Honeycomb-shaped PLGA (poly (lactic-co-glycolic acid)) microsphere with chitosan ambroxol coating and preparation method of honeycomb-shaped PLGA microsphere

Abstract

The invention provides a honeycomb PLGA (poly (lactic-co-glycolic acid)) microsphere with a chitosan ambroxol coating and a preparation method of the honeycomb PLGA microsphere with the chitosan ambroxol coating. The honeycomb PLGA microsphere with the chitosan ambroxol coating can be loaded with drugs such as andrographolide, colistin, amphotericin B and the like. Meanwhile, by modifying the ambroxol chitosan coating on the surface of the PLGA microsphere, the respiratory tract mucus penetrating ability of the PLGA microsphere is greatly enhanced, the sequential release of the loaded medicine can be realized, the ambroxol released in the first stage can break through the respiratory tract mucus barrier of the lung, and the release effect of the ambroxol chitosan coating on the surface of the PLGA microsphere is improved. The medicine released in the second stage can play anti-inflammatory and antifungal roles and is used for preventing and treating lung infectious diseases.

Description

Technical Field

[0001] The invention relates to the field of drug delivery, in particular to a chitosan ambroxol coated honeycomb PLGA microsphere and a preparation method thereof. Background Art

[0002] Due to the abundant capillaries in the lungs, the large total area of ​​alveoli, and the short distance for substance transport between epithelial cells and blood vessels, pulmonary drug delivery preparations deliver drugs directly to the lung tissue and can achieve higher local concentrations, making them the preferred choice for many drugs for treating lung diseases. However, drugs that enter the lungs are easily cleared by the lung tissue and are difficult to break through the respiratory mucus barrier and the biofilm formed by bacteria and fungi, which reduces the drug's retention time in the lungs and affects the efficacy. Although particles with smaller sizes (1-5 μm) are easily inhaled into the deep lungs, they are also easily phagocytosed and cleared by lung macrophages.

[0003] PLGA (polylactic-glycolic acid) is a biomedical polymer material certified by the FDA, with properties such as high biocompatibility and biodegradability. The polymer PLGA porous microspheres with large geometric particle size and low density can not only effectively escape the phagocytosis of alveolar macrophages, but also have a high effective lung deposition rate due to the appropriate aerodynamic diameter. The polymer is biodegradable and has no toxic side effects, so it can become a good lung drug delivery carrier. However, for many pulmonary infectious diseases or other chronic diseases, PLGA porous microspheres are difficult to break through the respiratory mucus barrier and the biofilm formed by bacteria and fungi, which reduces the drug's lung retention time and affects the efficacy.

[0004] Therefore, it is necessary to study and develop more effective drug delivery methods for lung infectious diseases or other chronic diseases. Summary of the invention

[0005] The present invention first provides a method for preparing chitosan ambroxol coated honeycomb PLGA microspheres, which comprises the following steps:

[0006] (1) mixing a 15 mg / mL PLGA dichloromethane solution with a 1% by weight polyvinyl alcohol (PVA) aqueous solution containing 1-10% by weight NaHCO3 (preferably 3-5%), and emulsifying in an ice bath for 2 minutes to form a primary emulsion; wherein the volume ratio of the PLGA dichloromethane solution to the PVA aqueous solution is 1-3:1;

[0007] The emulsification is carried out by a cell disruptor (SCIENTZ-IID, Japan) at 25-50W (preferably 30-35W) for 1-5 minutes (preferably 2-3 minutes);

[0008] (2) then adding the primary emulsion to a 1% PVA aqueous solution; homogenizing at 5000 rpm in an ice bath for 5 minutes to prepare an emulsion; wherein the volume ratio of the primary emulsion to the PVA aqueous solution is 1:1-3;

[0009] (3) the prepared emulsion was magnetically stirred at room temperature overnight; the solidified PLGA microspheres (HCLplga) were collected by centrifugation, washed with deionized water, then resuspended in deionized water, and finally freeze-dried to obtain HCLplga dry powder;

[0010] If necessary, the desired drugs, such as andrographolide, colistin, etc., can be loaded into the obtained HCLplga;

[0011] (4) Ambroxol was added to a chitosan acetic acid solution (chitosan was dissolved in a 2% by weight acetic acid solution, wherein the weight percentage content of chitosan was 0.25%), and the final concentration of ambroxol was 25 mg / mL; the solution was mixed with HCLplga or HCLplga loaded with drugs, and stirred continuously at room temperature for 4 hours; the microspheres (HCLplga-Ab) were collected by centrifugation at 3000 rpm, and then washed and freeze-dried.

[0012] The obtained chitosan-ambroxol coated PLGA microspheres were stored in a desiccator for later use.

[0013] The invention also provides HCLplga microspheres coated with chitosan and ambroxol prepared by the above method, wherein the geometric particle size of the microspheres is 10-15 μm and the median aerodynamic particle size is 2.57 μm.

