Artificial simulation of pathological airway mucus, its preparation method and pathological airway mucus model

Abstract

Artificially simulated pathological airway mucus, its preparation method and pathological airway mucus model precisely reproduce the rheological behavior, pH response characteristics, inflammatory factor profile and oxidative stress characteristics of asthmatic mucus through precisely designed mucin concentration gradients, dynamic crosslinking processes and multi-component synergistic effects, and have important research and application values. It can not only be used for the study of the pathological mechanism of asthma, but also be widely applied to the improvement of mucus clearance efficiency, the test of drug penetration efficiency, the optimization of inhaler design and the study of pathological mechanisms, and has the potential for standardized production and clinical transformation.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to an artificial simulated pathological airway mucus, a preparation method thereof, and a pathological airway mucus model. Background Art

[0002] Currently, traditional mucus models (such as animal mucus and healthy human mucus) have certain limitations and cannot accurately simulate the characteristics of airway mucus in asthma patients. The specific defects are as follows:

[0003] Rheological defect: Existing models fail to accurately reproduce the abnormal rheological properties of asthmatic mucus, especially the inability to reproduce high storage modulus and low loss factor. These parameters are crucial for the clearance of mucus plugs, drug delivery systems, and inhaler design. For example, the airway mucus of asthma patients has higher viscosity and elasticity than that of healthy people, and existing models cannot accurately simulate this change.

[0004] Incomplete composition: Traditional mucus models lack specific components in the airway mucus of asthma patients, especially the inflammatory factor profile (such as Th2 cytokines like IL-4, IL-13, IL-5, etc.), oxidative stress markers (such as 8-isoprostaglandin), and bioactive molecules such as eosinophil-derived granule protein (ECP). This makes it difficult for existing models to fully reflect the pathological mechanism of asthma.

[0005] Lack of standardization: Natural mucus samples cannot achieve standardized and reproducible production due to large individual differences, difficult acquisition, and biosafety risks, severely limiting their promotion in scientific research and clinical applications.

[0006] Limitations of cross-linking agents: Aldehyde cross-linking agents such as glutaraldehyde (PGA) have the risk of biological toxicity, and the cross-linking network is irreversible, unable to simulate the dynamic rheological response characteristics of natural mucus.

[0007] pH insensitivity: The pH value of asthmatic mucus abnormally increases (7.8 - 8.2), and traditional models cannot dynamically respond to the rheological property changes caused by pH changes.

[0008] Characteristics of asthmatic mucus:

[0009] Rheological behavior: There are significant differences in rheological behavior between the airway mucus of asthma patients and that of normal healthy people. Asthmatic mucus exhibits higher viscoelasticity, that is, the storage modulus (G') increases significantly, which means that the mucus can store more energy under pressure, leading to easier airway blockage. In addition, the viscous part of asthmatic mucus is stronger and the fluidity is poorer, which is in contrast to the higher fluidity and lower viscous part of normal airway mucus.

[0010] These rheological differences cause the airway mucus of asthma patients to exhibit stronger rheological rigidity under external forces, which has important implications for the onset and exacerbation of asthma. Existing animal mucus models and healthy human mucus models cannot accurately simulate these characteristics, thus affecting the research of asthma and the development of treatment methods.

[0011] High concentrations of mucins: Airway mucus in asthma patients usually has high concentrations of mucins (such as MUC5AC / MUC5B). The excessive secretion of these mucins leads to airway obstruction, further exacerbating asthma symptoms.

[0012] Eosinophil-derived granule protein (ECP) is an important biomarker in the airway mucus of asthma patients, and its level is positively correlated with the severity of the pathological state.

[0013] This mucus also contains excessive Th2 cytokines (such as IL-4, IL-13, IL-5), which play important roles in the immune response of asthma, promoting the persistence and exacerbation of airway inflammation.

[0014] The pH value of asthma airway mucus is usually abnormally elevated, which affects the microbial environment and immune response in the airway.

[0015] The abnormal elevation of oxidative stress markers (such as 8-isoprostaglandin) is also a significant feature of asthma mucus, which is closely related to airway inflammatory responses and damage.

