An inhaled pharmaceutical composition for treating inflammatory airway disease and a method of preparing the same

Abstract

This invention belongs to the field of traditional Chinese medicine, and more specifically relates to an inhaled pharmaceutical composition for treating inflammatory respiratory diseases and its preparation method. The inhaled pharmaceutical composition comprises an active ingredient and nanoliposomes, the surface of which is modified with mannose; the active ingredient comprises total alkaloids of Fritillaria cirrhosa and platycodon saponin D; the nanoliposomes are made from the following raw materials: phospholipids, cholesterol, and cholesterol-polyethylene glycol 1000-mannose ester. Through the surface-modified mannose ligand, the liposomes can be specifically recognized by mannose receptors highly expressed on the surface of alveolar macrophages, thereby initiating receptor-mediated endocytosis and efficiently delivering the encapsulated total alkaloids of Fritillaria cirrhosa and platycodon saponin D into the cell interior. The inhaled pharmaceutical composition of this invention collectively shifts macrophages from a pro-inflammatory M1 phenotype to a reparative M2 phenotype. Therefore, the overall therapeutic effect of the combined use far exceeds that of either ingredient used alone.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, and more specifically relates to an inhaled pharmaceutical composition for treating inflammatory respiratory diseases and its preparation method. Background Technology

[0002] Chronic inflammatory respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are characterized by a persistent abnormal immune response in the lung microenvironment. Alveolar macrophages, as the first line of immune defense in the respiratory tract, play a dual role in this process: their classic M1 phenotype drives the inflammatory response, while their alternative activated M2 phenotype promotes inflammation resolution and tissue repair. Imbalance in the phenotype of this cell population is a key factor contributing to the protracted nature of the disease. Currently, inhaled corticosteroids remain a first-line clinical treatment, but their mechanism of action primarily involves diffusion into cells and binding to cytoplasmic receptors, thereby non-specifically regulating gene transcription, making it difficult to precisely regulate the function of specific immune cells such as macrophages. Furthermore, long-term use of corticosteroids can lead to adverse reactions such as local immunosuppression and oral candidiasis, and their efficacy is limited in some severely ill patients.

[0003] In the search for alternative therapies, the classic herbal pair of Fritillaria cirrhosa and Platycodon grandiflorus from the treasure trove of traditional Chinese medicine has demonstrated clear clinical efficacy in "moistening the lungs and resolving phlegm, and promoting lung qi." Modern pharmacological studies have confirmed that the total alkaloids of Fritillaria cirrhosa have significant anti-inflammatory and antitussive activities, while Platycodon grandiflorus saponin D has been shown to promote respiratory mucus secretion and expulsion, and may increase the distribution of other drugs in lung tissue. However, traditional Chinese medicine dosage forms (such as decoctions and pills) have two major technical bottlenecks: First, after oral administration, the drug undergoes degradation in the gastrointestinal tract and the first-pass effect in the liver, resulting in a significant reduction in bioavailability and a negligible amount reaching the lungs; second, even if the drug enters the systemic circulation, it lacks targeting of diseased cells and cannot achieve effective accumulation in alveolar macrophages. Therefore, a large dose is often required to achieve therapeutic concentrations, increasing the potential risk of systemic side effects.

[0004] Although liposomes, as a mature nanodelivery system, offer advantages in improving drug solubility and delaying release, conventional inhaled liposome delivery still primarily relies on passive targeting, i.e., achieving deposition in the lungs through particle size control. However, liposomes deposited in the alveolar region, without active recognition capabilities, suffer limited uptake by alveolar macrophages and cannot effectively interact with specific intracellular targets. Therefore, developing an intelligent delivery system capable of actively recognizing and efficiently delivering drugs to the interior of alveolar macrophages is crucial to overcoming current therapeutic bottlenecks. Summary of the Invention

[0005] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an inhaled pharmaceutical composition for treating inflammatory respiratory diseases and a method for preparing the same, which solves the technical problem that it is difficult to precisely regulate the function of specific immune cells such as macrophages, mainly by diffusion into cells and binding to cytoplasmic receptors.

