Epimedium flavonoid phospholipid complex cell-targeting inhalation preparation and preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical preparations, and provides a cell-targeting inhalation preparation of phospholipid complex of icariin flavonoids and a preparation method and application thereof. The preparation of the application forms a core by compounding icariin flavonoids as active ingredients with PC phospholipids, introduces non-PC auxiliary phospholipids to optimize the delivery performance, further coats a cell membrane or a blank liposome membrane, and finally modifies a specific targeting peptide to realize multi-level targeted delivery. The application utilizes phospholipid complex to improve the drug loading capacity and bioavailability, combines membrane coating and active targeting modification, significantly delays drug release, enhances the enrichment of drugs in the lung lesion site, and precisely delivers to the lung microvascular endothelium, lung alveolar epithelium or inflammation-related cells; the preparation process is mild and controllable, and has good atomization performance; the preparation of the application can effectively prevent and treat chronic obstructive pulmonary disease, acute respiratory distress syndrome and pulmonary fibrosis and the like, and has important clinical application prospects.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical preparation technology. Specifically, this application provides a cell-targeted inhalation formulation of icariin flavonoid phospholipid complex, its preparation method, and its application. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), and pulmonary fibrosis are respiratory diseases that seriously threaten human health. While their causes differ, they share a pathological continuum of "inflammation-damage-repair / fibrosis." COPD is characterized by persistent airflow limitation, with its pathological basis including chronic small airway inflammation, airway remodeling, and emphysema. ARDS is an acute diffuse lung injury syndrome induced by various factors such as infection, sepsis, aspiration, trauma, and shock. Its core pathological features include alveolar-capillary barrier disruption, inflammatory cell infiltration, pulmonary edema, hyaline membrane formation, and impaired gas exchange. If acute damage is not controlled in time, it can progress to the fibrotic proliferative phase, characterized by fibroblast activation, collagen deposition, and lung tissue remodeling, ultimately leading to pulmonary fibrosis and even respiratory failure. Pulmonary fibrosis is characterized by abnormal fibroblast proliferation, excessive extracellular matrix deposition, lung tissue structure destruction, and decreased lung compliance. It can be induced by various factors, including environmental factors, drugs, radiation therapy, or persistent ARDS. Therefore, the pathological transformation of "inflammation-fibrosis" runs through the disease process of COPD, ARDS and pulmonary fibrosis, and intervention targeting this pathological link has important clinical significance.

[0003] Icariin, icariin C, and epimedin are the main flavonoid active components of the traditional Chinese medicine Epimedium, possessing multi-target and multi-pathway synergistic anti-inflammatory and anti-fibrotic effects, suitable for the prevention and treatment of inflammatory and fibrotic lung diseases. However, these components generally suffer from low bioavailability, insufficient exposure to lung tissue, and short retention time at lesion sites. Conventional suspensions, solutions, or ordinary preparations are unable to achieve effective drug concentrations in lung tissue and lesion-related cells such as pulmonary vascular endothelial cells and alveolar epithelial cells, thus limiting their full therapeutic potential.

[0004] Phospholipid complex technology is an effective strategy for improving the pharmaceutical properties of the aforementioned flavonoids. Phospholipid complexes are composite systems formed by non-covalent bonding between drugs and phospholipids through hydrogen bonds, hydrophobic interactions, van der Waals forces, and electrostatic interactions. Compared to traditional liposomes, phospholipid complexes offer advantages such as simple preparation processes, high drug loading capacity, good stability, and ease of hydration and dispersion to form nano-suspensions for further preparation into nebulized inhalation formulations, making them suitable for pulmonary drug delivery.

[0005] However, the target sites and target cells of icariin flavonoids differ in the treatment of COPD, ARDS, and pulmonary fibrosis, while phospholipid complex formulations themselves lack the ability to accumulate at target sites and on target cells. Therefore, developing targeted delivery systems that address specific disease pathological characteristics to achieve precision treatment has significant clinical value. Summary of the Invention

[0006] The purpose of this invention is to provide an icariin flavonoid phospholipid complex and its cell-targeting formulation to improve the formulation stability, lung delivery performance and lesion tissue exposure of icariin, icariin C and icariin.

[0007] Another objective of this invention is to provide a cell membrane biomimetic formulation or a blank liposome membrane-coated formulation. By coating the surface of a phospholipid complex suspension with a cell membrane or a blank liposome membrane, the formulation can improve its efficiency in recognizing, enriching, and delivering cells related to lung lesions; on the other hand, it can slow down the drug release rate and prolong the drug's residence time in the lungs and its target sites.

[0008] Another objective of this invention is to provide a cell-targeting formulation modified with a targeted peptide. By introducing specific targeting peptides onto the surface of phospholipid complexes, cell membrane biomimetic formulations, or blank liposome-coated formulations, precise targeted delivery to key lesion cells such as pulmonary vascular endothelial cells, inflammatory endothelial cells, and alveolar epithelial cells can be achieved, thereby significantly improving the therapeutic effects against COPD, ARDS, and pulmonary fibrosis. This targeted modification strategy can endow drugs with active targeting capabilities without relying on cell membrane coating, simplifying the preparation process and reducing transformation barriers, which is beneficial for the clinical promotion and industrial application of the product.

[0009] Specifically: On one hand, this application provides a cell-targeted inhalation formulation of an icariin flavonoid phospholipid complex, the formulation comprising: (a) A phospholipid complex core, wherein the phospholipid complex core is formed by intermolecular non-covalent bonding of icariin flavonoids and PC-type phospholipids; wherein the icariin flavonoids are selected from one or more of icariin, icariin C and icariin; And optional: (b) Auxiliary phospholipids, wherein the auxiliary phospholipids are selected from one or more of PG phospholipids, PI phospholipids, PS phospholipids and PEGylated PE phospholipids; (c) A coating layer, wherein the coating layer is a cell membrane component or a blank liposome membrane component; the cell membrane component is derived from neutrophils, apoptotic macrophages, platelets, alveolar epithelial cells, pulmonary vascular endothelial cells, exosomes, or extracellular vesicles; the blank liposome is a DPPC blank liposome. (d) Targeting peptides, wherein the targeting peptides are selected from pulmonary vascular endothelial targeting peptides, inflammation targeting peptides, alveolar epithelial targeting peptides, neutrophil targeting peptides or macrophage targeting peptides.

