355, GSE14905) revealed that the mRNA levels of CD177, NETs-related genes ELANE, and MPO were significantly upregulated in psoriatic lesions. Figure 2 Furthermore, CD177 expression is positively correlated with MPO expression. Figure 3 A), the overall NETs-related gene set in patients with high CD177 expression showed an upregulated trend ( ). Figure 3 B).

[0087] Animal model validation: In an imiquimod (IMQ)-induced mouse model of psoriasis, CD177 was detected in the skin lesions. + Neutrophil infiltration and MPO deposition were significantly higher in wild-type control mice than in wild-type control mice. Figure 1 B).

[0088] The above results clarify that "CD177" + Neutrophil-NETs are a key pathogenic pathway in psoriasis, and this invention targets CD177. + The design of neutrophils provides a solid theoretical basis and solves the core problems of unclear identification of pathogenic neutrophil subsets in psoriasis and lack of specificity in targeting in existing technologies.

[0089] Example 2: Evaluation of the efficacy of membrane-modified liposomes targeting CD177-positive neutrophils I. Experimental Methods 1. Animal model construction (IMQ-induced psoriasis model) 1) Animals and modeling: 8-10 week old C57BL / 6J mice were selected and acclimatized for 1 week. 5% IMQ cream (62.5 mg / mouse) was evenly applied to the hairless area on the back of the mouse every day for 5 consecutive days to induce psoriasis-like lesions.

[0090] 2) Grouping and administration: Animals were randomly divided into 6 groups (n=8 / group), and different components of nanomaterials were injected into the tail vein (3mg / kg, administered once on days 1, 3 and 5 respectively); normal control (NC, no modeling) and model control (MC, modeling only) were set up, and equal volumes of physiological saline were injected. 3) Sample collection: Mice were sacrificed on day 5 of the experiment, and skin tissue was collected. Skin erythema, scaling, and thickening were observed and recorded daily during the experiment, and scored according to the Psoriasis Area and Severity Index (PASI, 0–12 points).

[0091] 2. In vivo and in vitro functional testing methods 2.1 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=4 / 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.

[0092] 2.2 Histology and Immunostaining 1) Histological analysis: Heart, liver, spleen, lung, kidney and skin target organs were collected, and skin of the psoriasis model was stained with HE. 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 with primary antibody (anti-mouse MPO, Ly6G, F4 / 80, iNOS, CD206) at 4℃; incubated with Alexa Fluor-conjugated secondary antibody at room temperature for 45 min, counterstained with Hoechst 33342, and observed under a confocal microscope (ZEISS).

[0093] 3. Biosafety assessment methods 3.1 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.

[0094] 3.2 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).

[0095] 3.3 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.

[0096] II. Experimental Results 1. Biosafety assessment results Cytotoxicity validation: The cytotoxicity of different concentrations of GNLC on neutrophils and RAW264.7 macrophages was detected using the CCK-8 assay and Calcein AM / PI double staining method. The results showed that within the effective therapeutic concentration range (≤100 μg / mL), the cell viability was >90%, and there was no significant difference compared with the blank control group. Figure 6 (A) This demonstrates that GNLC does not cause significant damage to target cells or normal immune cells.

[0097] Blood compatibility verification: Different concentrations (10~500 μg / mL) of GNLC were co-incubated with 4% mouse erythrocyte suspension. The results showed that its hemolysis rate was consistently <5%, a safe threshold, and significantly lower than that of naked liposomes without neutrophil membrane coating. Figure 6 (B) meets the blood safety requirements for intravenous administration.

[0098] 2. Results of in vivo and in vitro functional tests of IMQ-induced psoriasis model mice In an IMQ-induced mouse model of psoriasis, the targeted delivery capability of GNLC was validated using fluorescence imaging: In vitro imaging validation: In vitro organ imaging was performed on mice intravenously injected with different formulations of DiD fluorescently labeled (naked liposome L-DiD, LC-DiD conjugated only with CD177 peptide, NL-DiD covering only the neutrophil membrane, and NLC-DiD corresponding to GNLC). The results showed that the fluorescence intensity of NLC-DiD in the skin lesions was significantly higher than that in other control groups. Figure 7 (A, B) proves that it has the best enrichment efficiency at the lesion site.

[0099] In vivo imaging verification: Dynamic in vivo imaging showed that NLC-DiD began to accumulate at the lesion site 3-6 hours after injection, reaching a fluorescence peak at 12 hours, and the fluorescence signal highly overlapped with the lesion boundary, lasting for more than 24 hours. Figure 7 C).

[0100] In an IMQ-induced mouse model of psoriasis, GNLC treatment was significantly superior to existing control formulations (naked GSK199, drug-free NLC, and GNL without a target peptide): Macroscopic phenotypic improvement: Daily observation and recording of erythema, scaling, and thickening of the dorsal skin of mice were performed. Assessment based on the Psoriasis Area and Severity Index (PASI) score (0–12 points) showed that the model control group (MC) mice exhibited typical psoriasis-like changes on their dorsal skin, including severe erythema, sclerosis, and scaling. The GNLC treatment group showed rapid and significant improvement in skin lesions; by day 5, the skin had essentially returned to smoothness. Its total PASI score and scores for sclerosis, scaling, and erythema were the lowest, significantly superior to the bare GSK group, the GNL group without a targeted peptide (GSK199@NM-LNP), and the unloaded NLC group (NM-LNP-CD177). Figure 8 B); Macroscopic photographs visually demonstrate the significant regression of skin lesions after GNLC treatment ( Figure 8 A).