[0016] Therefore, the existing technology cannot provide a standardized artificial synthesis method that can accurately simulate the physicochemical properties of asthma mucus and reflect the characteristics of inflammatory factors and oxidative stress. Summary of the Invention

[0017] To solve the technical deficiencies of traditional mucus models in the existing technology, the present invention provides an artificial simulation of pathological airway mucus, its preparation method, and a pathological airway mucus model, which can highly simulate the physicochemical properties, inflammatory factor profiles, and cellular components of asthma mucus, and are widely used in pathological mechanism research, mucus embolism clearance, drug delivery system testing, and treatment method development. By accurately simulating the airway mucus of asthma patients, a standardized and reproducible model can be provided, which provides strong support for drug development, disease research, and treatment method optimization.

[0018] The technical solution adopted by the present invention is as follows: an artificial pathological airway mucus mimic, which contains the following substances: porcine gastric mucin type II, cross-linking agent polyethylene glycol, lipid module, and inflammation module. The inflammation module includes bovine serum albumin that mimics protein aggregation in the airways of asthma patients to reflect protein aggregation phenomena, cell debris that mimics the remnants of eosinophil disintegration to enhance the biological relevance of the model, and Th2 cytokines that mimic the inflammatory microenvironment. The pH value of the artificial pathological airway mucus mimic is 7.8 - 8.2, and within this range, the increase in G' is ≥20%. The cell debris is obtained by inducing apoptosis / necrosis of cultured respiratory epithelial cells with hydrogen peroxide and then separating the debris by centrifugation.

[0019] Preferably, the inflammation module further includes an oxidative stress module that mimics oxidative damage under pathological conditions to induce oxidative damage markers and study the oxidative stress on the properties of asthma mucus.

[0020] Preferably, the oxidative stress module is hydrogen peroxide, and the amount of hydrogen peroxide in the oxidative stress module is 10 - 50 μM.

[0021] Preferably, the Th2 cytokines are one or more of IL-4, IL-13, or IL-5, and the amount of the Th2 cytokines is 10 - 50 ng / ml.

[0022] Preferably, the concentration of porcine gastric mucin type II is 3.5 - 5.0 g / 26 ml PBS.

[0023] Preferably, the concentration of the cross-linking agent polyethylene glycol is 5 - 15% w / w, and the molecular weight is 4000 - 8000 Da.

[0024] Preferably, the lipid module is porcine pulmonary surfactant Curosurf, and the concentration is 0.5 - 2 ml / 26 ml PBS.

[0025] Preferably, the concentration of bovine serum albumin in the inflammation module is 0.2 - 0.5 g / 26 ml PBS, and the concentration of cell debris is 0.003 g / 26 ml PBS.

[0026] The artificial pathological airway mucus mimic can be used in an in vitro model for evaluating the mucociliary clearance rate. The synthetic mucus is covered on human primary airway epithelial cells cultured at the air-liquid interface to simulate the mucus clearance process. The rheological properties of the artificial pathological airway mucus mimic are similar to those of airway mucus in clinical samples, and it can effectively simulate the biological behavior of ciliary clearance.

[0027] Artificially simulated pathological airway mucus can be applied in the testing of inhaled drug delivery systems, including simulating ciliary beating through frequency scanning to evaluate mucus clearance efficiency.

[0028] A method for preparing artificially simulated pathological airway mucus, comprising the following steps:

[0029] (1) Primary dispersion: Premix porcine pulmonary surfactant Curosurf with precooled PBS and stir magnetically until homogeneous.

[0030] (2) Mucin loading: Sequentially add porcine gastric mucin type II and bovine serum albumin to the homogeneous product obtained in step (1), and continuously stir at 37 °C to ensure uniform dissolution of porcine gastric mucin type II.

[0031] (3) Low-temperature pre-crosslinking: Add a crosslinking agent polyethylene glycol with a molecular weight of 6000 Da and let it stand at 4 °C for 24 hours to form a primary network.

[0032] (4) Physiological temperature curing: Vertically oscillate and mix at 37 °C for 168 hours to form a three-dimensional crosslinked network and enhance the viscoelastic properties of the mucus.

[0033] (5) Photo-crosslinking enhancement: Add 0.1% LAP photoinitiator and irradiate with ultraviolet light at 365 nm for 10 seconds.

[0034] (6) Dehydration curing: Dehydrate with acetone with a gradient of 50% → 90% → 100%, and dry with nitrogen to obtain a freeze-dried powder.