[0010] Furthermore, the PC-type phospholipids are selected from soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, distearate phosphatidylcholine, or combinations thereof.

[0011] Furthermore, the mass ratio of epimedium flavonoids to PC phospholipids in the core of the phospholipid complex is 1:1-10; preferably 1:2-6.

[0012] Further, in the formulation, the mass ratio of PC phospholipids to PG phospholipids is 1:0.03-0.4, preferably 1:0.05-0.15; the mass ratio of PC phospholipids to PI phospholipids is 1:0.01-0.15, preferably 1:0.02-0.10; the mass ratio of PC phospholipids to PS phospholipids is 1:0.01-0.15, preferably 1:0.02-0.8; and the mass ratio of PC phospholipids to PEGylated PE phospholipids is 1:0.002-0.02, preferably 1:0.005-0.02.

[0013] Furthermore, the cell membrane components are neutrophil cell membrane components, apoptotic macrophage cell membrane components, and platelet cell membrane components.

[0014] Furthermore, the mass ratio of the cell membrane component or blank liposome component to the epimedium flavonoid compound is 0.05:1-10:1, preferably 0.1:1-2:1, and more preferably 1:2.

[0015] Further, the targeting peptide is CGSPGWVRC, NNQKIVNLKEKVAQLEA, cyclo(1,12)PenITDGEATDSGC, VHPKQHR, VHPKQHRGGSKGC, RGD, cRGD, c(RGDfK), iRGD, CNGRC, NGR, GEBP11 peptides, CRKDKC and their conserved variants, truncated forms, cyclized forms or D-amino acid substitutes.

[0016] Furthermore, the targeting peptide is linked to other components of the formulation via a linker arm, wherein the linker arm is DSPE-PEG-, DPPE-PEG-, cholesterol-PEG-, fatty acid-PEG-, or maleimide-PEG-; the modification ratio of the targeting peptide is 0.01-20 mol% of the total lipid molar amount, preferably 0.1-10 mol%, more preferably 1-5 mol%.

[0017] Furthermore, the formulation is an atomized drug. When used, it forms an inhalable atomized aerosol through an air compressor nebulizer or a vibrating screen nebulizer.

[0018] On the other hand, this application provides the use of the above-mentioned formulation in the preparation of a medicament for treating chronic obstructive pulmonary disease, acute respiratory distress syndrome and / or pulmonary fibrosis.

[0019] On the other hand, this application provides a method for preparing the above-mentioned formulation, the method comprising: (1) Weigh out the flavonoids of Epimedium, PC phospholipids and auxiliary phospholipids, add organic solvent to dissolve and mix evenly, and react at 30-60℃ for 0.5-4 hours to form a phospholipid complex; after dissolution, filtration, rotary evaporation and vacuum drying, the phospholipid complex powder is obtained. (2) Add the phospholipid complex powder to deionized water or physiological saline, and then treat it by ultrasound or homogenization to obtain a phospholipid complex suspension.

[0020] Water-soluble phospholipids can also be added during hydration.

[0021] On the other hand, this application provides a method for preparing the above-mentioned formulation, the method comprising: (1) Weigh out the flavonoids of Epimedium, PC phospholipids and auxiliary phospholipids, add organic solvent to dissolve and mix evenly, and react at 30-60℃ for 0.5-4 hours to form a phospholipid complex; after dissolution, filtration, rotary evaporation and vacuum drying, the phospholipid complex powder is obtained. (2) Add the phospholipid complex powder to deionized water or physiological saline, and then treat it by sonication or homogenization to obtain a phospholipid complex suspension. (3) Dissolve the PEGylated targeting peptide in deionized water to prepare a stock solution, add it to the phospholipid complex suspension, and incubate at 20-40℃ for 0.5-4 hours.

[0022] On the other hand, this application provides a method for preparing the above-mentioned formulation, the method comprising: (1) Weigh the flavonoids of Epimedium, PC phospholipids and auxiliary phospholipids, add them to an organic solvent to dissolve and mix them evenly. Then, react them at 30-60℃ for 0.5-4 hours to form a phospholipid complex. After dissolving, filtering, rotary evaporation and vacuum drying, the phospholipid complex powder is obtained. Add the phospholipid complex powder to deionized water or physiological saline and treat it by sonication or homogenization to obtain a phospholipid complex suspension. (2) Extract cell membrane components or blank liposome membrane components; (3) The phospholipid complex suspension is mixed with cell membrane components or blank liposome membrane components, and then subjected to ultrasonic treatment, extrusion or incubation to coat the surface of the phospholipid complex with blank liposome membrane or cell membrane components. (4) Dissolve the PEGylated targeting peptide in deionized water to prepare a stock solution, add it to the product in step (3), and incubate at 20-40°C for 0.5-4 hours.

[0023] The method for preparing blank liposomes is as follows: DPPC and cholesterol are dissolved in ethanol at a molar ratio of 4:1, and blank liposomes are prepared by thin-film dispersion.

[0024] Beneficial effects: (1) For the first time, icariin flavonoids (icariin, icariin C, icariin) were prepared as phospholipid complexes, and membrane-coated formulations and targeted cell-targeted formulations were further constructed, forming a complete targeted drug delivery system.

[0025] (2) Phospholipid complexes can significantly improve drug dispersibility and formulation stability in hydration media.

[0026] (3) By using cell membrane biomimetic coating or blank liposome coating, drug release can be delayed, and the drug's residence time and duration of action in the lungs can be prolonged.

[0027] (4) By adding PG, PI and PS phospholipids, the uptake of drugs by lung epithelial cells, respiratory epithelial cells and lung immune cells can be promoted respectively.

[0028] (5) Cell membrane-coated biomimetic formulations endow the formulations with inflammatory chemotactic properties and cell recognition capabilities. For example, the icariin phospholipid complex coated on the neutrophil membrane achieved an uptake rate of 63.9% on pulmonary vascular endothelial cells, which is about twice that of the uncoated formulation (33.9%).

[0029] (6) Targeted peptide modification further enhances the targeting of the formulation to specific cells, which is significantly better than the unmodified formulation.