[0006] (II) Technical Solution In a first aspect, the present invention provides an inhaled pharmaceutical composition for treating inflammatory respiratory diseases, comprising an active ingredient and nanoliposomes, wherein the surface of the nanoliposomes is modified with mannose. The active ingredients include total alkaloids from Fritillaria cirrhosa and platycodon saponin D; the concentration of total alkaloids from Fritillaria cirrhosa is 8-10 μg / mL, and the concentration of platycodon saponin D is 4-5 μg / mL. The nanoliposomes are made from the following raw materials: phospholipids, cholesterol, and cholesterol-polyethylene glycol 1000-mannose ester.

[0007] Optionally, the average particle size of the nanoliposomes is 100-300 nm.

[0008] Furthermore, the average particle size of the nanoliposomes is 150-250 nm.

[0009] Optionally, the mass ratio of the total alkaloids of Fritillaria cirrhosa to Platycodon grandiflorus saponin D is (1-3):1.

[0010] Optionally, the mass ratio of the phospholipid to cholesterol is (3-10):1.

[0011] Optionally, the cholesterol-polyethylene glycol 1000-mannose ester accounts for 1%-10% of the total mass of the nanolipid material.

[0012] In a second aspect, the present invention provides a method for preparing an inhaled pharmaceutical composition for treating inflammatory respiratory diseases, comprising the following steps: S1. Phospholipids, cholesterol, cholesterol-polyethylene glycol 1000-mannose ester, total alkaloids of Fritillaria cirrhosa and Platycodon grandiflorus saponin D are dissolved together in an organic solvent, and the organic solvent is removed by rotary evaporation to form a lipid film. S2. Add hydration buffer to the lipid membrane and hydrate it at 40-60°C to obtain a crude liposome suspension. S3. The crude liposome suspension is extruded sequentially through polycarbonate membranes with different pore sizes to obtain a drug composition with uniform particle size.

[0013] Optionally, in S1, the organic solvent is chloroform or a mixture of chloroform and methanol.

[0014] Optionally, in S2, the hydration buffer is a phosphate buffer.

[0015] Optionally, in S3, the extrusion sequence is to pass through polycarbonate films with pore sizes of 0.8 μm, 0.4 μm, and 0.2 μm in sequence.

[0016] (III) Beneficial Effects This invention combines total alkaloids from Fritillaria cirrhosa with platycodin D and encapsulates them in the same liposome. Platycodin D, acting as a "guide drug," can direct the drug upwards and increase the distribution of total alkaloids from Fritillaria cirrhosa in the respiratory tract. The two work synergistically in the liposome carrier, achieving a synergistic effect of expectorant and antitussive while guiding the drug to the lungs.

[0017] This invention utilizes surface-modified mannose ligands, enabling liposomes to be specifically recognized by mannose receptors highly expressed on the surface of alveolar macrophages. This triggers receptor-mediated endocytosis, efficiently delivering total alkaloids from Fritillaria cirrhosa and Platycodon grandiflorus saponin D encapsulated in the liposomes into the cell interior. In vitro cell experiments show that this targeted liposome exhibits over 70% higher uptake in macrophage lines compared to unmodified ordinary liposomes, and achieves targeted drug release at lysosomes and other target sites.

[0018] This invention delivers the drug via inhalation, allowing it to directly reach the lesion and avoiding the first-pass effect and systemic distribution associated with oral medications, thus significantly improving local bioavailability. Real-time imaging studies in animals have confirmed that the DiR-labeled targeted liposomes accumulate specifically in the lungs for an extended period after inhalation and exhibit high co-localization with alveolar macrophages. Furthermore, uptake by the reticuloendothelial system in organs such as the liver and spleen is significantly lower than that of ordinary liposomes, suggesting a lower risk of systemic toxicity.

