Nanoparticles for clearing mucus from the lungs and methods of making and using the same

Abstract

The application discloses a kind of nanoparticles for removing lung mucus and its preparation method and application, it is related to the field of biological medicine.The nanoparticles of the application are composed of inner core and shell coated on the surface of the inner core, wherein the inner core contains citric acid and photoluminescent polymer-aliphatic polyester copolymer;The shell contains deoxyribonuclease and hydrophilic polymer.The nanoparticles provided by the application have excellent biocompatibility and multifunctional integration characteristics, which realizes effective penetration of lung mucus barrier, specific degradation of DNA network, down-regulation of pro-inflammatory factors and inhibition of mucus production in sequence through cascade response mechanism, breaking through the limitations of traditional single therapy.In addition, the in vivo experimental results show that it can significantly reduce the thickness of lung mucus and the protein concentration of lavage fluid, and effectively relieve airway obstruction, and can be used for removing lung mucus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanoparticle for clearing lung mucus, its preparation method, and its application. Background Technology

[0002] Excessive secretion of pulmonary mucus is a defensive response of lung tissue to infection, injury, or autoimmune abnormalities, involving multiple cellular and molecular mediators. Key mechanisms include: (1) pathogens or injury signals activate immune cells such as macrophages and neutrophils, releasing pro-inflammatory factors (such as TNF-α, IL-1β, IL-6, etc.), leading to vasodilation, increased permeability, and infiltration of inflammatory cells; (2) excessive expression of reactive oxygen species (ROS) in the inflammatory microenvironment, causing tissue damage and disruption of the mucus barrier; (3) after receiving pro-inflammatory factor signals, goblet cells in the trachea and bronchi express large amounts of mucins such as MUC5AC, leading to excessive mucus secretion. Excessive airway mucus secretion leads to mucus accumulation, airway obstruction, accelerated decline in lung function, and repeated airway infection, obstruction, and remodeling, forming a vicious cycle.

[0003] Currently, in the treatment of mucus-associated lung disease (MALD), traditional drugs such as mucolytics (e.g., N-acetylcysteine), expectorants, anti-inflammatory drugs (e.g., inhaled corticosteroids), and recombinant human deoxyribonuclease (alfachain enzyme) are often used alone or in combination. However, these preparations have significant limitations. For example, although alfachain enzyme can effectively degrade the DNA network in mucus, its single mechanism of action results in poor permeability and retention in the mucin-rich mucus layer, and it is easily degraded by proteases. It also has a short half-life, requiring frequent administration, and its efficacy is limited or even has side effects in non-CF (compulsory fibrosis) mucus hypersecretion diseases. Furthermore, small molecule drugs and biomolecules have difficulty diffusing in mucus, making it difficult to effectively reach deep lesions and act on target cells. Frequent use of high-dose drugs may also cause local irritation or systemic adverse reactions.

[0004] Nebulized inhalation therapy, as a highly efficient and locally effective drug delivery method, has shown promise in the treatment of various lung diseases. Although this therapy has demonstrated some benefits in clinical and preclinical studies, particularly for asthma and chronic obstructive pulmonary disease (COPD), its effectiveness is limited in treating mucus-prone lung diseases such as cystic fibrosis (CF), chronic bronchitis, and severe asthma due to its inability to effectively penetrate the mucus layer, given the complex mucus barrier and physiochemical barriers of the pulmonary microenvironment. With the development of nanotechnology, inhalable nanoparticles have been used to treat mucus-related lung diseases. Compared to traditional drugs, inhalable nanoparticles offer several advantages in the treatment of respiratory diseases, including reduced dosage, increased solubility of active pharmaceutical ingredients, targeted drug delivery to lung lesions, enhanced absorption in epithelial cells, and longer lung retention. Due to these advantages, inhalable nanoparticles can ensure better therapeutic effects even when patient conditions (e.g., unconsciousness, insufficient inspiratory flow, breath-holding problems, and inadequate coordination with the inhalation device) lead to poor inhalation outcomes. Therefore, inhalable nanoformulations are considered to have broad applications in the treatment of chronic obstructive pulmonary disease, asthma, lung cancer, COVID-19 and other lung diseases.

[0005] In related technologies, nanomaterials used for lung drug delivery mainly include lipid nanocarriers (such as liposomes and solid lipid nanoparticles), polymer nanocarriers (such as polymer nanoparticles and polymer micelles), protein nanocarriers (such as albumin and engineered proteins), inorganic nanocarriers (such as gold nanoparticles and calcium phosphate nanoparticles), and biomimetic nanocarriers (such as cell membranes and exosomes). However, these nanomaterials have certain safety issues. For example, liposomes, as one of the first nanocarriers approved by the FDA, have good biocompatibility and safety; however, cationic liposomes still pose cytotoxicity problems when delivering sensitive compounds such as nucleic acids, requiring further solutions to improve their safety. Dendritic polymers increase the number of cations by branching with many terminal amino groups, but excessive cations can lead to cell membrane rupture and apoptosis, resulting in severe cytotoxicity. In addition, the non-degradability of inorganic nanocarriers leads to their continuous accumulation in the reticuloendothelial system, thereby triggering inflammation and other adverse reactions.

[0006] Based on this, researchers further discovered that nanoparticle carrier systems constructed using biodegradable polymers (such as citric acid polymers, polylactic acid (PLA), polyglycolic acid (PGA), and their copolymers (PLGA)) can effectively overcome the limitations of traditional formulations. These polymers have attracted widespread attention due to their excellent biocompatibility, degradability, and non-immunogenicity. However, implementing nanoparticle-based nebulization therapy using these biodegradable polymers still faces significant challenges, including the stable preparation and large-scale production of drug-loaded nanoparticles, the potential impact of the nebulization process on the structural integrity of nanoparticles and drug activity, the difficulty in predicting and controlling their actual penetration and distribution behavior in complex human airway mucus, and the insufficient comprehensive assessment of their in vivo biodistribution, metabolic fate, and potential long-term safety. Therefore, there is an urgent need to develop a multi-effect nanoparticle nebulizer spray with high penetration efficiency, good safety and therapeutic effect, and the ability to clear mucus caused by lung inflammation. Summary of the Invention

[0007] The first aspect of the present invention is to provide a nanoparticle.

[0008] The second objective of this invention is to provide a method for preparing nanoparticles.

[0009] A third aspect of the present invention is to provide a nano-atomized spray.

[0010] A fourth aspect of the present invention is to provide a pharmaceutical composition.

[0011] The fifth aspect of this invention aims to provide the use of nanoparticles, nano-atomized sprays, or pharmaceutical compositions in the preparation of related pharmaceuticals.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a nanoparticle comprising a core and a shell covering the surface of the core, wherein: The core contains citric acid and a photoluminescent polymer-aliphatic polyester copolymer; The outer shell contains deoxyribonuclease and a hydrophilic polymer.

[0013] The nanoparticles according to embodiments of the present invention have at least the following beneficial effects: The nanoparticles provided by this invention possess excellent biocompatibility and multifunctional integration properties. Through a cascade response mechanism, they sequentially achieve effective penetration of the pulmonary mucus barrier, specific degradation of the DNA network, downregulation of pro-inflammatory factors, and inhibition of mucus production, thus overcoming the limitations of traditional single-therapy approaches. Furthermore, in vivo experimental results show that they can significantly reduce pulmonary mucus thickness and lavage fluid protein concentration, and effectively alleviate airway obstruction, making them suitable for clearing pulmonary mucus.

[0014] In some embodiments of the present invention, in the photoluminescent polymer-aliphatic polyester copolymer, the molar ratio of the photoluminescent polymer to the monomer of the aliphatic polyester is 1:10~30.

[0015] In some embodiments of the present invention, the aliphatic polyester is selected from at least one of poly(L-lactide), polyglycolic acid, polycaprolactone, and polylactic-co-hydroxyacetic acid.

[0016] In some embodiments of the present invention, the photoluminescent polymer-aliphatic polyester copolymer is selected from any one of the following: photoluminescent polymer-poly(L-lactide) copolymer (such as BPLP-PLLA), photoluminescent polymer-poly(glycolic acid) copolymer, photoluminescent polymer-polycaprolactone copolymer, and photoluminescent polymer-polylactic acid-glycolic acid copolymer (such as BPLPL-PLGA). Preferably, it is a photoluminescent polymer-poly(L-lactide) copolymer.

[0017] In some embodiments of the present invention, the photoluminescent polymer is prepared from citric acid, aliphatic diol and amino acid in a molar ratio of 1:1 to 1.2:0.2 to 1.

[0018] Preferably, the aliphatic diol includes 1,8-octanediol.

[0019] In this invention, citric acid, as the core component of the photoluminescent polymer, has the following three functions: (1) As the building block of polymer backbone. Citric acid is the most basic synthetic monomer, which forms the basic backbone of PLPL through polycondensation reaction with aliphatic diols (such as 1,8-octanediol). This provides a chemical reaction platform for the subsequent introduction of amino acids and the generation of fluorescent properties. Moreover, the hydroxyl or carboxyl groups at the end of the BPLP prepolymer synthesized with the participation of citric acid can act as macromolecular initiators to initiate the ring-opening polymerization of lactide, thereby generating BPLP-PLLA block copolymers, which greatly expands the processability and application range of the material.

