Artificial simulation pathological airway mucus, preparation method thereof and pathological airway mucus model

Abstract

According to the artificially simulated pathological airway mucus, the preparation method thereof and the pathological airway mucus model, through precisely designed mucoprotein concentration gradient, a dynamic cross-linking process and a multi-component synergistic effect, the rheological behavior, the pH response characteristic, the inflammatory factor spectrum and the oxidative stress characteristic of the asthma mucus are precisely represented, the important research and application value is achieved, and the application prospect is wide. The method not only can be used for studying the pathological mechanism of asthma, but also can be widely applied to improving the mucus removal efficiency, testing the medicine permeation efficiency, designing and optimizing an inhaler and studying the pathological mechanism, and has standardized production and clinical transformation potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular 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: Rheological deficiencies: Existing models fail to accurately reproduce the abnormal rheological properties of asthmatic mucus, especially the high storage modulus and low loss factor. These parameters are critical for the removal of mucus plugs, drug delivery systems, and inhaler design. For example, the airway mucus of asthmatic patients has higher viscosity and elasticity than that of healthy people, and existing models cannot accurately simulate this change.

[0003] Incomplete composition: Traditional mucus models lack specific components in the airway mucus of asthma patients, especially the spectrum of inflammatory factors (such as Th2 cytokines such as IL-4, IL-13, IL-5), oxidative stress markers (such as 8-isoprostaglandins), and bioactive molecules such as eosinophil-derived granule protein (ECP), which makes it difficult for existing models to fully reflect the pathological mechanism of asthma.

[0004] Lack of standardization: Natural mucus samples cannot be standardized and produced reproducibly due to large individual differences, difficulty in obtaining and biosafety risks, which seriously limits their promotion in scientific research and clinical applications.

[0005] Limitations of cross-linking agents: Aldehyde-based cross-linking agents such as polyglutaraldehyde (PGA) have biotoxicity risks, and the cross-linking network is irreversible and cannot simulate the dynamic rheological response characteristics of natural mucus.

[0006] pH insensitivity: The pH of asthma mucus is abnormally elevated (7.8-8.2), and traditional models cannot dynamically respond to changes in rheological properties caused by pH changes.

[0007] Characteristics of Asthma Mucus: Rheological behavior: There are significant differences in the rheological behavior of airway mucus from asthma patients and normal healthy people. Asthma mucus shows higher viscoelasticity, that is, the storage modulus (G') is significantly increased, which means that mucus can store more energy under pressure, making the airways more susceptible to blockage. In addition, asthma mucus has a stronger viscous part and poorer fluidity, which contrasts with the higher fluidity and lower viscous part of normal airway mucus.

[0008] This rheological difference makes the airway mucus of asthma patients show stronger rheological rigidity under external forces, which has an important impact on the onset and exacerbation of asthma. Existing animal mucus models and healthy human mucus models cannot accurately simulate these properties, thus affecting the research and development of treatment methods for asthma.

[0009] High concentration of mucin: Asthma patients usually have high concentrations of mucin (such as MUC5AC / MUC5B) in their airway mucus. Excessive secretion of these mucins leads to airway obstruction, which in turn aggravates asthma symptoms.

[0010] 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.

[0011] The mucus also contains excessive amounts of Th2 cytokines (such as IL-4, IL-13, IL-5), which play an important role in the immune response of asthma and promote the persistence and aggravation of airway inflammation.

[0012] The pH of airway mucus in asthma is often abnormally elevated, which affects the microbial environment and immune response within the airways.

[0013] Abnormal increase of oxidative stress markers (such as 8-isoprostane) is also a prominent feature of asthmatic mucus and is closely related to airway inflammation and damage.

[0014] 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

[0015] In order to solve the technical defects of the traditional mucus model in the prior art, the present invention provides an artificial simulated pathological airway mucus and its preparation method and pathological airway mucus model, which can highly simulate the physicochemical properties, inflammatory factor spectrum and cell components of asthma mucus, and is widely used in pathological mechanism research, mucus embolism removal, drug delivery system testing and treatment development. By accurately simulating the airway mucus of asthma patients, a standardized and repeatable model can be provided, providing strong support for drug development, disease research and treatment optimization.

[0016] The technical solution adopted by the present invention is: an artificial simulated pathological airway mucus, the artificial simulated pathological airway mucus contains the following substances: porcine gastric mucin type II, cross-linking agent polyethylene glycol, lipid module, inflammation module, the inflammation module includes bovine serum albumin that simulates protein aggregation to reflect the protein aggregation phenomenon in the airway 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 pH value of the artificial simulated pathological airway mucus is 7.8-8.2, and the G' increase in this range is ≥20%. The cell fragments are obtained by inducing apoptosis / necrosis of cultured respiratory epithelial cells with hydrogen peroxide, and then separating the fragments by centrifugation.