[0030] (7) Different targeting strategies can provide precise intervention for the different pathological characteristics of COPD, ARDS and pulmonary fibrosis: CGSPGWVRC (Let peptide): Targets pulmonary vascular endothelial cells for anti-inflammatory treatment of ARDS and anti-fibrotic treatment of pulmonary fibrosis; NNQKIVNLKEKVAQLEA: Targets pulmonary epithelial cells for epithelial barrier repair in COPD and alveolar regeneration in pulmonary fibrosis; cyclo(1,12)PenITDGEATDSGC: Targets pulmonary fibroblasts, blocking the progression of pulmonary fibrosis by inhibiting fibroblast activation and collagen deposition; VHPKQHR / VHPKQHRGGSKGC: Targets vascular endothelial cells at sites of inflammation, for anti-inflammatory treatment and regulation of vascular permeability in the acute phase of ARDS; RGD / cRGD / c(RGDfK) / iRGD: Targets activated fibroblasts and abnormal vascular endothelial cells with high expression of integrin αvβ3, for anti-vascular remodeling in pulmonary fibrosis and airway remodeling in COPD. CNGRC / NGR: Targets inflammation-associated endothelial cells with high CD13 / APN expression, used to inhibit abnormal angiogenesis in COPD complicated with lung cancer or pulmonary fibrosis; GEBP11 peptide: Specifically recognizes pulmonary vascular endothelial cells, used to reduce pulmonary edema and protect pulmonary vessels in ARDS; CRKDKC: Targets pulmonary immune cells such as macrophages, used to regulate chronic inflammation in COPD and suppress inflammatory storms in ARDS, reducing tissue damage by promoting the polarization of M1 macrophages to M2.

[0031] Targeted peptides can be achieved through conserved variants, truncated forms, cyclized forms, D-amino acid substitutes, lipid derivatives, or PEGylated derivatives. Cyclolated forms utilize head-and-tail cyclization or intramolecular disulfide bond cyclization to enhance protease stability; D-amino acid substitutes replace some or all L-amino acids with D-type forms to resist enzymatic degradation and prolong half-life; lipid derivatives link fatty acid chains to the N- or C-terminus of the peptide chain to enhance membrane affinity and cellular uptake efficiency; PEGylated derivatives extend circulation time and reduce immune clearance by linking polyethylene glycol chains. Each modification can be used alone or in combination to optimize targeted delivery efficiency.

[0032] (8) The endothelial cell-targeting icariin phospholipid complex formulation of the present invention showed significant protective effects in ARDS mouse models, with a 7-day survival rate of 80%, which was superior to the 40% of the unmodified phospholipid complex formulation. Attached Figure Description

[0033] Figure 1 In vitro release curves of icariin phospholipid complex IPC, neutrophil membrane-coated icariin phospholipid complex NM@IPC, and Let-modified NM@IPC (n=3, mean ± standard deviation).

[0034] Figure 2In vitro cellular uptake of cell membrane biomimetic agents. (a) Representative fluorescence images of AM@IPC, PM@IPC, and NM@IPC uptake in LPS-stimulated neutrophils, MLE-12 lung epithelial cells, and HUVEC lung vascular endothelial cells (scale bar = 100 μm). Quantitative analysis of the mean fluorescence intensity of AM@IPC, PM@IPC, and NM@IPC in LPS-stimulated (b) neutrophils, (c) lung epithelial cells, and (d) HUVEC lung vascular endothelial cells (n = 3, mean ± standard deviation).

[0035] Figure 3 This study investigated the lung distribution of mice with acute lung injury after intrapulmonary administration of icariin phospholipid complex suspension (IPC) or neutrophil membrane-coated icariin phospholipid complex NM@IPC and Let-NM@IPC. (a) Representative fluorescence images of lung tissue sections showing the distribution of nanoparticles in the tissue (scale bar = 20 μm). (b) In vitro fluorescence images of lung tissue. (c) Quantitative analysis of the mean fluorescence intensity obtained 24 hours after administration (n = 4, mean ± standard deviation).

[0036] Figure 4 Cellular uptake analysis of lung tissue in ALI mice. Quantitative analysis of cellular uptake in neutrophils, epithelial cells, and pulmonary vascular endothelial cells in ALI mice after intratracheal instillation of IPC, AM@IPC, PM@IPC, and NM@IPC. (a)(e) . Quantitative uptake analysis of Let-NM@IPC (d)(h) (n=3, mean ± standard deviation).

[0037] Figure 5A The figure shows the anti-inflammatory efficacy of the tracheally administered Chaohuodin C phospholipid complex EPC and the original drug EC in mice with acute exacerbation of COPD (total protein content, n=6, mean ± standard deviation).

[0038] Figure 5B The figure shows the anti-inflammatory efficacy of the tracheally administered Chaohuodin C phospholipid complex EPC and the original drug EC in mice with acute exacerbation of COPD (number of immune cells, n=6, mean ± standard deviation).

[0039] Figure 5C The figure shows the anti-inflammatory efficacy of the tracheally administered Chaohuodin C phospholipid complex EPC and the original drug EC in mice with acute exacerbation of COPD (IL-6, n=6, mean ± standard deviation).

[0040] Figure 5D The figure shows the anti-inflammatory efficacy of the tracheally administered Chaohuodin C phospholipid complex EPC and the original drug EC in mice with acute exacerbation of COPD (TNF-α, n=6, mean ± standard deviation).

[0041] Figure 5E The figure shows the anti-inflammatory efficacy of the tracheally administered phospholipid complex EPC and the original drug EC in mice with acute exacerbation of COPD (IL-1β, n=6, mean ± standard deviation).

[0042] Figure 5F The anti-inflammatory efficacy of the tracheally administered Chaohuodin C phospholipid complex EPC and the original drug EC in mice with acute exacerbation of COPD is shown in the figure (H&E stained lung tissue sections, n=6, mean ± standard deviation).

[0043] Figure 6 The anti-inflammatory effects of icariin phospholipid complex IPC and its cell membrane biomimetic formulation, Let-NM@IPC, administered via endotracheal instillation in mice with acute pneumonia (ALI). (a) H&E-stained lung tissue sections (scale bar = 100 μm). (b) Lung injury score. (c) Percentage of neutrophils in bronchoalveolar lavage fluid (BALF). (d) Number of immune cells and levels of inflammatory factors, including (e) IL-6, (f) TNF-α, and (g) IL-1β, in BALF after endotracheal instillation of IPC, AM@IPC, PM@IPC, NM@IPC, and Let-NM@IPC (n = 6, mean ± standard deviation).