[0020] (2) As the origin of the fluorophore. The fluorescence of BPLP-PLLA does not originate from citric acid itself or its simple polymer with diol (BPLP), but rather from the specific reaction between amino acids and citric acid. The specific fluorescence mechanism is as follows: the amino group of the amino acid forms an amide bond with the carboxyl group of the citric acid side chain, and then the carboxyl group of the amino acid itself undergoes intramolecular cyclization with the hydroxyl group on the central carbon atom of the citric acid, forming a six-membered ring structure. This ring structure is a conjugated system and is considered to be the fluorophore that produces the intrinsic photoluminescence phenomenon.

[0021] (3) Photoluminescent polymers based on citric acid have good biocompatibility. Citric acid itself is an intermediate product of the tricarboxylic acid cycle in organisms and has good biocompatibility. The ester bond backbone it participates in ensures the biodegradability of the material.

[0022] In some embodiments of the present invention, the deoxyribonuclease includes deoxyribonuclease I (i.e., alfa chain enzyme).

[0023] Preferably, the deoxyribonuclease I includes recombinant human deoxyribonuclease I.

[0024] Alpha-chain enzyme, a type of DNA enzyme, can dilute airway mucus by degrading DNA in it.

[0025] In some embodiments of the present invention, the hydrophilic polymer includes hyaluronic acid.

[0026] In some embodiments of the present invention, the particle size of the nanoparticles is 100-800 nm, preferably 350-600 nm.

[0027] In some embodiments of the present invention, the core-shell ratio of the nanoparticles is 1 to 5:1; preferably 1.5 to 3:1, for example 2:1.

[0028] A second aspect of the present invention provides a method for preparing nanoparticles as described in the first aspect, comprising the following steps: S1. The oil phase containing the photoluminescent polymer-aliphatic polyester copolymer is mixed with the aqueous phase containing citric acid and reacted to obtain the citric acid nanoparticle core. S2. The citric acid nanoparticle core is reacted sequentially with deoxyribonuclease and a hydrophilic polymer to obtain the final product.

[0029] The preparation method of the present invention has at least the following beneficial effects: This invention synthesizes biodegradable citric acid nanoparticle cores (CA NPs) by reacting citric acid with a photoluminescent polymer-aliphatic polyester copolymer. These cores are then loaded with deoxyribonuclease (such as alfa chain enzyme) and surface-modified with a high-molecular-weight hydrophilic polymer (such as hyaluronic acid) to form stable citric acid nanoparticles (DH-CA NPs) loaded with alfa chain enzyme and modified with hyaluronic acid. This design achieves multi-target synergistic therapy through a cascade response release mechanism, sequentially penetrating the mucus barrier, degrading DNA networks, reducing the release of pro-inflammatory factors, and inhibiting mucus gene expression. This effectively solves the problems of poor drug permeability, limited efficacy of single mechanisms, and systemic toxicity caused by frequent administration in traditional nebulization therapy.

[0030] In some embodiments of the present invention, the photoluminescent polymer-aliphatic polyester copolymer is a photoluminescent polymer-L-lactide copolymer.

[0031] Preferably, the preparation method of the photoluminescent polymer-poly(L-lactide) copolymer includes: S11. Under an inert atmosphere, the citric acid, the aliphatic diol and the amino acid are mixed and subjected to a condensation reaction to obtain a photoluminescent polymer. S12. Under esterase catalysis, the photoluminescent polymer is mixed with L-lactide, and after polymerization, the product is obtained.

[0032] Preferably, the inert atmosphere includes a nitrogen atmosphere.

[0033] Preferably, the aliphatic diol is 1,8-octanediol.

[0034] Preferably, the amino acid is an amino acid containing a thiol, amino, or hydroxyl side chain; more preferably, it is cysteine ​​or lysine.

[0035] Preferably, the esterase comprises porcine pancreatic lipase. The molar ratio of the porcine pancreatic lipase to the L-lactide is 1:20 to 100, for example, 1:20, 1:30, 1:50, 1:80, or 1:100.

[0036] Preferably, the molar ratio of the citric acid, the aliphatic diol, and the amino acid is 1:1~1.2:0.2~1.

[0037] Preferably, the molar ratio of the photoluminescent polymer to the L-lactide is 1:10~30.

[0038] In some embodiments of the present invention, in step S1, the mass ratio of the photoluminescent polymer-aliphatic polyester copolymer to the citric acid is 1:3~8.

[0039] In some embodiments of the present invention, the solvent of the oil phase includes at least one of dichloromethane and ethyl acetate.

[0040] In some embodiments of the present invention, the solvent of the aqueous phase includes water.

[0041] In some embodiments of the present invention, step S2, wherein the sequential reaction with deoxyribonuclease and hydrophilic polymer specifically includes: S21. The citric acid nanoparticle core and the deoxyribonuclease are mixed and reacted to obtain deoxyribonuclease-coated core particles; S22. Mix solution 1 containing the core particles coated with the deoxyribonuclease and solution 2 containing the hydrophilic polymer, react, and then purify to obtain the final product.

[0042] DNase I is adsorbed onto the surface of CA NPs through weak interactions such as electrostatic adsorption.

[0043] In some embodiments of the present invention, the mass ratio of the citric acid nanoparticle core to the deoxyribonuclease is 10:0.1~3; Preferably, the solvent of solution 1 is dichloromethane and / or ethyl acetate; Preferably, the solvent of solution 2 comprises water and ethanol; more preferably, the solvent of solution 2 is a 45% to 55% aqueous ethanol solution. Preferably, it is a 50% aqueous ethanol solution.

[0044] Preferably, the mass ratio of the deoxyribonuclease-coated core particles to the hydrophilic polymer is 5:5~20.

[0045] Preferably, the volume ratio of solution 1 to solution 2 is 1:20~30.

[0046] A third aspect of the present invention provides a nano-atomizing spray comprising the nanoparticles described in the first aspect.

[0047] In some embodiments of the invention, the nano-atomized spray comprises a therapeutically effective amount of the nanoparticles and a pharmaceutically acceptable carrier.

[0048] In a fourth aspect, the present invention provides a pharmaceutical composition having an active substance comprising the nanoparticles described in the first aspect.

[0049] In some embodiments of the present invention, the pharmaceutical composition further includes substances related to the treatment of lung diseases, as well as pharmaceutically acceptable excipients.

[0050] A fifth aspect of the invention provides the use of the nanoparticles as described in the first aspect, the nano-atomized spray as described in the third aspect, or the pharmaceutical composition as described in the fourth aspect in any of the following: A) Prepare a drug to clear pulmonary mucus; B) Preparation of drugs to inhibit mucus secretion; C) Preparation of drugs that target and deliver nucleic acids to the lungs; D) Prepare drugs for treating lung diseases.

[0051] In some embodiments of the present invention, the nucleic acid includes, but is not limited to, DNA, mRNA, siRNA, microRNA, antisense nucleic acid, and circular RNA.

[0052] The nanoparticles of this invention have excellent mucus penetration effect. When responding to the inflammatory microenvironment of the lungs, they can sequentially complete multi-target synergistic therapy by penetrating the mucus barrier, degrading the DNA network, reducing the release of pro-inflammatory factors, and inhibiting mucus production. They can be used as drug delivery carriers to deliver relevant nucleic acid therapeutic drug targets to the lungs, achieving better therapeutic effects.

[0053] In some embodiments of the present invention, the lung diseases include, but are not limited to, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary hypertension, asthma, etc.

[0054] When the nanoparticles or nano-atomized sprays are used to treat lung diseases, the nanoparticles' ability to penetrate the mucus barrier, as well as their beneficial effects such as DNA network degradation, reduction of pro-inflammatory factor release, and inhibition of mucus production, can achieve specific targeted therapeutic effects on lung diseases by encapsulating drugs that target lung disease treatment and delivering them to the lungs via tracheal administration or atomization.

[0055] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram illustrating the synthesis of DH-CA NPs of the present invention.

[0057] Figure 2 This is a schematic diagram of the structure of DH-CA NPs of the present invention.

[0058] Figure 3 The images shown are SEM images of DH-CA NPs and physical images of DH-CA NPs dispersions of the present invention, where A is an SEM image and B is a physical image of DH-CA NPs dispersions.

[0059] Figure 4The NMR of BPLP-PLLA of this invention 1 HNMR spectrum.

[0060] Figure 5 The fluorescence spectrum of the DH-CA NPs of this invention is shown.

[0061] Figure 6 The images show fluorescence microscopy of the DH-CA NPs of this invention and SEM images of the DH-CA NPs before and after nebulization. In the images, A is a white light image of the DH-CA NPs (scale bar: 20 μm); B is a DAPI channel image of the DH-CA NPs (scale bar: 20 μm); C is a SEM image of the DH-CA NPs before nebulization; and D is a SEM image of the DH-CA NPs after nebulization (20000X).