[0019] The liposomes of this invention exhibit potent immunomodulatory functions, effectively promoting the transformation of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Specific biological evidence demonstrates that in inflammatory model macrophages treated with these liposomes, the gene and protein expression levels of M1 markers (such as iNOS and TNF-α) were significantly suppressed, while the expression of M2 markers (such as Arg-1 and IL-10) was significantly upregulated. This formulation not only alleviates symptoms but also fundamentally regulates pulmonary immune homeostasis, achieving a therapeutic effect on chronic respiratory diseases. Detailed Implementation

[0020] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0021] Total alkaloids of Fritillaria cirrhosa, as the core active substance group of Fritillaria cirrhosa, play multiple key roles in the treatment of respiratory diseases. Traditional Chinese medicine summarizes its efficacy as "clearing heat and moistening the lungs, resolving phlegm and relieving cough." First, it exhibits excellent central and peripheral antitussive effects, significantly prolonging the cough latency and reducing the frequency of coughs in mice. Its mechanism is closely related to directly inhibiting the medullary cough center and relieving bronchospasm. Second, this component has significant expectorant efficacy, promoting respiratory gland secretion, diluting the viscous phlegm retained in the trachea, making it easier to cough up and effectively clearing the airways. Total alkaloids of Fritillaria cirrhosa have clear anti-inflammatory activity. Furthermore, studies have shown that some of its alkaloid components have a certain inhibitory effect on various respiratory pathogens. In summary, the synergistic effect of total alkaloids of Fritillaria cirrhosa through multiple targets—antitussive, expectorant, anti-inflammatory, and even antibacterial—constitutes the modern pharmacological basis for its treatment of cough, excessive phlegm, and lung inflammation.

[0022] Platycodon saponin D is a characteristic saponin compound in the traditional Chinese medicine Platycodon grandiflorus, and the core substance responsible for its efficacy in "clearing the lungs, soothing the throat, eliminating phlegm, and draining pus." Its functional characteristics lie in its specific regulation of the respiratory mucosa and immune system. In terms of expectoration, Platycodon saponin D has a strong mucosal stimulating effect. After oral administration, it can significantly increase the secretion of respiratory mucosal glands through vagal nerve reflex, thinning sputum. Simultaneously, it enhances the movement frequency of tracheal epithelial cilia, thereby greatly promoting the upward transport and expulsion of sputum. Its expectorant efficacy is superior to many synthetic drugs. In the fields of anti-inflammation and immunomodulation, Platycodon saponin D exhibits a complex and sophisticated bidirectional regulatory capacity. It can not only inhibit the overactivation of classical inflammatory signaling pathways such as nuclear factor κB, reducing the production of inflammatory mediators, but also regulate the function of immune cells such as macrophages, helping to balance the body's immune response. Therefore, Platycodon saponin D is not only a potent expectorant and anti-inflammatory agent itself, but also has the potential to act as a synergistic component in drug delivery, enhancing the accumulation and therapeutic effects of other active substances in the lungs.

[0023] The synergistic effect of total alkaloids from Fritillaria cirrhosa and platycodon saponin D is rooted in the classic TCM theory of "resolving phlegm and relieving cough" and "promoting lung function and regulating qi." This synergy is not a simple additive effect, but a multi-target, multi-link functional complementarity and enhancement. The potent anti-inflammatory activity of total alkaloids from Fritillaria cirrhosa can directly inhibit the release of key pro-inflammatory factors in the lungs, such as TNF-α and IL-6, while effectively suppressing the cough reflex through central and peripheral antitussive effects. However, after the inflammation subsides, the airways are often still filled with viscous phlegm composed of necrotic cells and inflammatory mediators. At this time, platycodon saponin D stimulates the secretion of serous fluid from the respiratory mucosa and enhances ciliary movement, acting like a clear stream entering the airways and starting a conveyor belt, powerfully expelling these pathological products and cleaning the airway environment. Furthermore, the two components work synergistically in immune regulation: the total alkaloids of Fritillaria cirrhosa tend to inhibit excessive inflammatory responses, while Platycodon grandiflorus saponin D shows the potential to promote the production of anti-inflammatory factors such as IL-10, together pushing macrophages from the pro-inflammatory M1 phenotype to the reparative M2 phenotype. Therefore, the overall therapeutic effect of the combination of the two components far exceeds that of either component used alone.