[0035] A pathological airway mucus model, wherein the pathological airway mucus model contains the artificially simulated pathological airway mucus, and the chronic phase model and acute exacerbation phase model are simulated by adjusting the concentration of porcine gastric mucin type II in the artificially simulated pathological airway mucus; an oxidative stress factor hydrogen peroxide is added to simulate airway acute inflammatory response.

[0036] The concentration of porcine gastric mucin type II in the simulated chronic phase model is 3.5 g / 26 ml PBS (about 3.5 times the physiological concentration); the concentration of porcine gastric mucin type II in the simulated acute exacerbation phase model is 5.0 g / 26 ml PBS (about 5 times the physiological concentration).

[0037] The amount of oxidative stress factor hydrogen peroxide added to simulate airway acute inflammatory response is 10 - 50 μM.

[0038] The beneficial effects of the present invention are as follows: an artificial pathological airway mucus simulation, its preparation method, and a pathological airway mucus model can accurately reproduce the rheological behavior, pH-responsive characteristics, inflammatory factor profile, and oxidative stress characteristics of asthmatic mucus through precisely designed mucin concentration gradients, dynamic cross-linking processes, and multi-component synergistic effects. It has important research and application values and can be used not only for studying the pathological mechanisms of asthma but also for widely improving mucus clearance efficiency, testing drug penetration efficiency, optimizing inhaler design, and studying pathological mechanisms, with the potential for standardized production and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the dynamic response of the PEG cross-linked network.

[0040] Figure 2 For optical microscopy imaging: (a) Basal asthmatic mucus after cross-linking; (c) Mucus during acute exacerbation.

[0041] Figure 3 For atomic force microscopy microscopic analysis: (a) Basal asthmatic mucus; (b) Asthmatic mucus during acute exacerbation.

[0042] Figure 4 For the elastic modulus and viscous modulus of the synthetic mucus. DETAILED DESCRIPTION OF THE INVENTION

[0043] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0044] Core Components and Formulations

[0045] Mucin: Porcine gastric mucin type II (simulating MUC5AC / MUC5B), with a concentration of 3.5 - 5.0 g / 26 ml PBS, simulating the over-secretion of mucin in asthmatic patients. Mucin is the main component of asthmatic airway mucus and can accurately simulate the over-secretion phenomenon under the pathological state of asthma.

[0046] Cross-linking agent: The cross-linking agent used in previous studies was glutaraldehyde polymer (PGA, 25% w / w), 3 ml / 26 ml PBS. The network density was regulated through thiol-aldehyde cross-linking to form a highly cross-linked three-dimensional structure to simulate the high viscoelastic properties of mucus. However, considering issues such as biosafety, polyethylene glycol (PEG4000 - 8000 Da) is now used, with a concentration of 5 - 15% w / w, to form a dynamic reversible network through hydrogen bonds.

[0047] Lipid module: Use porcine lung surfactant Curosurf (0.5 - 2 ml / 26 ml PBS) to mimic the structure of the mucus lipid layer, provide necessary surface activity characteristics, and enhance the stability of airway mucus.

[0048] Inflammatory module:

[0049] Bovine serum albumin (BSA 0.2 - 0.5 g / 26 ml PBS) mimics protein aggregation and reflects the phenomenon of protein aggregation in the airways of asthma patients.

[0050] Cell debris: Add 0.003 g / 26 ml PBS to mimic the remnants of eosinophil disintegration and further enhance the biological relevance of the model.

[0051] Oxidative stress module: As needed, add oxidative stress factors (hydrogen peroxide 10 - 50 μM) to mimic oxidative damage under pathological conditions and induce markers of oxidative damage, which is crucial for studying the effects of oxidative stress on the mucus properties in asthma.

[0052] Th2 cytokines: IL-4, IL-13, or IL-5 (10 - 50 ng / ml) can be added exogenously to mimic the inflammatory microenvironment.

[0053] The above technical advantages:

[0054] Biosafety: Use FDA GRAS-certified PEG to replace aldehyde cross-linking agents, eliminating the risk of cytotoxicity; no aldehyde residue toxicity.

[0055] Dynamic network design: Based on reversible cross-linking of hydrogen bonds and hydrophobic interactions, mimic the shear-thinning and self-healing properties of natural mucus;

[0056] Dynamic rheological properties: Consistent with clinical samples of natural mucus and mimic the self-healing behavior of natural mucus.