[0062] Figure 7 The following are the experimental results related to the in vitro degradation of mucus by DH-CA NPs according to the present invention. A shows the results of the inclined plate flow test of DH-CA NPs degrading mucus; B is a schematic diagram of the Transwell mucus degradation experiment; C is an electrophoresis diagram of DH-CA NPs degrading mucus; D shows the concentration of DH-CA NPs in the lower SILF solution of Transwell at 0, 1 and 2 h; E shows the movement trajectories of BPLP, BPLP-PLLA, D-CA NPs and DH-CA NPs in mucus.

[0063] Figure 8 The results of the cytotoxicity assays of the DH-CA NPs of this invention on MLE-12, BEAS-2B and MH-S cells at 12 and 24 h are presented.

[0064] Figure 9 The results of laser confocal microscopy were used to determine the uptake of DH-CA NPs by MLE-12, BEAS-2B and MH-S cells in this invention.

[0065] Figure 10 The present invention uses flow cytometry to determine the uptake of DH-CA NPs by MLE-12, BEAS-2B and MH-S cells.

[0066] Figure 11 The results of this invention, using laser confocal microscopy to observe the intercellular transport capacity of DH-CA NPs, are presented.

[0067] Figure 12 The following are in vivo experimental results of the degradation of lung mucus by nebulized DH-CA NPs of the present invention, wherein A is a schematic diagram of the animal experimental design process; B is the total protein concentration of mouse lung lavage fluid; C is the results of Alcian blue-periodic acid Schiff staining (AB-PAS) analysis of mouse trachea; and D is the results of AB-PAS analysis of mouse bronchus. Detailed Implementation

[0068] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0069] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0070] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0071] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0072] In this embodiment of the invention, MLE-12 (mouse alveolar epithelial cells), BEAS-2B (human bronchial epithelial cells), and MH-S (mouse macrophages) were all purchased from the American Type Culture Collection (ATCC).

[0073] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0074] Example 1: Preparation of DH-CA NP nanoparticles This embodiment provides DH-CA NPs nanoparticles that can clear mucus caused by lung inflammation. Figure 1The synthesis process and spatial structure diagram of DH-CA NPs are shown, including: S1, synthesizing biodegradable photoluminescent polymers (BPLPs) via a condensation reaction (one-pot method) of citric acid and 1,8-octanediol; S2, preparing biodegradable photoluminescent polymer-L-lactide nanoparticles (BPLP-PLLA NPs) using nanoprecipitation technology; S3, reacting citric acid with BPLP-PLLA NPs to synthesize BPLP-PLLA nanoparticles coated with citric acid molecules (denoted as CA NPs); S4, coating the surface of CA NPs with deoxyribonuclease I (DNase I) to form DNase I-coated CANPs (denoted as D-CA NPs); S5, surface modification with high molecular weight hyaluronic acid to synthesize hyaluronic acid-coated D-CA NPs (DH-CA NPs).

[0075] The specific preparation process of the above-mentioned nanoparticles is as follows.

[0076] 1. Synthesis of photoluminescent polymers The photoluminescent prepolymer (BPLP) with terminal hydroxyl groups in this experiment was synthesized by reacting citric acid, 1,8-octanediol, and L-cysteine ​​in a molar ratio of 1:1.1:0.2 to 1:1.1:1.0. The specific preparation method is as follows: Citric acid (CA) and 1,8-octanediol in a molar ratio of 1:1.1 were added to a 250 mL three-necked round-bottom flask. The mixture was melted under a constant flow of nitrogen, and then stirred in a silicone oil bath at 160 °C for about 15 min. The system temperature was then lowered to 140 °C, and L-cysteine ​​(Cys) was added, with a molar ratio of Cys to CA of 0.2. The mixture was stirred again at 140 °C for 30–60 min to form a prepolymer solution. This solution was then treated at 80 °C for 4 days to obtain photoluminescent polymers with different degrees of crosslinking (denoted as BPLP).

[0077] 2. Synthesis of photoluminescent polymer-poly(L-lactide) nanoparticles (1) Preparation of BPLP-PLLA polymer: The freeze-dried BPLP prepolymer was added to a dry 100 mL bottle, and L-lactide was added to the bottle at a molar ratio of 1:20. Next, porcine pancreatic lipase (PPL, purchased from MedChemexpress Biotechnology Co., Ltd.) was added, with a molar ratio of 1:20 to L-lactide. The bottle was vacuum treated and rinsed three times with nitrogen, then sealed and heated to 100 °C for 72 h. After the copolymer dissolved in chloroform, it was filtered through a filter membrane to remove PPL. The resulting polymer solution was concentrated under reduced pressure and then precipitated in cold ethanol to obtain the photoluminescent polymer-L-lactide polymer (denoted as BPLP-PLLA polymer).

[0078] (2) Preparation of BPLP-PLLA nanoparticles: prepared by nanoprecipitation technology. 5 mg of the BPLP-PLLA polymer prepared above was dissolved in 5 mL of tetrahydrofuran, and then the polymer solution was added dropwise to 50 mL of deionized water. The solution was stirred at 700 rpm until the solvent was completely evaporated at room temperature, thus obtaining biodegradable photoluminescent polymer-L-lactide nanoparticles (denoted as BPLP-PLLA NPs).

[0079] 3. Synthesis of citric acid-coated BPLP-PLLA NPs The synthesis of CA NPs by reacting citric acid with BPLP-PLLA NPs is as follows: 1 mg of citric acid was dissolved in 100 μL of deionized water, and the solution was vortexed (700 rpm) to ensure complete dissolution of CA, yielding a CA solution. Under the same vortexing conditions, 5 mg of BPLP-PLLA NPs were dissolved in 1 mL of dichloromethane and shaken thoroughly to obtain a BPLP-PLLA NPs solution. The CA solution and the BPLP-PLLA NPs solution were then mixed and vortexed for 5 min to obtain an oil-in-water emulsion, thus yielding citric acid-coated BPLP-PLLA NPs (denoted as CA NPs).

[0080] 4. Synthesis of DNase I-coated CA NPs DNase I was reacted with the CA NPs prepared above to obtain D-CA NPs. Specifically, 50 mg of the CA NPs prepared above was stirred for 5 min, then 0.5 mL of DNase I (1 mg / mL, also known as alfa streptomycin) was added and stirred overnight. The resulting nanoparticles were then centrifuged at 10,000 rpm for 30 min and resuspended in 1 mL of PBS to obtain DNase I-coated CA NPs (denoted as D-CA NPs).

[0081] 5. Synthesis of DH-CA NPs nanoparticles DH-CA NPs were obtained by reacting hyaluronic acid with the D-CA NPs prepared above. Specifically, 5 mg of the hydrophilic polymer hyaluronic acid was dissolved in 25 mL of a water-ethanol solution (50% v / v) and stirred evenly at room temperature using a magnetic stirrer. Next, 5 mg of D-CA NPs were dissolved in 1 mL of dichloromethane and added to the water-ethanol solution containing hyaluronic acid (50% v / v), immediately resulting in the precipitation of nanoparticles. After stirring the dispersion at room temperature for 10 min, the organic solvents, such as dichloromethane and ethanol, were removed by evaporation under rotating vacuum at 30°C. The precipitate was collected by centrifugation at 10,000 rpm and 4°C for 1 h, yielding the DH-CA NPs nanoparticles capable of clearing mucus caused by lung inflammation.

[0082] Figure 2 A schematic diagram of the structure of DH-CA NPs is shown, which is a shell-like spherical structure with a core-shell mass ratio of approximately 2:1. The above-mentioned DH-CA NP nanoparticles were resuspended in 5 mL of ultrapure water for later use.

[0083] Example 2: Characterization of DH-CA NPs This embodiment characterizes the synthesized DH-CA NPs to determine their properties, morphology, particle size, structure, and stability, specifically including the following:

[0084] 1. Observation using scanning electron microscopy Observation was performed using scanning electron microscopy (SEM). Specifically, a high-purity single-crystal silicon wafer with sides of 5 mm × 5 mm was selected as the sample carrier. Before use, the silicon wafer was ultrasonically cleaned sequentially for 10 min each in acetone, anhydrous ethanol, and ultrapure water to thoroughly remove surface organic contaminants and dust. After cleaning, the silicon wafer was dried with high-purity nitrogen in a clean bench. Approximately 2.0 mg of the aforementioned DH-CA NPs powder was accurately weighed and dispersed in 1.0 mL of anhydrous ethanol. Subsequently, the mixture was ultrasonically treated in an ice bath for 5 min until a homogeneous and stable suspension was formed, with no obvious particle agglomerates visible. 20 μL of the suspension was slowly and steadily added dropwise to the center of the pretreated silicon wafer using a micropipette. The silicon wafer with the sample suspension was placed horizontally in a clean dust cover and allowed to stand at room temperature for at least 2 h to ensure complete evaporation of the ethanol. After the silicon wafer was completely dry, it was firmly adhered to the metal post of the SEM sample stage using conductive adhesive, ensuring good electrical contact.