[0024] This invention creatively employs mannose as a target, modifying the surface of liposomes. This design is based on the high expression of mannose receptors on the surface of alveolar macrophages. This allows the liposomes to be actively recognized and internalized by macrophages, thereby achieving precise intracellular drug delivery, a fundamental difference from conventional inhaled formulations that rely on simple diffusion. By strictly controlling the liposome particle size within the 100-300 nm range, it ensures efficient deposition in the alveolar region via inhalation, perfectly meeting the specific requirements of pulmonary drug delivery.

[0025] In constructing this macrophage-targeted aspiration liposome, phospholipids, cholesterol, and cholesterol-polyethylene glycol 1000-mannose ester each play an indispensable role, working together to form a structurally stable and functionally advanced delivery system. Phospholipids, as the basic framework material of the entire liposome, have an amphiphilic molecular structure that determines the formation of the lipid bilayer. The self-assembly behavior of the hydrophilic head and hydrophobic tail creates a closed vesicle structure capable of simultaneously encapsulating both hydrophilic and hydrophobic drugs. This phospholipid-derived component further endows the liposome with excellent biocompatibility and degradability.

[0026] The cholesterol acts as a key membrane stabilizer embedded in the lipid bilayer composed of phospholipids. Its rigid steroidal ring structure can effectively fill the gaps between the fatty acid chains of phospholipid molecules. By regulating the microfluidic fluidity and density of the liposome membrane, it significantly reduces the permeability of liposomes in the complex environment in vivo, thereby preventing premature leakage of the encapsulated drug before it reaches the target site. This reinforcement effect can also enhance the ability of liposomes to resist the adsorption of pulmonary surfactant and proteins, and prolong their retention time in lung tissue.

[0027] The cholesterol-polyethylene glycol 1000-mannose ester is the core functional component for achieving active targeting in this invention. This chimeric molecule is firmly anchored in the lipid bilayer through its hydrophobic cholesterol end, while its hydrophilic polyethylene glycol chain extends outwards, forming a hydration protective layer on the liposome surface. This spatial barrier effectively reduces non-specific adsorption of plasma proteins, preventing premature clearance of the liposomes by the body's immune system, thereby prolonging the retention time of the liposomes in the body and achieving a long-circulation effect. Most importantly, the mannose ligand located at the end of the PEG chain can be specifically recognized by the mannose receptor highly expressed on the surface of alveolar macrophages. Like a precise navigation head, it guides the entire liposome to be efficiently taken up into the target cells through receptor-mediated endocytosis, thereby directly delivering the total alkaloids of Fritillaria cirrhosa and Platycodon grandiflorus saponin D to the core region of disease regulation, achieving a leap from passive diffusion to active targeting.

[0028] Cholesterol-PEG-Mannose provides immediate targeting by directly binding to receptors (such as MRC1) via its terminal free mannose. Cholesterol-PEG-Mannose Ester, on the other hand, employs a smarter prodrug strategy: its mannose ester bond requires esterase hydrolysis to activate its targeting function. This design aims to utilize the resulting delayed effect to allow for a longer in vivo circulation time for the drug-loaded liposomes.