[0017] Preferably, the inflammation module also includes an oxidative stress module for simulating oxidative damage under pathological conditions to induce oxidative damage markers to study the effect of oxidative stress on the characteristics of asthmatic mucus.

[0018] Preferably, the oxidative stress module is hydrogen peroxide, and the amount of hydrogen peroxide in the oxidative stress module is 10-50 μM.

[0019] Preferably, 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.

[0020] Preferably, the concentration of porcine gastric mucin type II is 3.5-5.0 g / 26 ml PBS.

[0021] Preferably, the concentration of the cross-linking agent polyethylene glycol is 5-15% w / w and the molecular weight is 4000-8000 Da.

[0022] Preferably, the lipid module is porcine lung surfactant Curosurf, with a concentration of 0.5-2 ml / 26 ml PBS.

[0023] 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.

[0024] Artificial simulated pathological airway mucus can be used as an in vitro model for evaluating mucociliary clearance, where the synthetic mucus is coated on primary human airway epithelial cells cultured at the air-liquid interface to simulate the mucus clearance process. The rheological properties of artificial simulated pathological airway mucus are similar to those of airway mucus in clinical samples, and can effectively simulate the biological behavior of ciliary clearance.

[0025] Artificial simulation of pathological airway mucus can be used in the testing of inhaled drug delivery systems, including simulating ciliary beating through frequency scanning to evaluate mucus clearance efficiency.

[0026] A method for preparing artificial simulated pathological airway mucus comprises 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.

[0027] A pathological airway mucus model, wherein the pathological airway mucus model comprises the artificial simulated pathological airway mucus, wherein the pathological airway mucus model simulates a chronic phase model and an 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 an acute airway inflammatory response.

[0028] The concentration of porcine gastric mucin type II in the simulated chronic phase model is 3.5 g / 26 ml PBS (approximately 3.5 times the physiological concentration); the concentration of porcine gastric mucin type II in the simulated acute attack model is 5.0 g / 26 ml PBS (approximately 5 times the physiological concentration).

[0029] The amount of hydrogen peroxide, an oxidative stress factor added to simulate acute airway inflammation, is 10-50 μM.

[0030] The beneficial effects of the present invention are: an artificial simulation of pathological airway mucus and its preparation method and pathological airway mucus model, which accurately reproduces the rheological behavior, pH response characteristics, inflammatory factor spectrum and oxidative stress characteristics of asthma mucus through precisely designed mucin concentration gradient, dynamic cross-linking process and multi-component synergy, and has important research and application value. It can not only be used for the study of the pathological mechanism of asthma, but also can be widely used in improving mucus clearance efficiency, drug penetration efficiency testing, inhaler design optimization and pathological mechanism research, and has the potential for standardized production and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the dynamic response of PEG cross-linked network.

[0032] Figure 2 Optical microscopy images of (a) basal asthma mucus after cross-linking; (c) mucus during an acute attack.

[0033] Figure 3 Atomic force microscopy analysis of (a) basal asthma mucus and (b) acute asthma mucus.

[0034] Figure 4 are the elastic modulus and viscous modulus of the synthetic mucus. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Core ingredients and formula Mucin: Porcine gastric mucin type II (simulating MUC5AC / MUC5B), with a concentration of 3.5-5.0 g / 26 ml PBS, simulates the excessive secretion of mucin in asthmatic patients. Mucin is the main component of asthmatic airway mucus and can accurately simulate the excessive secretion phenomenon in the pathological state of asthma.

[0037] Cross-linking agent: The cross-linking agent used in previous studies was polyglutaraldehyde (PGA, 25% w / w), 3 ml / 26 ml PBS, which regulated the network density 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 at a concentration of 5-15% w / w to form a dynamic reversible network through hydrogen bonds.

[0038] Lipid module: Use porcine lung surfactant Curosurf (0.5-2 ml / 26 ml PBS) to simulate the structure of the mucus lipid layer, provide the necessary surfactant properties, and enhance the stability of airway mucus.

[0039] Inflammation Module: Bovine serum albumin (BSA 0.2-0.5 g / 26 ml PBS) simulates protein aggregation and reflects the protein aggregation phenomenon in the airways of asthmatic patients.

[0040] Cell debris: 0.003 g / 26 ml PBS was added to simulate the remnants of eosinophil disintegration and further enhance the biological relevance of the model.

[0041] Oxidative stress module: As needed, oxidative stress factors (hydrogen peroxide 10-50 μM) are added to simulate oxidative damage in pathological conditions and induce oxidative damage markers, which is crucial for studying the effects of oxidative stress on asthma mucus properties.

[0042] Th2 cytokines: IL-4, IL-13 or IL-5 (10-50 ng / ml) can be added exogenously to simulate the inflammatory microenvironment.

[0043] Advantages of the above technologies: Biosafety: FDA GRAS-certified PEG is used to replace aldehyde cross-linking agents to eliminate the risk of cytotoxicity; there is no residual aldehyde toxicity.