[0085] The specific parameters are as follows: using high-purity platinum as the target material, under the conditions of a working current of 10 mA and a vacuum degree of less than 10 Pa, the spraying time is approximately 20 seconds, ultimately forming a continuous platinum conductive film of uniform thickness of approximately 10-20 nm on the sample surface. The prepared sample stage is placed in the scanning electron microscope sample chamber, and a high vacuum of better than 5 × 10⁻⁶ Pa is applied. -3 Pa. Secondary electron imaging mode was selected for observation. To balance image resolution and sample damage, the accelerating voltage was set to 5 kV, and the working distance to approximately 8 mm. Representative areas were located and images were captured at different magnifications.

[0086] Figure 3 Image A in the figure is a SEM image of DH-CA NPs, showing that DH-CA NPs exhibit good monodispersity on the silicon wafer surface, with no obvious agglomerates present. This proves the effectiveness of the solvent selection and ultrasonic dispersion process. High-magnification images clearly show that the vast majority of particles have a regular spherical morphology, smooth surface, and clear boundaries.

[0087] Furthermore, particle size analysis was performed on at least 100 randomly selected particles using image analysis software. The results showed that their diameters were mainly distributed in the range of 340 nm to 530 nm, with an average particle size of approximately 420 nm, indicating that the synthesis process has excellent repeatability and controllability. The uniformity of particle shape and the smoothness of the surface suggest that the nucleation and growth stages of the nanoparticles were well controlled during the synthesis process, and that the preparation process did not damage the particle structure. This uniform spherical morphology and suitable particle size are beneficial for the material to achieve uniform load distribution, stable release kinetics, and good biocompatibility in subsequent applications as a drug carrier. In addition, no particle breakage or deformation caused by solvent evaporation or electron beam irradiation was observed in the images, indicating that the material itself possesses certain mechanical strength and stability.

[0088] The above results show that the DH-CA NPs prepared by this invention have a regular spherical morphology, smooth surface, clear boundary, diameter mainly distributed in the range of 340 nm to 530 nm, average particle size of about 420 nm, and good monodispersity.

[0089] 2. Dispersion effect test The nanoparticles DH-CA NPs prepared above were dissolved in pure water and allowed to stand for 1 h. After standing, it was observed whether stratification or precipitation occurred.

[0090] Figure 3B in the figure is a physical image of DH-CA NPs dispersed in pure water. It shows that the aqueous solution of DH-CA NPs is a uniform milky white solution without layering or precipitation, indicating that the nanomaterial has good dispersibility in pure water solute and there is no aggregation or layering.

[0091] 3. Nuclear magnetic resonance hydrogen spectrum detection Before testing, the DH-CA NPs (purity >98%) were dehydrated in a vacuum drying oven at 40 °C for 12 h to remove residual solvent or moisture. Deuterated dimethyl sulfoxide (DMSO-d6) was used as the solvent, which was pre-treated with activated 4Å molecular sieves to ensure a moisture content below 50 ppm. Internal standard addition: Tetramethylsilane (TMS) was added as a chemical shift internal standard, and its single-peak signal was used to calibrate the instrument frequency drift. 10.0 mg of DH-CA NPs powder was accurately weighed and dissolved in 0.5 mL of DMSO-d6. The mixture was vortexed for 5 min, followed by sonication for 10 min until the solution became clear and transparent, indicating complete dispersion or dissolution of the nanoparticles. The solution was transferred to a standard 5 mm NMR tube using a Pasteur pipette, maintaining a column height of 4.0 cm (approximately 0.6 mL) to avoid uneven radio frequency field or incomplete signal acquisition due to a low liquid level. NPs were determined using a 400 MHz Bruker Avance III NMR spectrometer.

[0092] Figure 4 The NMR 1H spectral results for the determined DH-CA NPs showed the presence of both BPLP and L-lactide peaks, including a peak at 1.02 ppm (from L-cysteine ​​-CH2SH), 1.23 ppm, 1.40 ppm and 4.05 ppm (from 1,8-octanediol -CH2-), 2.76 ppm (from citric acid -CH-), 1.45 ppm (from L-lactide -CH3), and 5.2 ppm (from L-lactide -CH-), indicating that the DH-CA NPs prepared by the method of the present invention are consistent with expectations.

[0093] 4. Fluorescence spectroscopy detection Accurately weigh 5.0 mg of DH-CA NPs lyophilized powder and resuspend in 1.0 mL of ultrapure water. Sonicate the mixture in an ice bath for 30 min until a homogeneous milky-white suspension is formed, with no visible particle aggregation. Let stand for 5 min after sonication to avoid bubble interference. A blank control group (ultrapure water only) was set up to subtract solvent and instrument background noise. A BIOTEK Synergy H1 multi-functional microplate reader, equipped with a xenon lamp and monochromator, was used. The fluorescence scanning mode was selected, and the parameters were set as follows: Excitation wavelength range: 300-500 nm (5 nm step), medium scan speed (100 nm / min) to balance signal-to-noise ratio and resolution. Emission wavelength acquisition: fixed emission slit width of 10 nm, scan range covering 300-600 nm to ensure capture of all fluorescence signals. Gain setting: adjust the photomultiplier tube (PMT) voltage to 600 V based on the pre-scan results to avoid signal saturation or excessively low gain. Temperature control: Experiments were conducted at 25°C, using an onboard temperature control system to maintain temperature stability and minimize the impact of thermal fluctuations on fluorescence intensity. Plate well selection: Black 96-well plates were used, with 100 μL of sample suspension added to each well to avoid cross-contamination and edge effects.

[0094] Figure 5 The fluorescence spectrum of the DH-CA NPs was measured, showing that they emit fluorescence in the excitation wavelength range of 300-500 nm. The strongest excitation signal was observed at 360 nm, indicating that the DH-CA NPs have the highest photon absorption efficiency in this energy range. When excited at a fixed wavelength of 360 nm, the emission spectrum exhibits a broad peak, with the peak value located around 430-460 nm, consistent with the typical characteristics of organic fluorophores. Furthermore, these DH-CA NPs possess stable autofluorescence properties, with an optimal excitation wavelength of 360 nm and an emission peak at 430 nm. This characteristic not only simplifies the biological tracing process of the material but also avoids interference introduced by exogenous markers, providing crucial technical support for subsequent studies on its distribution, retention, and metabolic kinetics during lung-targeted delivery.

[0095] 5. Fluorescence microscopy observation Accurately weigh 5.0 mg of DH-CA NPs lyophilized powder and resuspend it in 1.0 mL of ultrapure water. Sonicate the mixture in an ice bath for 30 min until a homogeneous milky-white suspension forms with no visible particle agglomeration. After sonication, allow it to stand for 5 min to eliminate air bubbles and avoid interference with observation. Then, use a micropipette to slowly add 500 μL of the homogeneous suspension to the wells of a 24-well plate (a glass-bottomed or ultra-thin plastic-bottomed plate is recommended to enhance light transmittance). To avoid evaporation affecting the concentration, the sample addition should be completed just before observation. Instrument operation: First, turn on the main switch of the microscope's transmitted white light source and push the light path switch to eyepiece observation mode. Select the 4x objective lens for initial focusing, then switch to the 20x high-power objective lens for detailed observation. If digital imaging is required, pull the light path switch to CCD camera mode and set the exposure parameters using the accompanying software, while simultaneously performing white balance calibration to eliminate background chromatic aberration.

[0096] Figure 6 In the image, A represents the microscopic morphology of DH-CA NPs, showing that the DH-CA NP nanoparticles are dispersed spherical structures with a relatively uniform distribution and good dispersibility, exhibiting almost no aggregation. Combined with the SEM images and dispersion analysis of the DH-CA NPs above, it can be concluded that the DH-CA NPs product synthesized in this invention possesses good uniformity and dispersibility.

[0097] Furthermore, the microscope filter was switched from white light to the DAPI channel, with the excitation filter at 360 / 50 nm, the dichroic mirror at 400 nm, and the emission filter at 460 / 50 nm, ensuring accurate capture of the DAPI channel signal. The morphology of DH-CA NPs in the DAPI channel was observed. The results are as follows: Figure 6 As shown in Figure B, DH-CA NPs emit bright blue fluorescence under 360 nm excitation light, with the signal uniformly distributed across the entire field of view. The particle boundaries are clear, and there is no significant aggregation or diffusion. This figure, combined with... Figure 2 The F in the figure demonstrates that DH-CA NPs particles can emit significant fluorescence at an excitation wavelength of 360 nm, indicating that the material has good fluorescence properties.