[0101] Histopathological repair: H&E staining of the skin showed typical pathological changes in the skin of mice in the model control group, including significant epidermal thickening (acanthosis), parakeratosis, and a large number of inflammatory cell infiltrations in the dermis; the GNLC treatment group effectively reversed the above pathological changes, the epidermal thickness returned to normal, the parakeratosis disappeared, the number of inflammatory cell infiltrations in the dermis was significantly reduced, and the repair effect was better than that of other control groups. Figure 9 ).

[0102] Targeted inhibition of NETs: The formation of NETs and the "Munro microabscesses" formed by neutrophil aggregation in psoriatic lesions are classic pathological features and key drivers of persistent inflammation. Immunofluorescence staining of skin lesions showed that a large amount of Ly6G was present in the skin lesions of the model control mice. + Neutrophil infiltration and MPO + NET formation; GNLC treatment group significantly reduced intralesional Ly6G. + The number of neutrophil infiltrations, while fundamentally inhibiting MPO. + The formation of NETs was significantly reduced, and the inhibitory effect was significantly better than that of the bare GSK group and the GNL group without the target peptide. Figure 10 A) demonstrates that it can specifically act on CD177. + Neutrophils, blocking NETs-related pathological processes.

[0103] Immune microenvironment remodeling: In chronic inflammation of autoimmune diseases, macrophage phenotypic balance is crucial. Pro-inflammatory M1 macrophages drive tissue damage, while anti-inflammatory M2 macrophages promote inflammation resolution and tissue repair. Immunofluorescence staining directly confirmed that after GNLC treatment, the expression of the M1 marker iNOS in skin lesions was decreased, while the expression of the M2 marker CD206 was significantly increased. Figure 10 B).

[0104] 3. In vivo safety assessment results Healthy C57BL / 6J mice were given repeated tail vein injections of GNLC over 10 days, with a total of 5 high doses (6 mg / kg), to assess its systemic toxicity. Blood routine and biochemical indicators: After the administration, there were no significant differences in the levels of white blood cells (WBC), red blood cells (RBC), and hemoglobin (HGB) in the GNLC group mice compared with the PBS control group. Figure 11 A); Serum liver function indicators ALT, AST, kidney function indicators, and UREA were all within the normal physiological range, and there was no statistically significant difference compared with the PBS control group. Figure 11 B).

[0105] Organ pathological examination: H&E staining of the heart, liver, spleen, lungs, and kidneys showed that the tissue structure of each organ in the GNLC group was intact, without inflammatory infiltration, cell necrosis, fibrosis, or other pathological changes, consistent with the PBS control group. Figure 11 C).

[0106] 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. Application of membrane-modified liposomes targeting CD177-positive neutrophils in the preparation of drugs for the prevention and / or treatment of psoriasis; The drug targets CD177-positive neutrophils via membrane-modified liposomes, which have 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 application according to claim 1, characterized in that: The prevention and / or treatment of psoriasis is manifested in at least one of the following aspects: 1) Improves skin damage caused by psoriasis; 2) Repairing skin pathological damage caused by psoriasis; 3) Lower the PASI score; 4) Reduce Ly6G in skin lesions caused by psoriasis + The number of neutrophil infiltrations; 5) Inhibits MPO caused by psoriasis + The formation of NETs.

3. The application according to claim 2, characterized in that: The skin lesions caused by psoriasis include erythema, hardening of the skin, and scaling.

4. The application according to claim 2, characterized in that: The repair of skin pathological damage caused by psoriasis is further reflected in: the epidermal thickness returning to normal, the disappearance of parakeratosis, and a significant reduction in the number of inflammatory cells infiltrating the dermis.

5. The application according to any one of claims 1-4, 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. The PADI4 inhibitors include, but are not limited to, GSK199, GSK484, and Cl-amidine; The mass ratio of the PEG lipid to the steroid is 10:

1.

6. The application according to any one of claims 1-5, characterized in that: The amino acid sequence of the CD177 targeting peptide is CGGGTIRLNPMPKYFD.

7. The application according to any one of claims 1-6, characterized in that: The mass ratio of the CD177 targeting peptide to the PEG lipid is 1:(1-1.5). And / or, the total lipids in the liposomes loaded with the PADI4 inhibitor have a mass ratio of 11:1 to the native neutrophil membrane.

8. A medicament for the prevention and / or treatment of psoriasis, wherein the active ingredient comprises a membrane-modified liposome targeting CD177-positive neutrophils as described in any one of claims 1-7.

9. The medicament according to claim 8, characterized in that: The drug also contains pharmaceutically acceptable carrier materials.

10. A method of treating and / or preventing a disease or condition in a subject, comprising administering the drug of claim 8 or 9 to a subject in need; in, The disease is psoriasis; the symptoms are those caused by psoriasis.

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