[0029] Example 1

[0030] The preparation method of the pharmaceutical composition for treating respiratory diseases in this embodiment includes the following steps: the concentration of total alkaloids from Fritillaria cirrhosa in this embodiment is 1.3 mg / mL, and the concentration of Platycodon grandiflorus saponin D is 0.8 mg / mL. Mix 150 mg soybean lecithin (SPC), 30 mg cholesterol, 20 mg cholesterol-polyethylene glycol 1000-mannose ester, 15 mg total alkaloids of Fritillaria cirrhosa (calculated as fritillary alkaloids), and 10 mg platycodon saponin D in a clean round-bottom flask; add 50 mL of chloroform, shake or sonicate thoroughly to completely dissolve it, forming a homogeneous and transparent organic phase.

[0031] Connect the round-bottom flask to the rotary evaporator. In a constant temperature water bath at 40°C, slowly rotate the flask at 60 rpm while simultaneously starting the vacuum pump to slowly evaporate at -0.09 MPa. After the solvent has completely evaporated, a thin and uniform lipid film will form on the inner wall of the flask. Continue to evacuate for 45 minutes to ensure complete removal of trace amounts of organic solvent.

[0032] Add 10 mL of phosphate-buffered saline (PBS, pH 7.4), preheated to 50°C, to the flask containing the dried lipid film. Without rotating, manually shake the flask for 2-5 minutes to allow the lipid film to fully swell and detach from the flask wall. Reconnect the flask to a rotary evaporator (without vacuum) and rotate at 150 rpm for 1 hour at 50°C. This process ensures complete hydration of the lipid film, forming a milky white coarse suspension of multilocular liposomes.

[0033] The crude liposome suspension was treated in a water bath ultrasonic bath for 10 minutes to initially reduce the particle size and the number of liposome layers. Using a liposome extruder, the suspension was extruded sequentially through polycarbonate membranes: first through a membrane with a pore size of 0.8 μm, 10 times; then through a membrane with a pore size of 0.4 μm, 10 times; and finally through a membrane with a pore size of 0.1 μm, 10 times. This yielded bilayer liposomes with uniform particle size, primarily consisting of single chambers.

[0034] The extruded liposome suspension was ultracentrifuged at 12,000 rpm for 30 minutes at 4°C to precipitate the liposomes. The supernatant (containing unencapsulated free drug) was carefully discarded. The precipitate was resuspended in fresh, pre-cooled PBS to obtain the purified targeted inhalation liposome final product.

[0035] The product should be stored at 4°C, protected from light, and under nitrogen conditions, or freeze-dried after adding a freeze-drying protectant (such as 5% trehalose) for long-term preservation.

[0036] The synthesis method of cholesterol-polyethylene glycol 1000-mannose ester is as follows: 1 mmol of cholesterol succinate was dissolved in anhydrous dichloromethane, and 1.2 mmol of N,N'-dicyclohexylcarbodiimide (DCC) and 1.2 mmol of N-hydroxysuccinimide (NHS) were added under ice bath conditions, and the mixture was activated for 4 hours. Subsequently, 1 mmol of monomethoxy-amino-polyethylene glycol 1000 was added, and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, the precipitate was removed by filtration, and the solvent was removed by rotary evaporation to obtain the crude product of cholesterol-polyethylene glycol 1000-amino. This product was reacted with 1.5 mmol of activated mannose-valerate succinimide ester in PBS buffer for 12 hours. The reaction solution was purified by dialysis and lyophilized to obtain a white flocculent solid, which was the target product.

[0037] Extraction method of total alkaloids from Fritillaria cirrhosa: Take the crude powder of Fritillaria cirrhosa, add 8 times the amount of 70% ethanol solution, and extract twice by heating and reflux, 1.5 hours each time. Combine the two extracts and filter through a 200-mesh filter cloth to obtain the ethanol extract.

[0038] The alcohol extract was concentrated under reduced pressure at 60°C until no alcohol odor remained, yielding an extract. An appropriate amount of purified water was added to the extract and stirred to dissolve. The pH was adjusted to 2-3 with 10% hydrochloric acid solution, and the mixture was allowed to stand for 12 hours to allow the alkaloids to fully dissolve and form salts. The extract was then centrifuged, and the supernatant was collected.