[0098] 6. Scanning electron microscopy observation of DH-CA NPs before and after nebulization Approximately 2.0 mg of DH-CA NPs powder was accurately weighed and dispersed in 1.0 mL of anhydrous ethanol. The mixture was then sonicated in an ice bath for 5 min until a homogeneous and stable suspension was formed, with no visible particle agglomerates. Using a vibrating sieve nebulizer, the DH-CA NPs suspension was nebulized at a flow rate of 0.2 mL / min with compressed air (flow rate 10 L / min) as the carrier gas and a nebulization pressure of 0.5 bar to simulate clinical inhalation conditions. A pretreated silicon wafer was placed 15 cm from the nebulizer outlet and exposed for 5 min to collect settled droplets. The silicon wafer was then placed horizontally in a clean dust cover and allowed to stand at room temperature for at least 2 h to ensure complete ethanol evaporation. After the silicon wafer was completely dry, it was firmly adhered to the metal post of the SEM sample stage using conductive adhesive, ensuring good electrical contact. The specific parameters are as follows: using high-purity platinum as the target material, under the conditions of a working current of 10 mA and a vacuum degree of less than 10 Pa, the spraying time is approximately 20 s, ultimately forming a continuous platinum conductive film of uniform thickness of approximately 10-20 nm on the sample surface. The prepared sample stage is placed in the scanning electron microscope sample chamber, and a high vacuum of better than 5 × 10⁻⁶ Pa is applied. - ³ Pa. Secondary electron imaging mode was selected for observation. To balance image resolution and sample damage, the accelerating voltage was set to 5 kV, and the working distance to approximately 8 mm. Representative areas were captured at different magnifications. Structural changes in DH-CA NPs before and after nebulization were observed using the above method.

[0099] Figure 6 Images C and D in the figure show SEM images of DH-CA NPs before and after atomization, respectively. The images show that DH-CA NPs maintain a complete spherical structure after atomization, and the particle size distribution does not change significantly (mainly within the range of 600–700 nm), confirming that the atomization process did not induce particle breakage, fusion, or morphological distortion. The atomized particles exhibit a radial sputtering distribution, a phenomenon stemming from the hydrodynamic behavior of the atomized droplets impacting the silicon wafer surface. According to the atomization model, when the droplets contact the substrate at high speed, their kinetic energy is converted into spreading energy, causing the NPs on the carrier to diffuse along the impact direction, forming a ring-shaped pattern. The small interparticle gaps are due to the high NP concentration used during atomization (5 mg / mL), increasing the number of particles deposited per unit area, thus reducing the distance between them. This phenomenon is directly related to atomizer parameters such as gas-liquid ratio and flow rate. Compared with before nebulization, the morphology of DH-CA NPs particles did not change significantly after nebulization. This indicates that the nebulization process did not have a destructive effect on the DH-CA NPs particles, which not only verified the mechanical robustness of DH-CA NPs, but also highlighted their reliability as a carrier for inhaled formulations.

[0100] The above results demonstrate that the DH-CA NPs prepared in this invention are core-shell spherical particles with an average particle size of approximately 420 nm. They exhibit regular morphology and good dispersibility. SEM images before and after atomization show that the particle morphology and size remain stable, proving their structural integrity and mechanical robustness as an atomizing formulation. Furthermore, fluorescence microscopy observations reveal that this material possesses significant autofluorescence properties, with optimal excitation / emission wavelengths of 360 / 430 nm, making it suitable for label-free tracer applications.

[0101] Example 3: Detection of the mucus degradation ability of DH-CA NPs This embodiment uses artificially synthesized mucus to determine the degradation ability of DH-CA NPs nanoparticles on mucus through slope mucus flow experiments, Transwell cross-mucus experiments, and mucus migration trajectory experiments. Specifically, it includes the following:

[0102] 1. Slope viscous flow experiment Add 25 μL of sterile egg yolk milk, 25 mg of type II porcine gastric mucin, 20 mg of DNA, 30 μL of 1 mg / mL DTPA, 25 mg of NaCl, 11 mg of KCl, and 100 μL of RPMI 1640 to 5 mL of water, and stir well to obtain artificial mucus. Mix 100 μL of artificial mucus with either 100 μL of PBS or 100 μL of DH-CA NPs suspension (5 mg / mL). Incubate the mixture at 37°C on a shaker at 200 rpm for 30 min to ensure sufficient interaction between the NPs and the mucus components. Use a 10 cm × 5 cm tilting glass plate with a fixed tilt angle of 30° (based on preliminary experiments, this angle can balance gravity and mucus cohesion, preventing premature droplet slippage). Treat the plate surface with a plasma cleaner for 5 min to eliminate surface energy differences and ensure consistent contact angles. The movement trajectory of the mucus front over 60 seconds was recorded using a high-definition camera, and the maximum flow distance was analyzed using ImageJ software. Each experiment was repeated three times, and the mean ± standard deviation was taken. Since the flow distance of the mucus is negatively correlated with its viscosity, and the viscosity decreases when the mucus is degraded, the viscosity of the mucus can be determined by measuring the relative distance of the mucus flow to determine whether the viscosity of the mucus decreased after co-incubation with NPs.

[0103] Figure 7Figure A shows the results of the mucus tilt plate flow test after co-incubation with DH-CANPs. The results indicate that the artificial mucus in the DH-CANPs-treated group flowed a distance of 8.1 cm within 60 seconds, significantly higher than the 3.2 cm in the PBS control group (p<0.001). This increased flow distance directly reflects a decrease in apparent viscosity and enhanced shear thinning behavior of the mucus. It is speculated that this is due to the specific degradation of the mucus DNA backbone by deoxyribonuclease I (DNase I, alfa chain enzyme) in DH-CANPs, disrupting its cross-linking network. Simultaneously, the hyaluronic acid coating layer disrupts the hydrogen bonds of mucins through steric hindrance, collectively leading to the transformation of the mucus from a gel state to a sol state.

[0104] 2. Transwell transmucus assay The Transwell diffusion cell system was used as the experimental setup, and its schematic diagram is shown below. Figure 7 As shown in Figure B, the entire experimental setup is a double-layer structure, separated by a polycarbonate membrane with a pore size of 0.8 μm. The upper layer consists of an artificial mucus layer and atomized DH-CA NPs; the lower layer is simulated interstitial lung fluid. The amount of DH-CA NPs that have penetrated the polycarbonate membrane was determined by measuring the fluorescence intensity of the lower simulated interstitial lung fluid. This experiment aims to quantitatively evaluate the degradation ability of different nanomaterials on DNA in artificial mucus using agarose gel electrophoresis. The specific procedure is as follows: In the experimental group, 100 μL of artificial mucus was mixed with an equal volume of test solution (citric acid solution, BPLP-PLLA, H-CA NPs, or DH-CA NPs) at a concentration of 10 mg / mL. In the control group, PBS was used instead. H-CA NPs represent citric acid nanoparticles modified with hyaluronic acid. The difference between H-CA NPs and DH-CA NPs is that H-CA NPs are not coated with DNase I. The other preparation conditions are the same.

[0105] The above mixtures were incubated separately at 37 °C on a shaker (200 rpm) for 30 min to ensure complete reaction. A 1.5% agarose gel was prepared using TAE buffer (40 mM Tris-acetic acid and 1 mM EDTA, pH 8.0) as the matrix, dissolved by microwave heating, and then the nucleic acid dye GelRed was added. The gel was poured into a mold, a comb was inserted, and it was allowed to solidify at room temperature for 30 min. The gel was placed in an electrophoresis tank, and TAE buffer was added (the liquid level was 1-2 mm above the gel). Electrophoresis was performed at a constant voltage of 90 V for 60 min, stopping when bromophenol blue migrated to 2 / 3 of the gel. After electrophoresis, the gel was photographed using a Bio-Rad ChemiDoc imaging system under UV excitation (302 nm).

[0106] Figure 7In the figure, C represents the DNA content determination results of the mucus after co-incubation with different types of NPs. The results show that the DNA bands in the artificial mucus treated with DH-CA NPs completely disappeared, while the DNA band intensities in the citric acid, BPLP-PLLA, and H-CA NPs groups were not significantly different from the control group. This indicates that the DNA in the mucus was completely degraded after co-incubation with DH-CA NPs. DH-NPs can efficiently degrade DNA in artificial mucus, and their action is enzyme-specific. This result, along with previous characterization data (such as SEM morphology and fluorescence properties), supports the core advantage of the material of this invention: synergistic treatment achieved through a cascade mechanism of first penetrating the mucus, then degrading DNA, and finally reducing viscosity.

[0107] 3. Mucus layer penetration test This experiment, referencing the Transwell transmucus experiment described above, used a Transwell diffusion cell system as the experimental setup. The polycarbonate membrane had a pore size of 0.4 μm and a diameter of 12 mm. 200 μL of artificial mucus (approximately 500 μm thick) was uniformly spread in the upper chamber, while the lower chamber was filled with 600 μL of simulated lung interstitial fluid (composition: 137 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 5 mM glucose, pH 7.4). The system was pre-equilibrated at 37 ℃ for 30 min to ensure the stability of the mucus layer.