[0014] The present invention also provides HCLplga microspheres with chitosan ambroxol coating loaded with drugs, such as HCLplga microspheres with chitosan ambroxol coating loaded with andrographolide; HCLplga microspheres with chitosan ambroxol coating loaded with colistin or amphotericin B;

[0015] The prepared chitosan ambroxol coated HCLplga microspheres (HCLplga-Ab) freeze-dried particles are in the form of white powder, have excellent dispersibility, and can produce uniform mist when sprayed using a dry powder aerosol generator.

[0016] Among them, the above-mentioned HCLplga microspheres can be loaded with various drugs for the treatment of lung diseases, such as andrographolide, colistin, amphotericin B, etc., which are one or more of andrographolide, colistin, and amphotericin B; the drug can be added to a 1% PVA solution, or the prepared uncoated PLGA microspheres can be mixed with an aqueous solution containing the drug to obtain the drug-loaded PLGA microspheres, and then the chitosan ambroxol coating is added. The specific method of loading the drug is as follows:

[0017] In the above step (1), andrographolide is added to a 1% PVA solution containing 1-10% by weight of NaHCO3, and prepared according to the method for preparing HCLplga microspheres coated with chitosan and ambroxol; wherein the concentration of andrographolide in the PVA aqueous solution is 6 mg / mL;

[0018] For the preparation of the other two drugs (colistin, amphotericin B) loaded into microspheres: at room temperature, on the basis of HCLplga microspheres, 10 mg / mL colistin or 10 mg / mL amphotericin B was added to the HCLplga solution under magnetic stirring for 6 hours. The solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL), and finally freeze-dried by freeze drying to obtain drug-loaded HCLplga.

[0019] The drug-loaded PLGA microspheres are then coated with chitosan ambroxol in the above step (4). When used for the treatment of pulmonary infectious diseases, they can be administered by atomization or intravenous drip, or in other pharmaceutically acceptable dosage forms.

[0020] The innovation of the honeycomb PLGA microspheres provided by the present invention and the beneficial technical effects brought about by them:

[0021] The present invention uses an emulsification-solvent volatilization method to prepare a safe and efficient honeycomb porous PLGA microsphere delivery system (HCLplga), and adjusts the porosity, particle size, and pore size of the microspheres to adjust their aerodynamic diameter, so that the drug deposition rate in the lungs is the highest. At the same time, by modifying the surface of the microspheres with an ambroxol chitosan coating, the respiratory mucus penetration ability is greatly enhanced, and the sequential release of drugs can be achieved. The ambroxol released in the first stage can break through the respiratory mucus barrier of the lungs, and the drugs released in the second stage can play an anti-inflammatory and antifungal role.

[0022] The honeycomb PLGA microspheres provided by the present invention are inhalable honeycomb PLGA dry powder microspheres that avoid being phagocytosed by alveolar macrophages and can penetrate respiratory mucus. As a good lung drug delivery platform, it can be combined with traditional Chinese medicine and other drugs to treat a variety of lung diseases. For example, andrographolide, which is one of the traditional Chinese medicines, has a variety of therapeutic effects, such as anti-inflammatory, antioxidant, and antifungal. However, andrographolide has poor solubility, low permeability, and poor gastrointestinal stability, resulting in low bioavailability. Moreover, andrographolide also lacks a pulmonary inhalation dosage form. The honeycomb PLGA microspheres with ambroxol chitosan coating provided by the present invention can effectively transport andrographolide to the alveoli after loading andrographolide, and have a good therapeutic effect on lung diseases such as septic lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Parameters of the microspheres prepared in Example 2, such as the mirror image and particle size

[0024] Figure 2 Parameters of the prepared microspheres in Example 3: Mirror image and particle size

[0025] Figure 3 Parameters of the microspheres prepared in Example 4, such as the mirror image and particle size

[0026] Figure 4 Parameters of the prepared microspheres in Example 5: Mirror image and particle size

[0027] in, Figure 1-4 Middle, left: representative scanning electron micrograph of microspheres. Middle: fluorescence micrograph of porous microspheres labeled with Nile red. Right: geometric particle size distribution and porosity of microspheres.

[0028] Figure 5 Simulated lung distribution experiment

[0029] Figure 6 Results of the experimental study on the distribution of fluorescent microspheres in the lungs

[0030] Figure 7 Results of mucus penetration test DETAILED DESCRIPTION

[0031] The following examples are only used to illustrate and explain the present invention and are not intended to limit the scope of protection of the present invention.

[0032] Sources:

[0033] Poly(D,L-lactic acid-polyethylene glycol) [PLGA, lactic acid:polyethylene glycol (50:50), 30,000-60,000], polyvinyl alcohol (PVA), chitosan, colistin (CST), dichloromethane (DCM, CH2Cl2), porcine gastric mucin, and sodium bicarbonate were purchased from Sigma-Aldrich.