[0044] Dynamic network design: reversible cross-linking based on hydrogen bonding and hydrophobic interactions to mimic the shear thinning and self-healing properties of natural mucus; Dynamic rheological properties: consistent with natural mucus clinical samples and simulate the self-healing behavior of natural mucus.

[0045] pH response: The protonation state of the PEG chain segments changes with pH, ​​achieving dynamic regulation of mucus rheology and accurately reflecting the pathological characteristics of asthmatic mucus.

[0046] Oxidative stress compatibility: After hydrogen peroxide treatment, the polyunsaturated fatty acids in the lipid modules (Curosurf and cell fragments) in the artificial mucus were oxidized to generate 8-isoprostane (32.5±4.1 pg / ml) through free radical-mediated lipid peroxidation reactions, thereby simulating the oxidative stress characteristics of asthma patients, which matches the clinical data of moderate to severe asthma.

[0047] Preparation process Primary dispersion: Premix Curosurf with pre-cooled PBS (4°C) and stir magnetically (500 rpm, 25°C) until homogenized.

[0048] Mucin loading: Add mucin and BSA successively and stir continuously at 37°C for 12 hours to ensure that the mucin is evenly dissolved.

[0049] Dynamic cross-linking: Low-temperature pre-crosslinking: PEG 6000 (10% w / w) was added and allowed to stand at 4°C for 24 h to form a primary network; Curing at physiological temperature: vertical oscillation mixing at 37°C (frequency 2 Hz, amplitude 5 mm, 25°C) for 168 hours to form a three-dimensional cross-linked network, construct a dynamic hydrogen bond network, enhance the viscoelastic properties of the mucus, and the rheological parameters of the synthetic mucus meet the following requirements: elastic modulus greater than or equal to 9 Pa, viscous modulus greater than or equal to 1.94 Pa (1 Hz);.

[0050] Photocrosslinking enhancement: Add 0.1% LAP photoinitiator and irradiate with 365 nm UV for 10 seconds.

[0051] Dehydration and solidification: Use gradient acetone for dehydration (50%→90%→100%) and blow dry with nitrogen to obtain freeze-dried powder (active ingredient retention rate after reconstitution>97%) to achieve model standardization and long-term preservation.

[0052] Rheological behavior matching Test equipment: The strain-controlled ARES-G2 rotational rheometer from TA of the United States was used. The fixtures used were upper and lower fixtures, and the fixture surface was sandblasted to prevent errors caused by test slippage. During the test, the temperature was controlled by gradient temperature change, gradually increasing from room temperature to physiological environment temperature, simulating the physiological conditions of the airway of asthma patients. Key rheological parameters: including yield stress, dynamic modulus, and nonlinear response to match natural mucus.

[0053] Example 1 Basic Asthma Mucus Synthesis Formula: porcine gastric mucin type II 4.2 g, PEG6000 (10% w / w), BSA 0.3 g, Curosurf 1 ml, cell fragments 0.003 g, PBS 26 ml.

[0054] Steps: Prepare according to the above process and dynamically crosslink for 168 hours.

[0055] Results: The elastic modulus was 9.07±0.26 Pa, and the viscous modulus was 1.94±0.20 Pa.

[0056] Example 2: Acute attack model optimization Formula adjustment: porcine gastric mucin type II 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.

[0057] Rheological properties: G' increased to 19.13±0.52 Pa, and the viscous modulus was 3.26±0.33 Pa.

[0058] Example 3: pH dynamic control model Formula: porcine gastric mucin type II 4.2 g, PEG 6000 (10% w / w), IL-13 (20 ng / ml), PBS 26 ml.

[0059] Procedure: Adjust pH to 8.0 and equilibrate at 37°C for 2 hours.

[0060] Results: The rheological results responded dynamically with pH, ​​and the viscoelastic modulus increased significantly.

[0061] Example 4: Microstructure Characterization Optical microscopy: Pathological mucus showed a dense fiber network (fiber diameter > 200 nm), while normal mucus had a loose structure (fiber diameter < 100 nm). This structural difference reflects the pathological characteristics of asthmatic airway mucus. Atomic force microscopy (AFM): The network gap of pathological mucus (50 ± 2.1 nm) was significantly higher than that of normal mucus (150 ± 1.2 nm), which was positively correlated with the elastic modulus and yield stress, further demonstrating the consistency of the structure and rheological properties of the simulated mucus.

[0062] Technical personnel should note that although the present invention has been explained through the above specific implementation methods, the inventive concept of the present invention is not limited thereto. Any improvement or variation based on the inventive concept belongs to the protection scope of this patent right.

[0063] The above is only a preferred embodiment 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 ordinary technicians in this technical field, without departing from the purpose and scope of the present invention, any modification or equivalent replacement should be regarded as part of the protection 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 inflammatory module. The inflammatory 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.

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 method for preparing artificial simulated pathological airway mucus according to claim 1, characterized in that: The following steps are involved: (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.

10. 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.

Images