[0108] During the experiment, the prepared artificial mucus was spread on the top layer of a polycarbonate membrane. DH-CA NPs were dispersed using physiological saline and then atomized using a nebulizer to disperse them onto the surface of the artificial mucus. At 0, 1, and 2 h after nebulization, 100 μL of simulated interstitial lung fluid was collected, and the fluorescence intensity at an excitation wavelength of 360 nm was measured using an ELISA reader. The mass of DH-CA NPs in the simulated interstitial lung fluid was calculated using the relationship between the fluorescence and concentration of DH-CANPs. 10 mg of lyophilized DH-CANPs powder was resuspended in 5 mL of physiological saline (0.9% NaCl) and sonicated in an ice bath for 30 min to form a homogeneous suspension (concentration 5 mg / mL). The suspension was atomized using a vibrating sieve nebulizer at a flow rate of 0.3 mL / min (median particle size 3-5 μm). The aerosol was vertically sprayed onto the surface of the mucus layer through a conduit, with the atomization time controlled at 5 min to ensure uniform NP coverage. At three time points—0 h, 1 h, and 2 h—after nebulization, 100 μL of simulated lung interstitial fluid was precisely aspirated from the lower chamber, while an equal volume of fresh fluid was simultaneously added to maintain hydraulic equilibrium. The fluorescence intensity of the samples was detected using an ELISA reader. The excitation wavelength was set to 360 nm, the emission wavelength scanning range to 400-600 nm, the bandwidth to 5 nm, and the gain was automatically adjusted.

[0109] A fluorescence standard curve for DH-CA NPs was established: NPs were diluted with physiological saline to concentration gradients (0, 25, 50, 100, 200, and 400 μg / mL), and the fluorescence intensity under 360 nm excitation (emission peak at 430 nm) was measured. The linear regression equation was fitted to obtain Y = 37.388X + 136.87 (R² = 0.994), where Y is the fluorescence intensity and X is the NP concentration (μg / mL). Each sample was measured three times, and the average value was taken, with the background signal from the blank (physiological saline) subtracted.

[0110] Figure 7 D in the figure represents the mass of DH-CA NPs across the mucus barrier at 0, 1, and 2 hours. The figures show that the mass of DH-CA NPs in the lower simulated lung interstitial fluid at 0, 1, and 2 hours was 0 μg, 19.3 μg, and 28.4 μg, respectively, indicating that the NPs can penetrate the mucus barrier within 1 hour, and the cumulative penetration increases by approximately 47.2% within 2 hours. This result confirms that DH-CA NPs possess the ability to actively penetrate the mucus barrier and can be used in lung delivery systems. Their efficient penetration ensures drug targeting to the deep alveoli, while their sustained-release characteristics (the penetration rate continues to increase after 2 hours) help maintain long-term therapeutic concentrations, demonstrating their ability to deliver across the mucus barrier.

[0111] 4. Mucus Movement Trajectory Experiment This experiment aims to quantitatively evaluate the mobility of different nanomaterials in artificial mucus using real-time tracking technology via laser confocal microscopy, thereby indirectly reflecting their potential to penetrate the mucus barrier. The specific methods are as follows: 10 μL of 5 mg / mL BPLP, BPLP-PLLA, H-CA NPs, and DH-CA NPs were added to 100 μL of artificial mucus, respectively, and sonicated in an ice bath for 15 min to ensure uniform dispersion and avoid introducing air bubbles. Using a specially designed chambered slide, 20 μL of the mixed suspension was added to each well, and a coverslip was placed to form a sealed sample chamber to prevent evaporation. Laser confocal microscopy parameters: 40x oil immersion lens, FITC channel excitation wavelength 488 nm, emission collection range 500-550 nm. The time resolution was set to 1 frame per second, with continuous acquisition for 20 s, tracking at least 50 particles per sample. The ImageJ plugin TrackMate was used to automatically identify and track particle trajectories, with filtering conditions: trajectory length ≥ 10 frames, displacement signal-to-noise ratio > 5. The experimental environment was controlled in a 37℃ constant temperature chamber with humidity > 80% to reduce solution evaporation. Each experiment was repeated three times to ensure statistical significance.

[0112] Figure 7E in the diagram represents the representative trajectories of BPLP, BPLP-PLLA, H-CA NPs, and DH-CA NPs in mucus, showing that compared to the other three materials, the motion trajectory of DH-CA NPs in mucus is significantly more complex and covers a wider range. Compared to BPLPs, BPLP-PLLA, and H-CA NPs, the enhanced motility suggests that DH-CA NPs can penetrate the mucus layer more efficiently to reach epithelial cells, increasing local drug concentration. This study, through high-resolution real-time tracking, confirms that DH-CA NPs exhibit superior motility in artificial mucus due to their enzyme-degradation-assisted active permeation mechanism. This characteristic not only solves the bottleneck problem of limited diffusion of nanocarriers in the mucus barrier but also provides crucial experimental evidence for their targeted delivery application in mucus-rich sites such as the lungs and intestines.

[0113] The above results demonstrate that the DH-CA NPs prepared in this invention possess excellent mucus degradation and active penetration capabilities, significantly reducing the viscosity of artificial mucus and efficiently degrading DNA within it. Furthermore, Transwell experiments further prove their effective penetration of the mucus barrier and continuous delivery within 2 hours; particle motion trajectory analysis indicates that DH-CA NPs exhibit more active and wider-ranging motility in mucus, overcoming the diffusion limitations of traditional nanocarriers in mucus, and can be used for targeted delivery and treatment of the lungs.

[0114] Example 4: Cell compatibility test This embodiment tested the cytotoxicity of the above-mentioned DH-CA NPs nanoparticles to epithelial cells and macrophages. The specific method is as follows: MLE-12 (mouse alveolar epithelial cells), MH-S (human bronchial epithelial cells), and BEAS-2B (mouse macrophages) were seeded into RPMI-1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) and passaged in a 37 ℃, 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments.

[0115] DH-CA NPs were resuspended in sterile PBS and ultrasonically dispersed (100W, ice bath for 30 min) to form a homogeneous suspension. Six concentration gradients (0, 1, 5, 10, 20, and 40 μg / mL) were set up, with three replicates for each concentration. The original culture medium was discarded, and 100 μL of fresh culture medium containing different concentrations of NPs was added to each well. Incubation was performed at 12 h and 24 h. After incubation, 10 μL of CCK-8 solution was added to each well, avoiding the formation of air bubbles. The 96-well plate was gently shaken to mix, and incubation was continued for 2 h. The absorbance was measured at 450 nm using a microplate reader, with a reference wavelength of 600 nm to subtract background scattering. Before detection, the plate was shaken at 200 rpm for 30 s on an orbital oscillator to ensure homogeneity.

[0116] Figure 8 The results of cytotoxicity assays of DH-CA NPs on MLE-12, BEAS-2B, and MH-S cells at 12 h and 24 h were presented. The results showed that DH-CA NPs, within a concentration range of 1–40 μg / mL, did not significantly impair the survival of any of the three cell types (MLE-12, BEAS-2B, and MH-S) compared to the control group. p >0.05); among them, after 12 h of treatment, the cell viability remained at 95%-105% at all concentrations, with no concentration-dependent decreasing trend; after 24 h of treatment, at concentrations of 20 and 40 μg / mL, the viability of MH-S macrophages slightly decreased to 85%-90% ( p (<0.05), but MLE-12 and BEAS-2B cells still maintained a survival rate of over 90%.

[0117] The above results indicate that DH-CA NPs have excellent biocompatibility and good safety. The slight inhibition of MH-S cells may actually help regulate excessive inflammatory responses, such as inhibiting macrophage overactivation to reduce excessive mucus secretion.

[0118] Example 5: Cell uptake efficiency detection In this embodiment, laser confocal microscopy and flow cytometry were used to determine the uptake efficiency of DH-CA NPs by epithelial cells and macrophages, respectively, and the specific contents are as follows.

[0119] 1. Laser confocal microscopy inspection Cell lines MLE-12, BEAS-2B, and MH-S were cultured at 5 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per laser confocal microscopy culture dish and cultured at 37 ℃ in a 5% CO2 incubator for 24 h until the adhesion rate reached 80%-90%. For easier microscopic observation, DH-CA NPs were labeled with Cy5.5 fluorescent dye; 1 mg of nanomaterial was reacted with 10 μL of Cy5.5 under light-protected conditions for 2 h. The labeled DH-CA NPs were resuspended in sterile PBS buffer (pH 7.4) to a concentration of 5 mg / mL and sonicated on ice for 30 min to ensure uniform dispersion. 1 mL of culture medium containing DH-CA NPs (final concentration 10 µg / mL) was added to each cell culture dish, and the cells were incubated for 12 h and 24 h, respectively. A blank control (PBS treatment only) was also included to eliminate autofluorescence interference.