[0034] IR780 and Nile Red dye were purchased from Med Chemical Express.

[0035] Andrographolide (Andro), ambroxol (Ambroxol) and amphotericin B (AmB) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] Rhodamine phalloidin and 4',6-diamidino-2'-phenylindole (DAPI) were purchased from UELandy.

[0037] Deoxyribonucleic acid (DNA) and diethylenetriaminepentaacetic acid were purchased from Dalian Meilun Biotechnology Co., Ltd.

[0038] The percentage contents of the solutions used in the following examples are all by weight unless otherwise specified.

[0039] Example 1: Preparation of HCLplga microspheres

[0040] Preparation of HCLplga (honeycomb poly(lactic acid-co-glycolic acid)) microspheres: HCLplga-Ab microspheres were prepared using water-in-oil-in-water (W / O / W) double emulsions using solvent diffusion evaporation technology.

[0041] A PLGA dichloromethane solution (15 mg / mL, 10 ml) and a polyvinyl alcohol (PVA) aqueous solution (5 ml, 1%, weight percentage content, the same below) containing NaHCO3 (3%, weight percentage content, the same below) were emulsified in an ice bath using a cell disruptor (SCIENTZ-IID, Japan) at 35 W for 2 minutes to form a W / O primary emulsion.

[0042] The primary emulsion was then added to a 1% PVA aqueous solution (30 mL); the mixed solution was homogenized at 5000 rpm in an ice bath for 5 minutes to prepare a W / O / W emulsion.

[0043] The prepared emulsion was stirred magnetically overnight at room temperature to evaporate all the dichloromethane; the solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL). Finally, the HCLplga was obtained by freeze drying, and the freeze-dried microspheres were white powder with excellent dispersibility.

[0044] Example 2: Preparation of HCLplga-Ab microspheres

[0045] Preparation of HCLplga-Ab (honeycomb poly(lactic acid-co-glycolic acid)) microspheres: HCLplga-Ab microspheres were prepared using water-in-oil-in-water (W / O / W) double emulsions using solvent diffusion evaporation technology.

[0046] A PLGA dichloromethane solution (15 mg / mL, 10 ml) and a polyvinyl alcohol (PVA) aqueous solution (1%, 5 mL) containing NaHCO3 (1%) were emulsified in an ice bath using a cell disruptor (SCIENTZ-IID, Japan) at 35 W for 2 minutes to form a W / O primary emulsion.

[0047] The primary emulsion was then added to a 1% PVA aqueous solution (30 mL); the mixed solution was homogenized at 5000 rpm in an ice bath for 5 minutes to prepare a W / O / W emulsion.

[0048] The prepared emulsion was magnetically stirred at room temperature overnight to evaporate all the dichloromethane; the solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL).

[0049] Ambroxol was added to a dilute acetic acid solution containing 0.25% chitosan (the dilute acetic acid solution was 2% by weight), and the final concentration of ambroxol was 25 mg / mL; the solution was mixed with the HCLplga solution, and stirred continuously at room temperature for 4 hours; the microspheres were collected by centrifugation at a speed of 3000 rpm, and then washed and freeze-dried. Finally, the HCLplga-Ab with chitosan ambroxol coating was obtained by freeze drying, and the freeze-dried microspheres were white powder with excellent dispersibility.

[0050] Prepared microspheres (see Figure 1 )'s particle size, pores and other parameters: the particle size is approximately equal to 10 μm, and the porosity is 21.37%.

[0051] Example 3: HCLplga-Ab microspheres were prepared using a water-in-oil-in-water (W / O / W) double emulsion using solvent diffusion evaporation technology.

[0052] A PLGA dichloromethane solution (15 mg / mL, 10 ml) and a polyvinyl alcohol (PVA) aqueous solution (2%, 5 mL) containing NaHCO3 (2%) were emulsified in an ice bath using a cell disruptor (SCIENTZ-IID, Japan) at 35 W for 2 minutes to form a primary emulsion.

[0053] The primary emulsion was then added to a 1% PVA aqueous solution (30 mL). The mixed solution was homogenized at 5000 rpm for 5 minutes in an ice bath to prepare a W / O / W emulsion.

[0054] The prepared emulsion was stirred magnetically overnight at room temperature to evaporate all the dichloromethane. The solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL).

[0055] Ambroxol was added to a dilute acetic acid solution containing 0.25% chitosan (the dilute acetic acid solution was 2% by weight), and the final concentration of ambroxol was 25 mg / mL; the solution was mixed with the HCLplga solution, and stirred continuously at room temperature for 4 hours; the microspheres were collected by centrifugation at a speed of 3000 rpm, and then washed and freeze-dried. Finally, the HCLplga-Ab with chitosan ambroxol coating was obtained by freeze drying, and the freeze-dried microspheres were white powder with excellent dispersibility.