[0044] Figure 7 The effects of intratracheally administered icariin phospholipid complexes IPC, NM@IPC, and Let-NM@IPC on the survival rate of ARDS mouse models. (a) Timeline of model establishment, drug administration, and sample collection. (b) H&E-stained lung tissue sections (magnification: 10× and 20×; scale bar = 100 μm). (c) Lung injury score. (d) Survival curves of ARDS mice (n = 10, mean ± standard deviation).

[0045] Figure 8 Expression of fibrosis-related cytokines (a) IL-6, (b) α-SMA, (c) COL1A1 and (d) TGF-β in lung tissue of mice with bleomycin-induced pulmonary fibrosis. (e) H&E staining (top) (top 1 ×10, top 2 ×20) and Masson staining (bottom) (bottom 1 ×10, bottom 2 ×20) of lung tissue sections (n=6, mean ± standard deviation). Detailed Implementation

[0046] Example 1: Preparation of icariin phospholipid complex Icariin (ICA) and soybean lecithin (SPC) were weighed at a mass ratio of 1:3 and dissolved in a methanol-ethanol mixture at a volume ratio of 1:1 to achieve an icariin concentration of 0.5 mg / mL. The mixture was stirred in the dark at 50°C and 500 rpm for 1 hour. After the reaction was complete, cholesterol (cholesterol to phospholipid mass ratio of 1:3) was added. The organic solvent was then removed by rotary evaporation, and the mixture was vacuum dried overnight to obtain a dry powder of the icariin-phospholipid complex.

[0047] Example 2: Preparation of Icariin Phospholipid Complex (IPC) Suspension The icariin phospholipid complex dry powder prepared in Example 1 was ultrasonically hydrated with pure water or physiological saline in a 50°C water bath, and then extruded through a 0.45 μm polycarbonate membrane to form an icariin phospholipid complex suspension. The suspension had an average particle size of 189.3 ± 5.2 nm, a PDI of 0.305 ± 0.038, a potential of -17.4 ± 0.3 mV, an encapsulation efficiency of 91.78%, and a drug loading of 21.92%.

[0048] Example 3: Preparation of the astragalus C phospholipid complex (EPC) Epmedin C (EC), DPPC, and DSPE-PEG2000 were weighed and dissolved in anhydrous ethanol at a mass ratio of 1:4:0.04. The mixture was stirred in the dark at 50°C and 500 rpm for 1 hour. The organic solvent was removed by rotary evaporation, and the mixture was vacuum dried overnight to obtain EC phospholipid complex (EPC) powder. Subsequently, the powder was ultrasonically hydrated with pure water or physiological saline to form an Epmedin C phospholipid complex suspension. The average particle size of the suspension was 102.7 ± 0.8 nm, the PDI was 0.136 ± 0.03, and the potential was [not specified]. The value was 10.0 ± 0.7 mV, the encapsulation efficiency was 86.60%, and the drug loading was 19.84%.

[0049] Example 4: Preparation of a phospholipid complex containing PG-like phospholipids and phospholipids (PG@EPC) Astragalus C, DPPC, and DSPE-PEG2000 were weighed and dissolved in anhydrous ethanol at a mass ratio of 1:4:0.04. The mixture was stirred in the dark at 50℃ and 500 rpm for 1 hour. The organic solvent was removed by rotary evaporation, and the mixture was vacuum dried overnight to obtain astragalus C phospholipid complex powder. Subsequently, sodium DPPG (DPPG to DPPC mass ratio of 1:10) was dissolved in physiological saline, and the phospholipid complex powder was added and ultrasonically hydrated to form an astragalus C phospholipid complex suspension. The average particle size of the suspension was 102.0±0.4 nm, the PDI was 0.287±0.006, and the Zeta potential was -36.87±0.53.

[0050] Example 5: Preparation of icariin phospholipid complex Icariin (ICT) and soybean lecithin (SPC) were weighed at a mass ratio of 1:3 and dissolved in ethanol to achieve an ICT concentration of 1 mg / mL. The mixture was stirred in the dark at 50°C and 500 rpm for 1 hour, followed by rotary evaporation to remove the organic solvent. The mixture was then vacuum dried overnight to obtain an ICT phospholipid complex powder. Subsequently, the powder was ultrasonically hydrated with pure water or physiological saline to form an ICT phospholipid complex suspension. The average particle size of the suspension was 174.3 ± 0.7 nm, and the PDI was 0.243 ± 0.022.

[0051] Example 6: Preparation of a biomimetic icariin phospholipid complex (NM@IPC) for neutrophil membranes HL-60 cells were induced to differentiate into neutrophils by treatment with 1.3% (v / v) dimethyl sulfoxide (DMSO) for 4 days. Cells were collected, and membrane proteins were extracted using a cell membrane protein and cytoplasmic protein extraction kit. The extracted neutrophil membrane proteins were mixed with IPC nanosuspension at a mass ratio of 1:2 (based on membrane protein:ICA) and sonicated in an ice bath at 40 kHz and 100 W for 5 minutes to promote the self-assembly of membrane vesicles on the IPC surface, thus obtaining the neutrophil membrane biomimetic icariin phospholipid complex (NM@IPC). The average particle size of the suspension was 203.7 ± 4.5 nm, the PDI was 0.357 ± 0.010, and the Zeta potential was -27.9 ± 1.3.

[0052] Example 7: Preparation of a biomimetic icariin phospholipid complex (AM@IPC) for apoptotic macrophage membranes RAW264.7 cells were treated with 1 μg / mL lipopolysaccharide (LPS) for 24 hours to induce apoptosis. The apoptosis rate reached 92.4%, as confirmed by Annexin V-FITC / PI double staining, and the cell morphology changed from round to spindle-shaped. Apoptotic cells were collected, and membrane proteins were extracted using a cell membrane and cytoplasmic protein extraction kit. The extracted apoptotic macrophage membrane proteins were mixed with IPC nanosuspension at a mass ratio of 1:2 (based on membrane protein:ICA). The mixture was sonicated at 40 kHz and 100 W for 5 minutes in an ice bath to promote the self-assembly of membrane vesicles on the IPC surface, yielding the apoptotic macrophage membrane-inspired icariin phospholipid complex (AM@IPC). The average particle size of the suspension was 213.0 ± 2.1 nm, the PDI was 0.323 ± 0.047, and the Zeta potential was -29.9 ± 3.7.