[0120] After incubation, cells were gently washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.1% Triton X-100 for 10 min. DAPI staining solution (1 μg / mL) was then added and stained in the dark for 15 min. The slides were then mounted and stored in the dark. A Nikon AX / AX R laser confocal system was used, equipped with a 405 nm (DAPI channel) and a 660 nm (Cy5.5 channel) laser. A 60x oil immersion objective (NA=1.4) was used, with a scanning resolution of 1024 × 1024 pixels and a pixel dwell time of 2 μs. To reduce fluorescence crosstalk, sequential scanning mode was used to acquire multi-channel images. Five fields of view were randomly selected for each sample to ensure a cell count ≥ 30. Images were saved in TIFF format to avoid compression loss. Colocalization analysis was performed using ImageJ software to qualitatively determine overlapping regions.

[0121] Figure 9 To determine the uptake of DH-CA NPs by MLE-12, BEAS-2B, and MH-S cells using laser confocal microscopy, the results showed that DH-CA NPs were effectively internalized by all three cell types at both 12 and 24 h, with the uptake increasing over time. Specifically, clear NP fluorescence signals (red) were visible around the DAPI-labeled nuclei (blue), with the signal primarily enriched in the cytoplasmic region. Colocalization analysis further confirmed successful cellular uptake of DH-CA NPs. These results indicate that the DH-CA NPs of this invention possess excellent delivery potential, such as good biocompatibility (non-cytotoxicity) ensuring that the uptake process does not cause membrane damage; nanoscale particle size (600-700 nm) and surface hyaluronic acid coating promoting specific binding to cell membrane receptors; and time-dependent accumulation providing a time window for controllable drug release.

[0122] 2. Flow cytometry detection Cell lines MLE-12, BEAS-2B, and MH-S were respectively planted at 1 × 10⁶ cells per well.5 NPs were inoculated at a density of 10 μg / mL into 6-well plates containing RIPA 1640 medium and incubated at 37 °C in a 5% CO2 incubator for 24 h until the adhesion rate reached 80%-90%. Simultaneously, the lyophilized DH-CANPs were resuspended in sterile PBS to a concentration of 5 mg / mL and sonicated on ice for 30 min to ensure complete dispersion. An experimental group (with added NPs) and a negative control group (with an equal volume of PBS) were set up, with 3 replicates per group. The original medium was discarded, and 2 mL of medium containing NPs (final concentration 10 μg / mL) was added to each well of the experimental group, while an equal volume of PBS was added to each well of the control group.

[0123] Cells were incubated at 37°C for 12 h and 24 h, respectively. After incubation, the cells were gently washed three times with pre-cooled PBS to remove uninternalized DH-CA NPs. For adherent cells (MLE-12, BEAS-2B), 0.25% trypsin (containing EDTA) was added for digestion for 3 min, and digestion was terminated with serum-containing medium. Suspension cells (MH-S) were collected directly. The cell suspension was transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in PBS containing 2% BSA and the density was adjusted to 1×10⁶ cells / mL. 6 Cells / mL were filtered through a 40 μm cell sieve to remove clumping and ensure the quality of the single-cell suspension. A BDFACSCantoII flow cytometer (equipped with a 405 nm UV laser) was used, with DAPI channel settings: excitation light 355 nm, emission light collected through a 450 / 50 nm bandpass filter. 10,000 effective cell events were collected per tube, with the flow rate controlled at 300-500 cells / s to avoid overlapping events.

[0124] Figure 10 Flow cytometry results showed that, compared with the PBS control group, the proportion of DAPI channel fluorescently positive cells in the DH-CA NPs treatment group was significantly increased at both 12 h and 24 h (p<0.001). However, when comparing data from the same cell type at the two time points, there was no significant change in the proportion of cells taking up DH-CA NPs at 12 h, indicating that cells had already completed the uptake of DH-CA NPs by 12 h. High uptake efficiency (especially in macrophages) suggests that DH-CA NPs can effectively deliver anti-inflammatory or antibacterial drugs to immune cells, contributing to enhanced therapeutic effects against lung infections. This experiment quantitatively demonstrated by flow cytometry that DH-CA NPs can be effectively internalized by lung-derived cells, and that uptake is time-dependent and cell-type specific. These results, combined with previous morphological characterization (SEM, fluorescence microscopy) and biocompatibility data, fully reveal the core advantages of DH-CA NPs as a lung-targeted delivery system: highly efficient cell internalization and low toxicity.

[0125] The above results indicate that the DH-CA NPs prepared in this invention can be efficiently taken up by the main lung cells (MLE-12 alveolar epithelial cells, BEAS-2B bronchial epithelial cells, and MH-S macrophages).

[0126] Example 6: Detection of intercellular transport capacity This embodiment uses laser confocal microscopy to observe the intercellular transport capacity of DH-CA NPs. The specific experimental method is as follows: Cell lines MLE-12, BEAS-2B, and MH-S were used at 5 × 10⁶ cells per well. 4 The culture medium was seeded at a density of 100 cells / wells in a laser confocal glass-bottomed culture dish (such as ibidiμ-Dish) in a 24-well plate and incubated at 37 °C in a 5% CO2 incubator for 24 h until the adhesion rate reached 80%-90%. The bottom thickness of the culture dish was 0.17 mm to accommodate the working distance of high-magnification objectives.

[0127] To track NP transport, DH-CA NPs were covalently coupled with Cy5.5 fluorescent dye (excitation / emission: 660 / 710 nm). Specifically, 1 mg of NPs was reacted with 10 μL of Cy5.5 (1 mg / mL in DMSO) under light-protected conditions for 2 h, followed by dialysis with PBS to remove free dye. The labeled NPs were resuspended in PBS to a concentration of 100 μg / mL and sonicated on ice for 30 min to ensure uniform dispersion. The first batch of cells was incubated with Cy5.5-labeled DH-CA NPs for 6 h, washed three times with PBS, and then imaged using a confocal microscope. Next, 0.6 mL of fresh culture medium was added to the cells, and the cells were cultured for another 12 h. The culture medium was collected and used to culture a second batch of cells for 12 h. The second batch of cells was imaged, and then cultured for another 12 h in fresh culture medium. The culture medium was again separated and used to culture a third batch of fresh cells for 12 h, followed by imaging. Similarly, the third batch of cells was cultured, and then the culture medium was further used to culture a fourth batch of fresh cells for 12 h, followed by imaging. All cells were imaged using a laser confocal microscope with an excitation wavelength of 680 nm, with the Cy5.5 settings remaining unchanged.

[0128] Figure 11The results of the intercellular transport capacity assay for DH-CA NPs showed that Cy5.5-labeled DH-CA NPs exhibited strong fluorescence signals in the first batch of cells (positive cell rate exceeding 35%), the signal decreased to approximately 18% in the second batch, further decreased to 6% in the third batch, and showed no significant difference between the signal and background in the fourth batch (p>0.05). This result indicates that DH-CA NPs possess intercellular transport capacity, with efficiency decreasing progressively with each batch. This limited but present intercellular transport capacity can assist DH-CA NPs in forming a local diffusion network within lung tissue, enhancing drug distribution uniformity. The disappearance of the signal in the fourth batch suggests that DH-CA NPs do not accumulate indefinitely, reducing the risk of long-term toxicity.

[0129] The above results indicate that the DH-CA NPs prepared in this invention have high-efficiency cell internalization ability and can be used as a core component of a lung-targeted delivery system.

[0130] Example 7: In vivo experiment This embodiment tested the ability of DH-CA NPs to degrade lung mucus using a mouse pneumonia model. Figure 12 Figure A shows a schematic diagram of the experimental design for DH-CA NPs to degrade pulmonary airway mucus at the individual level. The specific experimental procedure is as follows.

[0131] 1. Experimental Methods (1) Establishment of an acute pneumonia model in mice: 8-10 week old C57BL / 6 mice (weighing 20-25 g) were used, with at least 6 mice per group to ensure statistical power. Animals were housed in an SPF-grade environment and fasted for 12 h (with free access to water) before the experiment to reduce metabolic interference. Lipopolysaccharide (LPS) was dissolved in sterile PBS to a concentration of 5 mg / mL, vortexed, and sonicated for 10 min to ensure complete dispersion. Mice were anesthetized by isoflurane inhalation and fixed at a 45° angle on a tilting plate. 50 μL of LPS solution was slowly injected into the trachea through a microsyringe. The mice were kept upright for 30 s after injection to ensure uniform distribution of the solution to the lungs, thus establishing an acute pneumonia model in mice.

[0132] (2) Drug administration: BPLP-PLLA, H-CA NPs, and DH-CA NPs were resuspended in physiological saline to a concentration of 5 mg / mL, and sonicated for 5 min to ensure uniform particle size distribution. Six hours after LPS injection, physiological saline, BPLP-PLLA, H-CA NPs, and DH-CA NPs were nebulized using a vibrating sieve nebulizer for 60 min, with a nebulization flow rate of 10 mL / min. Eighteen hours after LPS injection (i.e., 12 hours after drug administration), mice were euthanized by carbon dioxide asphyxiation. The thorax was immediately opened, and the heart was perfused with PBS to remove blood. The intact lung tissue was then removed for subsequent sectioning and staining.