[0056] Prepared microspheres (see Figure 2 ): Particle size is approximately equal to 10 μm, porosity is 70.26%.

[0057] Example 4: HCLplga-microspheres were prepared using a water-in-oil-in-water (W / O / W) double emulsion using solvent diffusion evaporation technology.

[0058] A PLGA dichloromethane solution (15 mg / mL, 10 ml) and a polyvinyl alcohol (PVA) aqueous solution (2%, 5 mL) containing NaHCO3 (3%) were emulsified in an ice bath using a cell disruptor (SCIENTZ-IID, Japan) at 35 W for 2 minutes to form a primary emulsion.

[0059] The primary emulsion was then added to a 1% PVA aqueous solution (30 mL). The mixed solution was homogenized at 5000 rpm for 5 minutes in an ice bath to prepare a W / O / W emulsion.

[0060] The prepared emulsion was stirred magnetically overnight at room temperature to evaporate all the dichloromethane. The solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL).

[0061] Ambroxol was added to a dilute acetic acid solution containing 0.25% chitosan (the dilute acetic acid solution was 2% by weight), and the final concentration of ambroxol was 25 mg / mL; the solution was mixed with the HCLplga solution, and stirred continuously at room temperature for 4 hours; the microspheres were collected by centrifugation at a speed of 3000 rpm, and then washed and freeze-dried. Finally, the HCLplga-Ab with chitosan ambroxol coating was obtained by freeze drying, and the freeze-dried microspheres were white powder with excellent dispersibility.

[0062] Prepared microspheres (see Figure 3 ): Particle size is approximately 12 μm, porosity 73.56%

[0063] Example 5: HCLplga-microspheres were prepared using a water-in-oil-in-water (W / O / W) double emulsion using solvent diffusion evaporation technology.

[0064] A PLGA dichloromethane solution (15 mg / mL, 10 ml) and a polyvinyl alcohol (PVA) aqueous solution (2%, 5 mL) containing NaHCO3 (10%) were emulsified in an ice bath using a cell disruptor (SCIENTZ-IID, Japan) at 35 W for 2 minutes to form a primary emulsion.

[0065] The primary emulsion was then added to a 1% PVA aqueous solution (30 mL). The mixed solution was homogenized at 5000 rpm in an ice bath for 5 minutes to prepare a W / O / W emulsion. The prepared emulsion was magnetically stirred overnight at room temperature to evaporate all the dichloromethane. The solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL)

[0066] Ambroxol was added to a dilute acetic acid solution containing 0.25% chitosan (the dilute acetic acid solution was 2% by weight), and the final concentration of ambroxol was 25 mg / mL; the solution was mixed with the HCLplga solution, and stirred continuously at room temperature for 4 hours; the microspheres were collected by centrifugation at a speed of 3000 rpm, and then washed and freeze-dried. Finally, the HCLplga-Ab with chitosan ambroxol coating was obtained by freeze drying, and the freeze-dried microspheres were white powder with excellent dispersibility.

[0067] Prepared microspheres (see Figure 4 ): The particle size is approximately equal to 10 μm and the porosity is 85.42%.

[0068] Example 6 Microsphere distribution experiment in simulated lungs:

[0069] Experimental procedures: To simulate the deposition of microspheres in the lungs, HCLplga and HCLplga-Ab were tested using an Anderson Cascade Impactor (ACI) manufactured by Copley, Nottingham, UK.

[0070] Individual hard gelatin capsules containing 25 mg of regular HCLplga (Example 1) and HCLplga-Ab (Example 4) dry powder were prepared and inserted into the sample chamber of the Rotahaler device near the inlet of the ACI instrument. After the device was started, the capsule was pierced to release the dry powder, which was then drawn through the impactor at a continuous airflow rate of 30 liters per minute. The ACI plate consists of different stages with different retention pore sizes corresponding to different anatomical regions of the respiratory tract. The powder collected from each stage of the ACI was separated and weighed. The weight of each plate was used to determine the proportion of powder to the total amount of initial dry powder.

[0071] Experimental results ( Figure 5 ): It can be seen that most of the microspheres are distributed in stage 4, stage 5 and stage 6, indicating that the microspheres prepared in Example 1 and Example 4 can be deposited in the distal lung area.

[0072] Example 7 Experiment on the distribution of fluorescent microspheres in lungs:

[0073] Preparation of fluorescently labeled HCLplga microspheres: The method of Example 4 was used, wherein in step (1), 0.02% Nile red was added to the PLGA solution to prepare Nile red fluorescently labeled HCLplga-Ab.

[0074] Experimental procedures: Mice were anesthetized with sodium pentobarbital (50 mg / kg). Nile red-labeled HCLplga-Ab (20 mg / kg) was atomized through the trachea. Mice were killed 2 hours later, lung tissues were collected and placed in liquid nitrogen, and frozen section tissue analysis was performed at BIOSSCI Biotechnology Co., Ltd. Lung tissue was stained with DAPI (4',6-diamidino-2-phenylindole).