[0057] pH response: The protonation state of the PEG segment changes with pH, enabling dynamic regulation of mucus rheology and accurately reflecting the pathological characteristics of asthma mucus.

[0058] Oxidative stress compatibility: After treatment with hydrogen peroxide, polyunsaturated fatty acids in the lipid module (Curosurf and cell debris) of the oxidized artificial mucus are oxidized to generate 8-isoprostaglandin (32.5 ± 4.1 pg / ml) through free radical-mediated lipid peroxidation, thus mimicking the oxidative stress characteristics of asthma patients and matching the clinical data of moderate to severe asthma.

[0059] Preparation process

[0060] Primary dispersion: Premix Curosurf with pre-cooled PBS (4 °C), and stir magnetically (500 rpm, 25 °C) until homogeneous.

[0061] Mucin loading: Add mucin and BSA successively, and continuously stir at 37 °C for 12 hours to ensure the uniform dissolution of mucin.

[0062] Dynamic crosslinking:

[0063] Low-temperature pre-crosslinking: Add PEG 6000 (10% w / w), and let it stand at 4 °C for 24 hours to form a primary network;

[0064] Curing at physiological temperature: Mix vertically with oscillation (frequency 2 Hz, amplitude 5 mm, 25 °C) at 37 °C for 168 hours to form a three-dimensional crosslinked network, construct a dynamic hydrogen bond network, enhance the viscoelastic properties of mucus, and the synthesized mucus rheological parameters meet: the elastic modulus is greater than or equal to 9 Pa, and the viscous modulus is greater than or equal to 1.94 Pa (1 Hz);

[0065] Photo-crosslinking enhancement: Add 0.1% LAP photoinitiator and irradiate with 365 nm ultraviolet light for 10 seconds.

[0066] Dehydration curing: Dehydrate using gradient acetone (50% → 90% → 100%), dry with nitrogen to obtain freeze-dried powder (the retention rate of active ingredients after reconstitution > 97%), and achieve the standardization and long-term preservation of the model.

[0067] Rheological behavior matching

[0068] Testing equipment: Use a strain-controlled ARES-G2 rotational rheometer from TA Instruments, USA. The fixtures used are upper and lower fixtures, and the surfaces of the fixtures are sandblasted to prevent errors caused by test slippage. During testing, the temperature is controlled by gradient temperature change, gradually increasing from room temperature to physiological environmental temperature to simulate the physiological conditions of the airways of asthma patients. Key rheological parameters: including yield stress, dynamic modulus, and non-linear response, which match those of natural mucus.

[0069] Example 1 Synthesis of basic asthma mucus

[0070] Formulation: 4.2 g of porcine gastric mucin type II, PEG6000 (10% w / w), 0.3 g of BSA, 1 ml of Curosurf, 0.003 g of cell debris, 26 ml of PBS.

[0071] Steps: Prepare according to the above process and perform dynamic crosslinking for 168 hours.

[0072] Results: The elastic modulus is 9.07 ± 0.26 Pa, and the viscous modulus is 1.94 ± 0.20 Pa.

[0073] Example 2: Optimization of Acute Attack Phase Model

[0074] Formulation adjustment: The type II porcine gastric mucin was increased to 5.0 g / 26 ml PBS, 25 μM hydrogen peroxide was added to simulate oxidative stress, and the PEG concentration was increased to 12% w / w.

[0075] Rheological properties: G' was increased to 19.13 ± 0.52 Pa, and the viscous modulus was 3.26 ± 0.33 Pa.

[0076] Example 3: pH Dynamic Regulation Model

[0077] Formulation: 4.2 g of type II porcine gastric mucin, PEG 6000 (10% w / w), IL-13 (20 ng / ml), 26 ml of PBS.

[0078] Steps: Adjust the pH to 8.0 and equilibrate at 37°C for 2 hours.

[0079] Results: The rheological results showed a dynamic response to pH, and the viscoelastic modulus increased significantly.