[0053] Example 8: Preparation of a biomimetic icariin phospholipid complex (PM@IPC) for platelet membranes Platelets were isolated from platelet-rich plasma (PRP), centrifuged at 800×g for 20 min to obtain platelet pellet, washed twice with PBS, and resuspended in PBS containing protease inhibitors. After three freeze-thaw cycles (liquid nitrogen / 37°C water bath), platelet membranes were collected by centrifugation at 4,000×g for 3 min, and membrane proteins were extracted using a cell membrane protein and cytoplasmic protein extraction kit. The extracted platelet membrane proteins were mixed with IPC nanosuspension at a mass ratio of 1:2 (based on membrane protein:ICA), and sonicated at 40 kHz and 100 W for 5 min in an ice bath to promote the self-assembly of membrane vesicles on the IPC surface, thus obtaining the platelet membrane biomimetic icariin phospholipid complex (PM@IPC). The average particle size of the suspension was 215.1±5.0 nm, the PDI was 0.366±0.024, and the Zeta potential was -16.3±3.4.

[0054] Example 9: Preparation of blank liposome-coated phospholipid complex Cholesterol and DPPC were weighed at a mass ratio of 1:4 and dissolved in ethanol. The organic solvent was then removed by rotary evaporation, followed by vacuum drying overnight. The resulting film was ultrasonically hydrated with pure water or physiological saline at 50°C and extruded through a 0.45 μm polycarbonate membrane to form blank DPPC liposomes. The blank liposomes were mixed with a phospholipid complex of choropoietin C or IPC at a phospholipid mass ratio of 1:1 and ultrasonically treated at 100 W for 5 minutes to promote the fusion of the liposome membrane and the phospholipid complex, forming blank liposome-coated choropoietin C or icariin phospholipid complexes (BL@EPC or BL@IPC). After coating, the particle size increased by approximately 20-40 nm, and the zeta potential remained negative.

[0055] Example 10: Preparation of Let-peptide-modified icariin phospholipid complex (Let-IPC) A stock solution was prepared by dissolving DSPE-PEG2000-Let in deionized water. This stock solution was then added to the IPC suspension at a mass ratio of DSPE-PEG2000-Let to ICA of 1:1. The solution was incubated in a 37°C water bath for 2 hours in the dark to allow DSPE-PEG2000-Let to insert into the phospholipid complex. Unbound DSPE-PEG2000-Let was removed by centrifugation, yielding the Let peptide-modified IPC. The Let modification ratio was 3.4 mol%.

[0056] Example 11: Preparation of Let peptide-modified NM@IPC (Let-NM@IPC) A stock solution was prepared by dissolving DSPE-PEG2000-Let in deionized water. This stock solution was then added to NM@IPC suspension at a mass ratio of DSPE-PEG2000-Let to ICA of 1:1. The solution was incubated in a 37°C water bath for 2 hours in the dark to allow DSPE-PEG2000-Let to insert into the neutrophil membrane. Unbound DSPE-PEG2000-Let was removed by centrifugation, yielding the Let peptide-modified neutrophil membrane biomimetic icariin phospholipid complex.

[0057] Example 12: Preparation of Let-peptide-modified BL@IPC (Let-BL@IPC) A stock solution was prepared by dissolving DSPE-PEG2000-Let in deionized water. This stock solution was then added to a blank DPPC liposome suspension at a mass ratio of DSPE-PEG2000-Let to ICA of 1:1. The solution was incubated in a 37°C water bath for 2 hours in the dark to allow DSPE-PEG2000-Let to insert into the blank liposome layer. Unbound DSPE-PEG2000-Let was removed by centrifugation, yielding the Let peptide-modified blank liposome-coated icariin phospholipid complex.

[0058] Experimental Example 1: In vitro release evaluation The in vitro release of phospholipid complex suspensions was evaluated using dialysis, and the differences in in vitro release were compared with those before and after nebulization. Specifically, 2 mL of the original drug solution and phospholipid complex suspensions with different coatings or modifications were placed in a dialysis bag (8000-12000 Da), and then placed in 200 mL of solution. The test solution was placed on a magnetic stirrer and released at 37°C and 100 rpm. At predetermined time points, 1 mL of the extravasated solution was collected and replaced with 1 mL of the solution. The solution was diluted with methanol, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The drug content in the supernatant was determined by HPLC.

[0059] The results showed that, compared with drug solutions, the phospholipid complexes of ICA, EC and ICT could delay the drug release rate. After nebulization, the drug release curves of the phospholipid complexes in the collected aerosols and the drug release curves of the pre-nebulization formulations had a similarity factor f2 greater than 60, indicating consistency.

[0060] The release rate of IPC, EPC, and ICT phospholipid complexes slowed down after coating with cell membranes or blank phospholipids. Taking IPC and coated IPC as examples, in vitro release studies showed that membrane coating improved the sustained-release behavior of IPC. Uncoated IPC released approximately 88% of the encapsulated icariin within 24 hours, while NM@IPC and Let-NM@IPC both showed significantly slower release rates, with a cumulative release of approximately 73% within the same time period. The release curves of the latter two formulations were highly consistent, indicating that the targeted peptide modification did not change the drug release characteristics. Figure 1 ).

[0061] Experimental Example 2: In vitro cell targeting properties The cellular targeting ability of phospholipid complexes with different modifications was evaluated using cellular uptake methods. Specifically, 2×10 5 Neutrophils, MLE-12 cells, and HUVEC cells were seeded in 24-well plates. After culturing at 37 °C and 5% CO2 for 24 h, 100 ng / mL LPS was added to each well, and incubation continued for 12 h. Then, 50 μL of different coumarin-labeled phospholipid complexes (with the same C6 fluorescence intensity) was added to each well, and incubation continued for 1 h. After incubation, the culture medium was aspirated from the wells, and the cells were washed three times with PBS buffer. After washing, 500 μL of 4% cell fixative was added to each well, and the cells were fixed at room temperature for 30 min. After removing the fixative, the cells were washed twice with PBS buffer, stained with DAPI for 10 min, rinsed twice with PBS, and then 1 mL of PBS was added before observation. Cells were observed and images were acquired using a fluorescence microscope. Finally, the average fluorescence intensity of the images was semi-quantitatively analyzed using ImageJ software.