[0039] Slowly add 10% sodium hydroxide solution dropwise to the supernatant to adjust the pH to 9-10, causing the alkaloids to precipitate out. Then extract with chloroform, adding chloroform equivalent to 1 / 3 volume of the aqueous phase each time, for 3 extractions; combine the chloroform layers.

[0040] The combined chloroform extracts were dehydrated with anhydrous sodium sulfate, and then concentrated under reduced pressure at 50°C to recover the chloroform, yielding a brownish-brown crude extract of total alkaloids from Fritillaria cirrhosa. Purification was then performed using macroporous adsorption resin, specifically D101 resin. Impurities were first eluted with water, followed by elution of the total alkaloids with 70% ethanol. Finally, the eluent was concentrated and dried to obtain a purified extract of total alkaloids from Fritillaria cirrhosa.

[0041] Extraction method of platycodon saponin D: Take coarse powder of Platycodon grandiflorus, add 10 times the amount of 70% methanol solution, and perform ultrasonic-assisted extraction twice at 50℃ (300W power), 30 minutes each time. Combine the extracts, filter, and obtain the methanol extract.

[0042] The methanol extract was concentrated under reduced pressure at 60°C to a paste-like consistency. A suitable amount of hot water was added to the paste for dispersion, followed by extraction three times with water-saturated n-butanol, each time using half the volume of the aqueous phase. The n-butanol layers were combined and concentrated under reduced pressure to obtain crude total saponins from Platycodon grandiflorus.

[0043] Column chromatography was performed using silica gel (200 mesh). Gradient elution was performed using a chloroform-methanol-water (lower layer) system (from 10:1:0.1 to 5:1:0.1). The fraction containing platycodin D was collected by thin-layer chromatography (TLC) (using 10% sulfuric acid ethanol solution as the colorimetric reagent, heated to 105°C until the spots were clear).

[0044] Further purification was performed using semi-preparative high-performance liquid chromatography (HPLC). Chromatographic conditions: C18 column; mobile phase: acetonitrile-water (25:75, v / v); detection wavelength: 210 nm. The target peak was collected, and acetonitrile was removed by rotary evaporation at low temperature. The resulting product was then freeze-dried to obtain a white powder of platycodon saponin D.

[0045] Example 2 The preparation method of the pharmaceutical composition for treating respiratory diseases in this embodiment includes the following steps: the concentration of total alkaloids from Fritillaria cirrhosa in this embodiment is 1.5 mg / mL, and the concentration of Platycodon grandiflorus saponin D is 0.9 mg / mL. Mix 240 mg soybean lecithin (SPC), 30 mg cholesterol, 30 mg cholesterol-polyethylene glycol 1000-mannose ester, 30 mg total alkaloids of Fritillaria cirrhosa (calculated as fritillary alkaloids), and 10 mg platycodon saponin D in a clean round-bottom flask; add 50 mL of chloroform, shake or sonicate thoroughly to completely dissolve it, forming a homogeneous and transparent organic phase.

[0046] The other steps are the same as in Example 1. Comparative Example 1 (Liposomes without a target) The preparation method of the pharmaceutical composition for treating respiratory diseases in this comparative example includes the following steps: the concentrations of total alkaloids from Fritillaria cirrhosa and Platycodon grandiflorus saponin D in this comparative example are the same as in Example 1. 20 mg of DSPE-PEG2000 (1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000) was used to replace cholesterol-polyethylene glycol 1000-mannose ester. The remaining steps, including lipid film formation, hydration, extrusion, and purification, were completely consistent with those in Example 1.