[0080] Example 4: Microstructural Characterization

[0081] Optical microscope: The pathological mucus showed a dense fiber network (fiber diameter > 200 nm), while the normal mucus had a loose structure (fiber diameter < 100 nm). This structural difference reflected the pathological characteristics of asthmatic airway mucus. Atomic force microscope (AFM): The network voids of the pathological mucus (50 ± 2.1 nm) were significantly higher than those of the normal mucus (150 ± 1.2 nm), showing a positive correlation with the elastic modulus and yield stress, further demonstrating the consistency of the structure and rheological properties of the simulated mucus.

[0082] Notice to all technicians: Although the present invention has been explained through the above specific embodiments, the inventive concept of the present invention is not limited thereto. Any improvement or variation based on the inventive concept of the present invention falls within the protection scope of the patent right of the present invention.

[0083] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments and experimental examples. Any technical solution that follows the concept of the present invention is included in the protection scope of the present invention. It should be emphasized that for those of ordinary skill in the art, any modification or equivalent replacement should be regarded as part of the protection scope of the present invention without departing from the spirit and scope of the present invention.

Claims

1. An artificial simulation of pathological airway mucus, characterized in that: The artificial simulated pathological airway mucus comprises the following substances: porcine gastric mucin type II, cross-linking agent polyethylene glycol, lipid module, and inflammation module. The inflammation module comprises bovine serum albumin that simulates protein aggregation to reflect the protein aggregation phenomenon in the airways of asthma patients, cell fragments that simulate the biological relevance of the eosinophil disintegration residual enhancement model, and Th2 type cytokines that simulate the inflammatory microenvironment. The artificial simulated pathological airway mucus is prepared by the following steps: (1) Primary dispersion: Premix porcine lung surfactant Curosurf with precooled PBS and stir magnetically until homogeneous; (2) Mucin loading: add porcine gastric mucin type II and bovine serum albumin successively to the homogenate obtained in step (1), and continue stirring at 37° C. to ensure that porcine gastric mucin type II is evenly dissolved; (3) Low-temperature pre-crosslinking: Add 6000 Da crosslinker polyethylene glycol and let stand at 4 °C for 24 hours to form a primary network; (4) Physiological temperature curing: vertical oscillation mixing at 37°C for 168 hours to form a three-dimensional cross-linked network and enhance the viscoelastic properties of the mucus; (5) Photocrosslinking enhancement: adding 0.1% LAP photoinitiator and irradiating with 365 nm UV for 10 seconds; (6) Dehydration and solidification: Dehydrate with acetone in a gradient of 50% → 90% → 100%, and blow dry with nitrogen to obtain a freeze-dried powder.

2. The artificial simulated pathological airway mucus according to claim 1, characterized in that: The inflammation module also includes an oxidative stress module for simulating oxidative damage under pathological conditions to induce oxidative damage markers to study the effects of oxidative stress on the properties of asthmatic mucus.

3. The artificial simulated pathological airway mucus according to claim 2, characterized in that: The oxidative stress module is hydrogen peroxide, and the amount of hydrogen peroxide in the oxidative stress module is 10-50 μM.

4. The artificial simulated pathological airway mucus according to claim 1, characterized in that: The Th2 type cytokine is one or more of IL-4, IL-13 or IL-5, and the amount of the Th2 type cytokine is 10-50 ng / ml.

5. The artificial simulated pathological airway mucus according to claim 1, characterized in that: The concentration of the porcine gastric mucin type II is 3.5-5.0 g / 26 ml PBS.

6. The artificial simulated pathological airway mucus according to claim 1, characterized in that: The concentration of the cross-linking agent polyethylene glycol is 5-15% w / w and the molecular weight is 4000-8000 Da.

7. The artificial simulated pathological airway mucus according to claim 1, characterized in that: The lipid module is porcine lung surfactant Curosurf, with a concentration of 0.5-2 ml / 26 ml PBS.

8. The artificial simulated pathological airway mucus according to claim 1, characterized in that: The concentration of bovine serum albumin in the inflammation module is 0.2-0.5 g / 26 ml PBS, and the concentration of cell fragments is 0.003 g / 26 ml PBS.

9. A pathological airway mucus model, characterized in that: The pathological airway mucus model comprises the artificial simulated pathological airway mucus as described in any one of claims 1-8, wherein the pathological airway mucus model simulates the chronic phase model and the acute attack phase model by adjusting the concentration of porcine gastric mucin type II in the artificial simulated pathological airway mucus; and adds the oxidative stress factor hydrogen peroxide to simulate the acute airway inflammatory response.

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