[0075] Experimental results ( Figure 6 ): Two hours after nebulization, it was observed that most of the aerosol particles were evenly dispersed in the lower region of the lung, especially in the terminal bronchi, bronchi and alveoli, while they were rarely found in the upper respiratory tract such as the alveoli, indicating that the microspheres can still reach the distal lung regions such as the alveoli in vivo.

[0076] Example 8 Mucus penetration experiment:

[0077] Synthesis of artificial mucus: 250 mg porcine gastric mucin, 500 mg DNA, 0.295 mg diethylenetriaminepentaacetic acid, 250 μl sterile egg yolk emulsion, 250 mg sodium chloride, 110 mg potassium chloride and 1 ml RPMI 1640 were mixed and suspended in DNase-free water to a final volume of 50 ml. The dispersion was equilibrated at 25°C for 2 hours.

[0078] Then, HCLplga prepared in Example 1 and HCLplga-Ab prepared in Example 3 were added to artificial mucus at a concentration of 2 mg / ml, respectively, and cultured at 37° C. for 1 hour.

[0079] The rheological properties of the artificial mucus were evaluated by rheological tests using a rotational rheometer (DHR20, TA, USA) equipped with a parallel plate configuration (60 mm diameter, 1000 μm gap) at a controlled temperature of 25° C. The viscosity of the artificial mucus was evaluated at shear rates ranging from 1 to 1000 / 1 s. The storage modulus (G′, Pa) and loss modulus (G″, Pa) of artificial mucus were determined by oscillatory frequency sweep tests. The experimental data obtained were processed and interpreted using TRIOS rheometer data analysis software. The plotted graphs show the changes in viscosity at different shear rates. The motion of individual particles was captured by acquiring time-lapse fluorescence overlay images. These motion data were merged into the motion trajectory of each monitored particle using ImageJ software. Experimental results: As shown in the figure below, due to the presence of chitosan, the coating layer of HCLplga-Ab aerosol was degraded and further released ambroxol in an acidic environment. At the same time, the released ambroxol can promote the penetration of particles into the highly viscous mucosa within the mucus layer on the airway surface. Multiple particle tracking technology (MPT) was used to evaluate the motion trajectory of aerosol particles in artificial mucus (AM). The movement of HCLplga was mainly trapped in a small range in AM, while HCLplga-Ab showed more active movement in a larger range. Similarly, the mean square displacement (MSD) value of HCLplga-Ab was much higher than that of HCLplga, indicating its excellent mucus penetration ability.

[0080] Experimental results: Figure 7 As shown in the figure, due to the presence of chitosan, the coating layer of HCLplga-Ab aerosol is degraded and further releases ambroxol in an acidic environment. At the same time, the released ambroxol can promote the particles to penetrate the highly viscous mucosa in the mucus layer on the airway surface. Multiple particle tracking technology (MPT) was used to evaluate the movement trajectory of aerosol particles in artificial mucus (AM). The movement of HCLplga is mainly trapped in a small range in AM ( Figure 7 Left), while HCLplga-Ab showed more active movement over a larger range ( Figure 7 Similarly, the mean square displacement (MSD) value of HCLplga-Ab is much higher than that of HCLplga ( Figure 7 right), which indicates its excellent mucus penetration ability.

[0081] Example 9 Preparation of HCLplga-Ab loaded with andrographolide (Ad@HCLplga-Ab)

[0082] 30 mg of andrographolide Andro was added to 5 ml of 1% PVA solution. PLGA dichloromethane solution (15 mg / mL, 10 ml) and andrographolide polyvinyl alcohol (PVA) aqueous solution (1%, 5 mL) containing NaHCO3 (3%) were emulsified in an ice bath using a cell disruptor (SCIENTZ-IID, Japan) at 35 W for 2 minutes to form a W / O primary emulsion.

[0083] The primary emulsion was then added to a 1% PVA aqueous solution (30 mL); the mixed solution was homogenized at 5000 rpm in an ice bath for 5 minutes to prepare a W / O / W emulsion.

[0084] The prepared emulsion was magnetically stirred at room temperature overnight to evaporate all the dichloromethane; the solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL).

[0085] Ambroxol is added to a dilute acetic acid solution containing 0.25% chitosan (the dilute acetic acid solution is 2% by weight), and the final concentration of ambroxol is 25 mg / mL; the solution is mixed with the HCLplga solution and stirred continuously at room temperature for 4 hours; the microspheres are collected by centrifugation at a speed of 3000 rpm, and then washed and freeze-dried. Finally, the HCLplga-Ab with chitosan and ambroxol coating loaded with andrographolide is obtained by freeze drying, and the freeze-dried microspheres are white powder with excellent dispersibility.