[0062] The results showed that PG@EPC enhanced drug uptake in MLE-12 cells due to the addition of PG, while the cell targeting and modification of the cell membrane by the biomimetic phospholipid complex were related. Taking biomimetic IPC as an example (… Figure 2 The modified IPCs (AM@IPC) on the apoptotic macrophage membrane increased drug uptake by epithelial cells, while the modified IPCs (NM@IPC) on the neutrophil membrane increased drug uptake by lung endothelial cells. However, the different modifications did not have a significant effect on the uptake by immune cells.

[0063] Experimental Example 3: Drug Retention in the Lungs To evaluate pulmonary drug retention of different phospholipid complexes administered to the lungs, a mouse model of pulmonary inflammation was first induced by intratracheal instillation of a low dose of lipopolysaccharide. Two hours after induction, different DiD-labeled phospholipid complexes (DiD equivalent of 0.1 mg / kg) were administered via the same route. Real-time fluorescence imaging of mice was performed using an IVIS imaging system at 1, 3, 6, 12, and 24 hours post-administration. At the 24-hour endpoint, mice were sacrificed, and lung tissue was harvested for in vitro imaging and quantitative fluorescence analysis. To further assess intrapulmonary localization, lung tissue sections were counterstained with DAPI and observed under a fluorescence microscope to determine their microscopic distribution.

[0064] The results showed that pulmonary retention of the drug was related to the in vitro drug release rate, and that coating with blank liposome membranes or biomembranes significantly improved pulmonary retention. Examples of pulmonary retention were given for IPC, NM@IPC, and Let-NM@IPC. Figure 3 After 24 hours, the drug was uniformly distributed in the isolated lung tissue sections, indicating that all three formulations had good lung tissue penetration. Semi-quantitative fluorescence analysis showed that the fluorescence intensity of Let-NM@IPC and NM@IPC was significantly higher than that of IPC. Notably, the fluorescence intensity of Let-NM@IPC was roughly equivalent to that of NM@IPC, indicating that Let modification did not bring any additional retention advantage.

[0065] Experiment Example 4: Distribution of Lung Cells in Vivo To investigate drug uptake by neutrophils, alveolar epithelial cells, and pulmonary vascular endothelial cells, mice with LPS-induced lung inflammation were intratracheally administered different DiD-labeled phospholipid complexes. Mice were sacrificed 12 hours post-administration, and lung tissue was harvested for comprehensive cellular analysis. To quantify lung distribution, single-cell suspensions were prepared using enzymatic digestion. Specifically, lung tissue was mechanically minced and digested with 1 mg / mL collagenase I and 0.2 mg / mL DNase I at 37°C for 10 minutes. Digestion was terminated with serum-free medium, and the resulting homogenate was filtered through a 100 μm cell sieve. After centrifugation, erythrocytes were removed with erythrocyte lysis buffer, and the remaining cells were resuspended in PBS for subsequent immunophenotypic analysis. Cells were stained with the following fluorescently labeled antibodies: FITC-labeled anti-Ly-6G antibody, PE-labeled anti-CD326 (Ep-CAM) antibody, PerCP / Cy5.5-labeled anti-CD11b antibody, APC / Cy7-labeled anti-CD45 antibody, and PE / Cy7-labeled anti-CD31 antibody. After incubation in the dark for 15 minutes, the cells were washed twice and analyzed by flow cytometry. Specific cell populations were defined using the following gating strategy, and the cellular uptake of DiD-labeled nanoparticles was quantified: neutrophils (CD11b... + Ly6G + ), alveolar epithelial cells (CD45) -CD326 + ), pulmonary vascular endothelial cells (CD45) - CD31 + ).

[0066] The results showed that the in vivo cell distribution and in vitro cell targeting of different phospholipid complexes were closely related. Taking different IPC results as an example ( Figure 4 Flow cytometry results showed that neutrophils exhibited strong phagocytic capacity, with an uptake rate exceeding 95%, and no significant differences in uptake rate among different formulations. However, in alveolar epithelial cells, the accumulation rate of AM@IPC (84.1%) was significantly higher than that of uncoated IPC (20.8%). In pulmonary vascular endothelial cells, the uptake rate of NM@IPC significantly increased to 63.9%, approximately twice that of unmodified IPC (33.9%). Let peptide modification further enhanced this endothelial affinity, with Let-NM@IPC achieving an uptake rate of 86.8%.

[0067] Experimental Example 5: Efficacy Evaluation of EPC in a COPD Model The effects of LPS on improving airway inflammation, emphysema, and lung tissue damage were evaluated using an inflammatory model simulating acute exacerbations of COPD. Specifically, a COPD model induced by LPS was established in mice via intratracheal instillation, and the pulmonary efficacy of EPC and EC (Epi) suspensions after intratracheal instillation was compared. Mice were randomly assigned to four groups: a saline control group (Ctrl), a model group (LPS), a budesonide positive control group (BUD), an EPC group, and an EC group. Each mouse in the Ctrl group received 50 μL of saline, mice in the budesonide group received commercially available inhaled budesonide suspension via intratracheal instillation at a dose of 0.5 mg / kg, and mice in the EPC / EC groups received intratracheal instillation at doses of 0.5, 1, and 2 mg / kg, respectively. In the LPS-induced model, except for the NS group, mice were instilled with LPS intratracheally 2 hours after administration. All mice were euthanized 6 hours after LPS stimulation, and samples were collected.

[0068] The results showed that low, medium, and high doses of the phospholipid complex EPC improved COPD symptoms in mice, specifically by reducing total protein, total cell count, and inflammatory factor levels in bronchoalveolar lavage fluid, and improving lung tissue pathological damage, all in a dose-dependent manner. Under the same high-dose conditions, EPC showed superior anti-inflammatory effects on the lungs compared to EC, particularly in reducing total cell count and TNF-α levels in bronchoalveolar lavage fluid. Figure 5A -F).