[0133] (3) Determination of total protein concentration in lung lavage fluid: After euthanizing the mice, the trachea was immediately exposed, and a sterile intravenous catheter was inserted into the trachea to slowly inject 800 μL of pre-cooled PBS buffer (pH 7.4, containing protease inhibitors to prevent protein degradation). After injection, the pleural cavity was gently massaged for 30 seconds to promote fluid distribution, and then about 1.5 mL of lavage fluid was aspirated. This process was repeated 3 times, and the lavage fluids were combined and centrifuged at 2000 rpm for 10 min at 4 ℃. The supernatant was collected and stored at -80℃ for later analysis. (2) The total protein concentration in mouse lung lavage fluid was determined using the BCA protein quantification kit.

[0134] The preparation of the protein standard curve included: preparing a concentration gradient of bovine serum albumin (BSA) (0, 0.1, 0.2, 0.5, 1.0, and 2.0 mg / mL), adding 25 μL of standard or sample (wash buffer supernatant) to each well, followed by the addition of 200 μL of LCA working solution. The reaction system was incubated at 37°C in the dark for 30 min, and the absorbance was then measured at 562 nm using a microplate reader. Three technical replicates were performed for each sample.

[0135] (4) AB-PAS analysis of lung trachea: After the mice were sacrificed, intact lung tissue was immediately removed and tracheal segments were separated. The tissue was fixed with 4% paraformaldehyde at 4℃ for 24 h to maintain the structural integrity of mucoglycoproteins. After fixation, the tissue was dehydrated by gradient sucrose (10%, 20%, and 30% sucrose solutions for 24 h each) and finally embedded in OCT embedding medium. Transverse frozen sections with a thickness of 10 μm were prepared using a cryostat. The sections were attached to polycysteine-treated slides and air-dried at room temperature for 2 h to ensure firm adhesion. Then, the sections were stained with AB-PAS using an AB-PAS kit. The specific operation was as follows: the sections were first stained with Alcian blue for 30 min, then rinsed with running water for 5 min to remove unbound dye; then immersed in 1% periodic acid solution for 10 min for oxidation, and rinsed with distilled water; finally, stained with Scheffler's reagent in the dark for 20 min, and separated with sulfite solution twice, 1 min each time, to reduce background staining. Finally, an optical microscope was used to capture cross-sectional images of the trachea under a 40x objective lens. Five fields of view were randomly selected for each sample to ensure that the entire circumference of the trachea was covered.

[0136] (5) AB-PAS analysis of lung bronchus: After the mice were sacrificed, the intact lung tissue was immediately removed, the lung lobes (containing terminal bronchi) were separated, and then processed according to the above-mentioned AB-PAS analysis method of lung bronchus. Finally, cross-sectional images of the terminal bronchi of the lung lobes were taken under a 40x objective lens using an optical microscope. Five fields of view were randomly selected for each sample to ensure that the complete area of ​​the circumference of the terminal bronchi was covered.

[0137] 2. Experimental Results Figure 12 Figure B represents the statistical results of total protein concentration in mouse bronchoalveolar lavage fluid. The results show that the total protein concentration in the DH-CA NPs treatment group was significantly reduced to 0.161 mg / mL, while the saline control group had a concentration of 0.196 mg / mL (p<0.05). The total protein concentrations in the BPLP-PLLA and H-CA NPs groups were 0.198 mg / mL and 0.185 mg / mL, respectively, showing no significant difference from the saline control group (p>0.05). This result confirms the unique mucus-degrading ability of DH-CA NPs. This result, together with previous in vitro data (tilt plate flow, gel electrophoresis), forms a complete chain of evidence, jointly supporting the therapeutic efficacy of DH-CA NPs at the individual level, and providing crucial in vivo validation for the claim in the patent application to "significantly reduce lung mucus viscosity."

[0138] Figure 12C represents the AB-PAS analysis results of the trachea. Compared with the blank control group, BPLP, BPLP-PLLA and H-CA NPs did not significantly reduce the thickness of the tracheal mucus layer, while the tracheal mucus layer thickness of the DH-CA NPs treatment group was significantly reduced, indicating that DH-CA NPs can significantly degrade the tracheal mucus layer and relieve airway obstruction symptoms.

[0139] Figure 12 D in the figure represents the results of AB-PAS analysis of mouse lung bronchus. Similar to the results of AB-PAS analysis of lung trachea, compared with the blank control group, BPLP, BPLP-PLLA and H-CA NPs did not significantly reduce the thickness of the mucus layer in the exhaust trachea, while the mucus layer thickness of the terminal bronchus in the DH-CANPs treatment group was significantly reduced, indicating that DH-CA NPs can significantly degrade the mucus layer of the bronchus and alleviate airway obstruction.

[0140] The above results indicate that in an LPS-induced mouse pneumonia model, nebulized DH-CA NPs can effectively treat excessive mucus secretion. Compared with saline and other control groups, the DH-CA NPs treatment group significantly reduced the total protein concentration in the bronchoalveolar lavage fluid and significantly reduced the thickness of the mucus layer in the trachea and bronchi, thus alleviating airway obstruction. This demonstrates that DH-CA NPs have a highly efficient ability to degrade and clear mucus, and can be used for the clinical treatment of inflammatory mucus-related diseases of the lungs.

[0141] In summary, this invention provides nanoparticles for clearing pulmonary mucus, their preparation method, and applications. The citric acid nanoparticles (DH-CA NPs) of this invention, loaded with alfa chain enzyme and modified with hyaluronic acid, are synergistically constructed using natural materials, exhibiting excellent biocompatibility and functional diversity. In response to the inflammatory microenvironment of the lungs, they can sequentially achieve multi-target synergistic therapy by penetrating the mucus barrier, degrading DNA networks, reducing the release of pro-inflammatory factors, and inhibiting mucus production, overcoming the limitations of single-drug therapy. Furthermore, in vivo experimental results show that they can significantly reduce pulmonary mucus thickness and lavage fluid protein concentration, and effectively alleviate airway obstruction, demonstrating promising application prospects in the treatment of mucus hypersecretion pulmonary diseases.

[0142] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A nanoparticle, characterized in that, It consists of a core and a shell covering the surface of the core, wherein: The core contains citric acid and a photoluminescent polymer-aliphatic polyester copolymer; The outer shell contains deoxyribonuclease and a hydrophilic polymer.

2. The nanoparticles according to claim 1, characterized in that, The aliphatic polyester is selected from at least one of poly(L-lactide), polyglycolic acid, polycaprolactone, and polylactic-co-glycolic acid; Preferably, the photoluminescent polymer-aliphatic polyester copolymer is selected from any one of the following: photoluminescent polymer-poly(L-lactide) copolymer, photoluminescent polymer-poly(glycolic acid) copolymer, photoluminescent polymer-polycaprolactone copolymer, and photoluminescent polymer-polylactic acid-glycolic acid copolymer.

3. The nanoparticles according to claim 1 or 2, characterized in that, The deoxyribonuclease includes deoxyribonuclease I; And / or, the hydrophilic polymer includes hyaluronic acid; And / or, the particle size of the nanoparticles is 100~800 nm.

4. A method for preparing nanoparticles as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. The oil phase containing the photoluminescent polymer-aliphatic polyester copolymer is mixed with the aqueous phase containing the citric acid and reacted to obtain the citric acid nanoparticle core. S2. The citric acid nanoparticle core is reacted sequentially with the deoxyribonuclease and the hydrophilic polymer to obtain the final product.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass ratio of the photoluminescent polymer-aliphatic polyester copolymer to the citric acid is 1:3~8; And / or, the solvent of the oil phase includes at least one of dichloromethane and ethyl acetate; And / or, the solvent of the aqueous phase includes water.

6. The preparation method according to claim 4, characterized in that, In step S2, the sequential reaction with the deoxyribonuclease and the hydrophilic polymer specifically includes: S21. The citric acid nanoparticle core and the deoxyribonuclease are mixed and reacted to obtain deoxyribonuclease-coated core particles; S22. Mix solution 1 containing the core particles coated with the deoxyribonuclease and solution 2 containing the hydrophilic polymer, react, and then purify to obtain the final product.

7. The preparation method according to claim 6, characterized in that, The mass ratio of the citric acid nanoparticle core to the deoxyribonuclease is 10:0.1~3; Preferably, the solvent of solution 1 is dichloromethane and / or ethyl acetate; Preferably, the solvent of solution 2 comprises water and ethanol; Preferably, the mass ratio of the deoxyribonuclease-coated core particles to the hydrophilic polymer is 5:5~20.

8. A nano-atomizing spray, characterized in that, It includes the nanoparticles as described in any one of claims 1 to 3.

9. A pharmaceutical composition, characterized in that, The active material includes the nanoparticles as described in any one of claims 1 to 3.

10. The use of the nanoparticles according to any one of claims 1 to 3, the nano-atomizing spray according to claim 8, or the pharmaceutical composition according to claim 9 in any one of the following: A) Prepare a drug to clear pulmonary mucus; B) Preparation of drugs to inhibit mucus secretion; C) Preparation of drugs that target and deliver nucleic acids to the lungs; D) Prepare drugs for treating lung diseases.

Images