[0086] Example 10 Construction of sepsis acute lung injury model and the effect of administering HCLplga-Ab loaded with andrographolide (Ad@HCLplga-Ab) prepared in Example 9

[0087] 1. Animal modeling: intratracheal instillation of lipopolysaccharide (10 mg / kg);

[0088] 2. 12 hours after modeling, the HCLplga loaded with andrographolide prepared in Example 9 was atomized and inhaled.

[0089] 3. After 48 hours, the inflammatory factors, total protein content, total cell content in the alveolar lavage fluid of mice and the inflammatory factors in the plasma were quantified.

[0090] 4. Analyze the survival of mice 168 hours after modeling.

[0091] The results are as follows:

[0092] In this example, a mouse model of severe sepsis acute lung injury (ALI) was used to study the in vivo therapeutic effect of Ad@HCLplga-Ab aerosol particles. 12 hours after LPS modeling, mice inhaled PBS, Andro (4 mg kg -1 )、Ad@HCLplga(10mg kg -1 )、Ad@HCLplga-Ab(10mg kg -1 ). After 48 hours, the levels of inflammatory factors and other indicators in the bronchoalveolar lavage fluid (BALF) and serum of these mice were analyzed. The survival rate of mice was analyzed after 168 hours. Compared with mice treated with PBS (20%), Andro (25%) and Ad@HCLplga (30%), mice treated with Ad@HCLplga-Ab had a higher survival rate (60%).

[0093] To verify the degree of pulmonary interstitial edema in the above treatments, the lung dry-to-wet weight ratio (W / D) and protein in BALF were also calculated. Mice treated with PBS showed the highest W / D ratio and protein content, while the group treated with Ad@HCLplga-Ab showed values ​​close to those of the negative control, indicating that Ad@HCLplga-Ab can effectively alleviate pulmonary edema and interstitial water leakage in mice. The number of cells in LPS-induced mice treated with Ad@HCLplga-Ab was significantly reduced, indicating that severe inflammation was alleviated.

[0094] The levels of proinflammatory cytokines TNF-α, IL-6, and interleukin-1β (IL-1β) in BALF and serum were significantly lower than those in other groups of mice. In order to better observe the changes in lung tissue of mice with severe ALI in different groups, the mice were euthanized after 48 hours, and lung tissues were collected for histological analysis. Hematoxylin and eosin (H&E) staining analysis of lung tissue sections showed that mice treated with Ad@HCLplga-Ab had reduced pulmonary edema, widened alveolar walls, and aggregated foamy macrophages. Myeloperoxidase (MPO) staining of lung sections also confirmed these observations, showing reduced neutrophil infiltration and inflammation levels.

[0095] In summary, the experimental results of this example show that Ad@HCLplga-Ab aerosol significantly alleviated the inflammation of mice with acute lung injury caused by sepsis, and its effect was better than that of simple inhalation of free andrographolide.

[0096] Example 11 Preparation of HCLplga-Ab loaded with polymyxin andrographolide (CST / Ad@HCLplga-Ab)

[0097] 30 mg of andrographolide Andro was added to 1% PVA solution. PLGA dichloromethane solution (15 mg / mL, 10 ml) and andrographolide polyvinyl alcohol (PVA) aqueous solution (1%, 5 mL) containing NaHCO3 (3%) were emulsified in an ice bath using a cell disruptor (SCIENTZ-IID, Japan) at 35W for 2 minutes to form a W / O primary emulsion.

[0098] The primary emulsion was then added to a 1% PVA aqueous solution (30 mL); the mixed solution was homogenized at 5000 rpm in an ice bath for 5 minutes to prepare a W / O / W emulsion.

[0099] The prepared emulsion was magnetically stirred at room temperature overnight to evaporate all the dichloromethane; the solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL).

[0100] 10 mg / mL colistin was added to the above HCLplga solution loaded with andrographolide under magnetic stirring for 6 hours. The solidified HCLplga was collected by centrifugation (3000 rpm, 5 minutes), washed three times with deionized water, and then resuspended in deionized water (10 mL);

[0101] Ambroxol was added to a 0.25% chitosan dilute acetic acid solution, and the final concentration of ambroxol was 25 mg / mL; the solution was mixed with HCLplga or HCLplga solution loaded with drugs, and stirred continuously at room temperature for 4 hours; the microspheres were collected by centrifugation at 3000 rpm, and then washed and freeze-dried. Finally, the HCLplga-Ab loaded with andrographolide and colistin was obtained by freeze drying, and the freeze-dried microspheres were white powder with excellent dispersibility.

[0102] Example 12 Construction of bacteria-mediated acute lung injury model and the effect of administering HCLplga-Ab loaded with colistin and andrographolide (CST / Ad@HCLplga-Ab) prepared in Example 11

[0103] 1. Animal modeling: Klebsiella pneumoniae (kp2648,10 8 CFU / Ml, 50ul) intratracheal instillation;

[0104] 2. 12 hours after modeling, CST / Ad@HCLplga-Ab loaded with colistin andrographolide was inhaled by nebulization.