[0069] Experimental Example 6: Evaluation of the anti-inflammatory efficacy of IPC in an ARDS model The anti-inflammatory effect of the formulation was evaluated using an LPS-induced mouse ARDS model. Specifically, Balb / c mice were randomly divided into a control group, a model group, a budesonide (positive control) group, an IPC group, an AM@IPC group, a PM@IPC group, a NM@IPC group, and a Let-NM@IPC group. Mice were administered the corresponding formulation, budesonide (0.5 mg / kg), or saline via intratracheal infusion at the same ICA dose (0.5 mg / kg) before administration. Except for the control group, all groups underwent LPS-induced ARDS modeling 1 hour after administration. Six hours after modeling, the mice were euthanized, and bronchoalveolar lavage fluid and lung tissue samples were collected. The results showed that LPS-induced ARDS mice exhibited neutrophil infiltration in the alveoli and interstitial spaces, alveolar wall thickening, patchy hemorrhage, and other significant features such as pulmonary fibrosis. Intratracheal instillation of IPC and its cell membrane biomimetic formulations (AM@IPC, PM@IPC, and NM@IPC) alleviated lung injury and significantly reduced lung injury scores to varying degrees. Figure 6 Part a). These pathological changes are also reflected in the lung injury score (part a). Figure 6 (Part b). The total BALF cell counts in the AM@IPC, PM@IPC, and NM@IPC groups decreased to 71.4%, 66.0%, and 61.0% of the LPS group, respectively, while the number of neutrophils also decreased accordingly. Figure 6 The cd portion indicates that neutrophils are the dominant cell type in BALF during acute inflammation, and this synchronous decrease reflects effective suppression of the inflammatory response. Inflammation suppression was further confirmed by a significant reduction in the levels of pro-inflammatory factors, including TNF-α, IL-6, and IL-1β. Specifically, IL-6 levels in AM@IPC, PM@IPC, and NM@IPC decreased by 38.2%, 43.6%, and 47.3%, respectively. Figure 6 (e part). TNF-α and IL-1β levels in BALF also showed a similar decreasing trend ( Figure 6 (f and g parts). Notably, the degree of improvement in lung injury and anti-inflammatory effect were positively correlated with the formulation's targeting of endothelial cells, but not with drug exposure to epithelial cells or neutrophils. To further enhance endothelial specificity, our Let peptide-based functionalized formulation, Let-NM@IPC, exhibited superior anti-inflammatory performance compared to NM@IPC. Specifically, Let-NM@IPC reduced the total cell count in BALF to 47.0% in the LPS group, a significant improvement compared to NM@IPC (61.0%). Figure 6 (part d). Furthermore, Let-NM@IPC reduced IL-6, TNF-α, and IL-1β levels by 57.5%, 50.2%, and 54.3%, respectively. Figure 6The efficacy of the neutrophil membrane (eg part) is comparable to that of budesonide. Compared with NM@IPC, its enhanced efficacy may be attributed to the synergistic targeting of neutrophil membrane and Let peptide, which together promote more efficient accumulation in inflamed lung endothelium, thereby enhancing anti-inflammatory effects and inhibiting vascular hyperpermeability. These findings suggest that precise endothelial targeting can effectively alleviate cytokine storms.

[0070] Experiment Example 7: Study on the effect of IPC on the survival rate of ARDS mice A mouse ARDS model was induced by intraperitoneal injection of LPS to compare and evaluate the effects of cell membrane biomimetic agents and targeted peptide-modified agents administered via tracheal instillation on mouse survival. Specifically, Balb / c mice were randomly divided into a healthy control group, an ARDS model group, an IPC group, an NM@IPC group, and a Let-NM@IPC group. Systemic inflammatory response and ARDS were induced by a single intraperitoneal injection of lipopolysaccharide (LPS, 30 mg / kg). In the intervention group, mice were administered the corresponding agent (0.5 mg / kg) via tracheal instillation 1 hour after LPS exposure, repeated every 24 hours for three consecutive days. Figure 7 (Part a). Survival rate and weight were recorded every 12 hours over 7 days to monitor survival outcomes.

[0071] The results showed that LPS-induced ARDS was characterized by alveolar wall thickening and pulmonary fibrosis. Figure 7 Part b). Intratracheal injection of IPC, NM@IPC, and Let-NM@IPC can alleviate lung injury and significantly reduce lung injury scores to varying degrees. Figure 7 (Part c). Therefore, mice in the model group died from ARDS within 2 days. The survival rate in the IPC group was only 40%, which may be attributed to drug retention in the lungs; NM@IPC increased the survival rate to 60%. Notably, Let-NM@IPC exhibited the strongest protective effect, achieving a survival rate of 80% on day 7. Figure 7 d). All deaths occurred within 1.5 to 3 days. By day 7, survivors in the Let-NM@IPC group exhibited macroscopic phenotypes similar to healthy mice, including smooth fur and restored motor function. These results suggest that protecting the integrity of pulmonary vascular endothelium is a key strategy for preventing death in severe acute respiratory distress syndrome (ARDS).

[0072] Experimental Example 8: In vivo anti-fibrotic efficacy study of icariin phospholipid complex A mouse model of pulmonary fibrosis induced by bleomycin intratracheal instillation was used to compare and evaluate the anti-fibrotic efficacy of oral administration and intratracheal instillation of icariin phospholipid complex suspension. Specifically, 30 C57 mice were randomly divided into four groups: a blank control group, a bleomycin (BLM) model group, a pirfenidone (PFD) group, an oral icariin phospholipid complex IPC (PO) group, and an intratracheal instillation icariin phospholipid complex IPC (IT) group, with 6 mice in each group. The blank control group was administered 50 μL of normal saline, while the model group and treatment group were administered BLM (1.5 mg / kg). After 14 days of modeling, the blank control group and model group were administered normal saline, the positive control group was administered PFD (300 mg / kg) orally, and the experimental groups were administered IPC orally (10 mg / kg) and intratracheally (1 mg / kg), respectively. After 14 days of continuous administration, the mice were euthanized with CO2, and lung tissue was collected. Bloodstains on the surface of the lung tissue were washed off with physiological saline, and the tissue was then soaked in a general-purpose tissue fixative for more than 24 hours. Subsequently, paraffin embedding was performed, and the paraffin block was cut into 4 μm thin slices using a microtome and mounted on glass slides. The slides were placed in a 45°C oven for 2 hours, followed by hematoxylin and eosin (H&E) staining and Masson's staining. The collected lung tissue was then analyzed.