[0047] Comparative Example 2 (Liposomes containing only total alkaloids from Fritillaria cirrhosa) The preparation method of the pharmaceutical composition for treating respiratory diseases in this comparative example includes the following steps: the concentration of total alkaloids from Fritillaria cirrhosa in this comparative example is the same as in Example 1. Mix 150 mg soybean lecithin (SPC), 30 mg cholesterol, 20 mg cholesterol-polyethylene glycol 1000-mannose ester, and 15 mg total alkaloids of Fritillaria cirrhosa (calculated as fritillary alkaloids) in a clean round-bottom flask; add 20 mL of chloroform, shake or sonicate thoroughly to completely dissolve it, forming a homogeneous and transparent organic phase.

[0048] The other steps are the same as in Example 1. Comparative Example 3 (single-component liposome containing only platycodin D) The preparation method of the pharmaceutical composition for treating respiratory diseases in this comparative example includes the following steps: the concentration of platycodon saponin D in this comparative example is the same as in Example 1. Mix 150 mg soybean lecithin (SPC), 30 mg cholesterol, 20 mg cholesterol-polyethylene glycol 1000-mannose ester, and 10 mg platycodon saponin D in a clean round-bottom flask; add 20 mL of chloroform, shake or sonicate thoroughly to completely dissolve it, forming a homogeneous and transparent organic phase.

[0049] The other steps are the same as in Example 1. Test case 1. This experimental example is used to verify the real-time distribution and targeting of the liposome inhalation drug composition of the present invention in vivo. Experimental animals: Asthma model mice were selected and divided into 3 groups.

[0050] Experimental methods: The liposomal drug compositions of Examples 1-2 and Comparative Example 1 were prepared by DiR (near-infrared fluorescent dye) labeling.

[0051] The liposomal drug compositions of Examples 1-2 and Comparative Example 1 were administered to three groups of mice using a small animal nebulizer.

[0052] Using a small animal in vivo imaging system, whole-body imaging of mice was performed at different time points after drug administration (e.g., 0.5h, 2h, 6h, 24h). The experimental results showed that the strongest and longest-lasting fluorescence signals were observed in the thoracic cavity (lungs) of mice in Examples 1 and 2 of this invention, while signals in other parts of the body (e.g., liver and spleen) were weak. This indicates that the liposomal drug provided by this invention has lung-targeting properties. In contrast, the lung signal in Comparative Example 1 was weaker, and there was significant fluorescence accumulation in the liver and spleen, indicating that the drug in Comparative Example 1 was captured and cleared by the systemic reticuloendothelial system.

[0053] 2. This experimental example is used to verify that the liposome inhaled drug composition of the present invention can shift macrophages from the pro-inflammatory M1 phenotype to the reparative M2 phenotype. Experimental animals: Mouse mononuclear macrophage leukemia cells (RAW264.7) were used.

[0054] Cell modeling: RAW264.7 cells were stimulated with 100 ng / mL lipopolysaccharide (LPS) for 24 hours to establish a stable M1 polarized inflammation model.

[0055] Collect cell culture supernatant and, following the instructions of the ELISA kit, detect the protein concentrations of TNF-α and IL-10 in the supernatant.

[0056] Table 1: Experimental Groups Used in the Test Examples of this Invention

[0057] Table 2: Phenotypic data of pharmaceutical compositions M2 in the embodiments and comparative examples of the present invention

[0058] Data showed that, compared with the LPS control group, the groups of Examples 1 and 2 significantly downregulated the protein expression of TNF-α and significantly upregulated the expression of IL-10. Although the comparative examples 1-3 also had similar effects, the downregulation of TNF-α and the upregulation of IL-10 in macrophages were significantly higher in Examples 1-2. This indicates that the liposomal drug composition provided by the present invention has excellent immunomodulatory function and can effectively promote the transformation of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.

[0059] The mannose-modified liposomes of this invention exhibit comprehensive and significant advantages in treating an asthma model, with airway resistance approaching normal levels, significant relief of pulmonary inflammatory infiltration, and a greater conversion of macrophages to the M2 phenotype, which promotes tissue repair. This is directly attributed to the mannose receptor-mediated active targeting mechanism: the mannose ligands on the liposome surface can be precisely recognized by the mannose receptors highly expressed on the surface of alveolar macrophages, thereby enabling the liposome-encapsulated total alkaloids of Fritillaria cirrhosa and Platycodon grandiflorus saponin D to be directly delivered to the core effector cells of these pulmonary inflammatory cells, achieving precise drug delivery and maximizing synergistic effects.