[0105] 3. After 48 hours, the inflammatory factors, total protein content, total cell content in the alveolar lavage fluid of the mice, the inflammatory factors in the plasma, and the bacteria in the lung homogenate were quantified.

[0106] 4. Analyze the survival of mice 168 hours after modeling.

[0107] The results are described below:

[0108] It is well known that when colistin (CST) is injected intravenously, its serum concentration in the lungs is insufficient to achieve a therapeutic effect in a clinical setting. In order to verify the wide applicability of the aerosol, this example applies the integration of the antibiotic CST into Ad@HCLplga-Ab to prepare CST / Ad@HCLplga-Ab aerosol particles. Based on the standard curve of CST dilution, the loading efficiency of CST is about 25.36%. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a major public health threat and has become the second most common bacterium isolated in clinical practice in China. Therefore, this example uses Kp2648 (10 8 CFU mL -1 , 50 μL) of CRKP strain and infected mice after receiving PBS, CST (2.5 mg kg -1 )、CST / Ad@HCLplga-Ab(10mg kg -1 ) After 48 hours of treatment, the therapeutic effect was evaluated.

[0109] In order to make the aerosol particles simulate clinical applications as much as possible, the cross-sectional penetration velocity and diffusion characteristics of Ad@HCLplga-Ab and Ad@HCLplga aerosol particles in airway mucus were first studied. Representative images showed that CST / Ad@HCLplga-Ab was able to penetrate airway mucus more actively at 0.5 hours and 2.0 hours compared with CST / Ad@HCLplga. In addition, examination of whole lung sections showed that Ad@HCLplga-Ab aerosol particles were evenly distributed in the deep lung due to its ability to penetrate the mucus barrier.

[0110] Eliminating pathogens and maintaining a dynamic balance of immune responses are two major goals in managing infectious diseases. CFU (colony forming unit) results showed that the residual Kp2648 load in the lungs of mice treated with CST / Ad@HCLplga-Ab aerosol was significantly reduced compared with the CST alone or PBS control groups, indicating that bacterial replication was effectively inhibited in the lung tissue. After CST / Ad@HCLplga-Ab treatment, the lung wet / dry ratio and protein levels in BALF of mice decreased, consistent with the reduction in the severity of pulmonary edema. In addition, the concentrations of proinflammatory cytokines, including IL-1β, IL-6, and TNF-α, in BALF and serum showed a downward trend compared with the PBS group or the CST alone group. In addition, mice treated with normal saline showed obvious systemic infection, as manifested by increased white blood cell (WBC) counts, while after Ad@HCLplga-Ab treatment, WBC levels were similar to those of healthy mice.

[0111] Immunohistochemical (IHC) staining of lung tissue sections showed that the untreated control group had inflammation in the lung lobes, as evidenced by a large number of neutrophil infiltrations and strong expression of IL-1β receptors. However, the IHC results of the lungs after CST / Ad@HCLplga-Ab treatment showed significant improvement compared with the control group treated with CST or PBS alone. To further investigate the relevant anti-inflammatory pathways, immunofluorescence staining (IF staining) of lung tissues was performed. Kp2648 infection resulted in increased fluorescence intensity of p-NF-κB p65, NLRP3, and Toll-like receptor 4 (TLR4) in lung tissue sections; however, CST / Ad@HCLplga-Ab downregulated the expression of these proteins, almost restoring them to normal levels.

[0112] The high efficiency of CST / Ad@HCLplga-Ab is also reflected in the higher survival rate (70%), compared with 40% in the PBS group and 50% in the CST alone group. These results provide evidence for the signaling pathway hypothesis proposed based on in vitro experiments. Overall, these findings support that inhalation of CST / Ad@HCLplga-Ab aerosol can effectively respond to the thick mucus layer on the airway surface, severe lung infection, and intense inflammatory response caused by CRKP infection.

Claims

1. A method for preparing a honeycomb PLGA microsphere coated with chitosan and ambroxol, comprising the following steps: (1) mixing a 15 mg / mL PLGA dichloromethane solution with a 1% by weight polyvinyl alcohol (PVA) aqueous solution containing 1-10% by weight NaHCO3, and emulsifying in an ice bath for 2 minutes to form a primary emulsion; The volume ratio of the PLGA dichloromethane solution to the PVA aqueous solution is 1-3:1; the emulsification is performed by a cell disruptor at 25-50W for 1-5 minutes; (2) then adding the primary emulsion to a 1% by weight PVA aqueous solution; homogenizing at 5000 rpm in an ice bath for 5 minutes to prepare an emulsion; wherein the volume ratio of the primary emulsion to the PVA aqueous solution is 1:1-3; (3) the prepared emulsion was magnetically stirred at room temperature overnight; the solidified PLGA microspheres HCLplga were collected by centrifugation, washed with deionized water, and then resuspended in deionized water, and freeze-dried to obtain HCLplga dry powder; (4) adding ambroxol to the chitosan acetic acid solution, the final concentration of ambroxol being 25 mg / mL; mixing the solution with HCLplga or HCLplga loaded with drugs, and stirring continuously for 4 hours at room temperature; collecting the microspheres HCLplga-Ab coated with ambroxol chitosan by centrifugation at 3000 rpm, and then washing and freeze-drying; The chitosan acetic acid solution is prepared by dissolving chitosan in a 2% by weight acetic acid solution, wherein the weight percentage content of chitosan is 0.25%.