[0073] The results showed that in mice induced by BLM, the levels of α-SMA, IL-6, TGF-β, and COL1A1 were significantly increased. While oral or tracheal administration of IPCs inhibited the expression of these factors, tracheal administration of IPCs showed a more significant inhibitory effect on several key factors. This indicates that direct delivery of the drug to the lungs via the trachea can concentrate the drug concentration in the lung tissue within a shorter time. This direct administration method reduces drug loss during gastrointestinal absorption and avoids the first-pass effect. Therefore, tracheal administration can provide a higher local drug concentration, thereby enhancing efficacy. Figure 8 ).

Claims

1. A cell-targeted inhalation formulation of an icariin flavonoid phospholipid complex, the formulation comprising: (a) A phospholipid complex core, wherein the phospholipid complex core is formed by the intermolecular non-covalent bonding of epimedium flavonoids and PC phospholipids; The icariin flavonoids are selected from one or more of icariin, icariin C, and icariin. And optional: (b) Auxiliary phospholipids, wherein the auxiliary phospholipids are selected from one or more of PG phospholipids, PI phospholipids, PS phospholipids and PEGylated PE phospholipids; (c) A coating layer, wherein the coating layer is a cell membrane component or a blank liposome membrane component; the cell membrane component is derived from neutrophils, apoptotic macrophages, platelets, alveolar epithelial cells, pulmonary vascular endothelial cells, exosomes, or extracellular vesicles; the blank liposome is a DPPC blank liposome. (d) Targeting peptides, wherein the targeting peptides are selected from pulmonary vascular endothelial targeting peptides, inflammation targeting peptides, alveolar epithelial targeting peptides, neutrophil targeting peptides or macrophage targeting peptides.

2. The formulation according to claim 1, wherein the PC-type phospholipid is selected from soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, distearate phosphatidylcholine, or a combination thereof.

3. In the formulation according to claim 1 or 2, the mass ratio of epimedium flavonoids to PC phospholipids in the core of the phospholipid complex is 1:1-10; preferably 1:2-6.

4. The formulation according to any one of claims 1-3, wherein the mass ratio of PC phospholipids to PG phospholipids in the formulation is 1:0.03-0.4, preferably 1:0.05-0.15; the mass ratio of PC phospholipids to PI phospholipids is 1:0.01-0.15, preferably 1:0.02-0.10; the mass ratio of PC phospholipids to PS phospholipids is 1:0.01-0.15, preferably 1:0.02-0.8; and the mass ratio of PC phospholipids to PEGylated PE phospholipids is 1:0.002-0.02, preferably 1:0.005-0.

02.

5. The formulation according to any one of claims 1-4, wherein the cell membrane component is a neutrophil cell membrane component, an apoptotic macrophage cell membrane component, or a platelet cell membrane component.

6. The formulation according to any one of claims 1-5, wherein the mass ratio of the cell membrane component or blank liposome component to the epimedium flavonoid compound is 0.05:1-10:1, preferably 0.1:1-2:1, and more preferably 1:

2.

7. The formulation according to any one of claims 1-6, wherein the targeting peptide is CGSPGWVRC, NNQKIVNLKEKVAQLEA, cyclo(1,12)PenITDGEATDSGC, VHPKQHR, VHPKQHRGGSKGC, RGD, cRGD, c(RGDfK), iRGD, CNGRC, NGR, GEBP11 peptides, CRKDKC and their conserved variants, truncated forms, cyclized forms or D-amino acid substitutes.

8. The formulation according to any one of claims 1-7, wherein the targeting peptide is linked to other components of the formulation via a linker arm, wherein the linker arm is DSPE-PEG-, DPPE-PEG-, cholesterol-PEG-, fatty acid-PEG-, or maleimide-PEG-; and the modification ratio of the targeting peptide is 0.01-20 mol% of the total lipid molar amount, preferably 0.1-10 mol%, more preferably 1-5 mol%.

9. The formulation according to any one of claims 1-8, wherein the formulation is an atomized drug.

10. The use of the formulation according to any one of claims 1-9 in the preparation of a medicament for treating chronic obstructive pulmonary disease, acute respiratory distress syndrome and / or pulmonary fibrosis.

11. The method for preparing the formulation according to any one of claims 1-9, characterized in that, The method includes: (1) Weigh out the flavonoids of Epimedium, PC phospholipids and auxiliary phospholipids, add organic solvent to dissolve and mix evenly, and react at 30-60℃ for 0.5-4 hours to form a phospholipid complex; after dissolution, filtration, rotary evaporation and vacuum drying, the phospholipid complex powder is obtained. (2) Add the phospholipid complex powder to deionized water or physiological saline, and then treat it by ultrasound or homogenization to obtain a phospholipid complex suspension.

12. The method for preparing the formulation according to any one of claims 1-9, characterized in that, The method includes: (1) Weigh out the flavonoids of Epimedium, PC phospholipids and auxiliary phospholipids, add organic solvent to dissolve and mix evenly, and react at 30-60℃ for 0.5-4 hours to form a phospholipid complex; after dissolution, filtration, rotary evaporation and vacuum drying, the phospholipid complex powder is obtained. (2) Add the phospholipid complex powder to deionized water or physiological saline, and then treat it by sonication or homogenization to obtain a phospholipid complex suspension. (3) Dissolve the PEGylated targeting peptide in deionized water to prepare a stock solution, add it to the phospholipid complex suspension, and incubate at 20-40℃ for 0.5-4 hours.

13. The method for preparing the formulation according to any one of claims 1-9, characterized in that, The method includes: (1) Weigh out the flavonoids of Epimedium, PC phospholipids and auxiliary phospholipids, add organic solvent to dissolve and mix evenly, and react at 30-60℃ for 0.5-4 hours to form a phospholipid complex; after dissolution, filtration, rotary evaporation and vacuum drying, the phospholipid complex powder is obtained. (2) Extract cell membrane components or blank liposome membrane components; (3) The phospholipid complex is mixed with cell membrane components or blank liposome membrane components, and then subjected to ultrasonic treatment, extrusion or incubation to coat the surface of the phospholipid complex with blank liposome membrane or cell membrane components. (4) Dissolve the PEGylated targeting peptide in deionized water to prepare a stock solution, add it to the product in step (3), and incubate at 20-40°C for 0.5-4 hours.

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