[0060] In contrast, while unmodified mannose-based liposomes in Comparative Example 1 showed some efficacy, the difference was significant. Their effect relied more on the passive physical deposition of nanoparticles in the lungs, followed by slow, passive diffusion and non-specific uptake by cells. This delivery efficiency was low, with a large amount of drug failing to enter target cells or being metabolized and cleared extracellularly. Consequently, despite carrying the same dose of active ingredient, they failed to achieve an effective therapeutic concentration at the lesion site, ultimately failing to achieve a good immunomodulatory effect.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An inhaled pharmaceutical composition for the treatment of inflammatory airway disease, characterized in that, It contains an active ingredient and nanoliposomes, and the surface of the nanoliposomes is modified with mannose; The active ingredients include total alkaloids from Fritillaria cirrhosa and platycodon saponin D; the concentration of total alkaloids from Fritillaria cirrhosa is 1.0-1.5 mg / mL, and the concentration of platycodon saponin D is 0.5-1.0 mg / mL. The nanoliposomes are made from the following raw materials: phospholipids, cholesterol, and cholesterol-polyethylene glycol 1000-mannose ester; The average particle size of the nanoliposomes is 100-300 nm. In S1, the preparation method of cholesterol-polyethylene glycol 1000-mannose ester is as follows: Cholesterol succinate is dissolved in anhydrous dichloromethane and subjected to carboxyl activation treatment under DCC and NHS, wherein the molar ratio of cholesterol succinate to DCC and NHS is 1:1.2:1.2; then, monomethoxy-amino-polyethylene glycol 1000 is added and reacted at room temperature; after the reaction is completed, the precipitate is removed by filtration and the solvent is removed by rotary evaporation to obtain the crude product of cholesterol-polyethylene glycol 1000-amino; the crude product is reacted with a relatively excess of activated mannose-valerate succinimide ester in PBS buffer, and the reaction solution is purified by dialysis and lyophilized to obtain a white flocculent solid, which is the target product.

2. The inhaled pharmaceutical composition according to claim 1, characterized in that, The mass ratio of total alkaloids from Fritillaria cirrhosa to Platycodon grandiflorus saponin D is (1-3):

1.

3. The inhaled pharmaceutical composition according to claim 1, characterized in that, The mass ratio of phospholipids to cholesterol is (3-10):

1.

4. The inhaled pharmaceutical composition according to claim 1, characterized in that, The cholesterol-polyethylene glycol 1000-mannose ester accounts for 1%-10% of the total mass of the nanolipid materials.

5. A method for preparing an inhaled pharmaceutical composition as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Phospholipids, cholesterol, cholesterol-polyethylene glycol 1000-mannose ester, total alkaloids of Fritillaria cirrhosa and Platycodon grandiflorus saponin D are dissolved together in an organic solvent, and the organic solvent is removed by rotary evaporation to form a lipid film. S2. Add hydration buffer to the lipid membrane and hydrate it at 40-60°C to obtain a crude liposome suspension. S3. The crude liposome suspension is extruded sequentially through polycarbonate membranes with different pore sizes to obtain a drug composition with uniform particle size.

6. The preparation method according to claim 5, characterized in that, In S1, the organic solvent is chloroform or a mixture of chloroform and methanol.

7. The preparation method according to claim 5, characterized in that, In S2, the hydration buffer is a phosphate buffer.

8. The preparation method according to claim 5, characterized in that, In S3, the extrusion sequence is to pass through polycarbonate films with pore sizes of 0.8μm, 0.4μm, and 0.2μm in sequence.

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