2. The method for preparing the cellular PLGA microspheres of chitosan ambroxol coating according to claim 1, wherein in step (1), the weight ratio of NaHCO3 in the PVA aqueous solution is 3-5%; wherein the emulsification is carried out by a cell disruptor at 30-35W for 2-3 minutes.

3. The method for preparing honeycomb PLGA microspheres coated with chitosan and ambroxol according to claim 1, wherein the loaded drug described in step (4) is one or more of andrographolide, colistin or amphotericin B.

4. The method for preparing the honeycomb PLGA microspheres of chitosan ambroxol coating according to claim 3, wherein the method for loading the drug is as follows: In the above step (1), andrographolide is added to a 1% PVA aqueous solution containing 1-10% NaHCO3, and then prepared according to the preparation method of HCLplga microspheres coated with chitosan and ambroxol; wherein the concentration of andrographolide in the PVA aqueous solution is 6 mg / mL; or, At room temperature, on the basis of the HCLplga microspheres prepared in step (3), 10 mg / mL colistin or 10 mg / mL amphotericin B was added to the HCLplga solution under magnetic stirring for 6 hours, and the solidified HCLplga was collected by centrifugation, washed three times with deionized water, and then resuspended in deionized water, and finally freeze-dried to obtain HCLplga loaded with colistin or amphotericin B.

5. Honeycomb PLGA microspheres coated with chitosan and ambroxol prepared by the preparation method according to any one of claims 1 to 4.

6. A honeycomb PLGA microsphere with chitosan ambroxol coating loaded with andrographolide, characterized in that It is prepared by the following method: (1) adding andrographolide to a 1% PVA solution, wherein the concentration of andrographolide is 6 mg / ml; mixing a 15 mg / mL PLGA dichloromethane solution with an andrographolide polyvinyl alcohol PVA aqueous solution containing 1-10% NaHCO3, and emulsifying in an ice bath for 2 minutes to form a primary emulsion; The volume ratio of the PLGA dichloromethane solution to the PVA aqueous solution is 1-3:1; the emulsification is performed by a cell disruptor at 25-50W for 1-5 minutes; (2) then adding the primary emulsion to a 1% by weight PVA aqueous solution; homogenizing at 5000 rpm in an ice bath for 5 minutes to prepare an emulsion; wherein the volume ratio of the primary emulsion to the PVA aqueous solution is 1:1-3; (3) the prepared emulsion was stirred magnetically at room temperature overnight; the solidified PLGA microspheres HCLplga were collected by centrifugation, washed with deionized water, and then resuspended in deionized water, and freeze-dried to obtain HCLplga dry powder loaded with andrographolide; (4) adding ambroxol to the chitosan acetic acid solution, the final concentration of ambroxol being 25 mg / mL; mixing the solution with HCLplga loaded with andrographolide, and stirring continuously at room temperature for 4 hours; collecting the microspheres HCLplga-Ab coated with ambroxol chitosan by centrifugation at 3000 rpm, and then washing and freeze-drying; The chitosan acetic acid solution is prepared by dissolving chitosan in a 2% by weight acetic acid solution, wherein the weight percentage content of chitosan is 0.25%.

7. A honeycomb PLGA microsphere loaded with chitosan ambroxol coating and andrographolide and colistin, characterized in that It is prepared by the following method: (1) adding andrographolide to a 1% PVA solution, wherein the concentration of andrographolide is 6 mg / ml; mixing a 15 mg / mL PLGA dichloromethane solution with an andrographolide polyvinyl alcohol PVA aqueous solution containing 1-10% NaHCO3, and emulsifying in an ice bath for 2 minutes to form a primary emulsion; The volume ratio of the PLGA dichloromethane solution to the PVA aqueous solution is 1-3:1; the emulsification is performed by a cell disruptor at 25-50W for 1-5 minutes; (2) then adding the primary emulsion to a 1% by weight PVA aqueous solution; homogenizing at 5000 rpm in an ice bath for 5 minutes to prepare an emulsion; wherein the volume ratio of the primary emulsion to the PVA aqueous solution is 1:1-3; (3) the prepared emulsion was stirred magnetically overnight at room temperature; the solidified PLGA microspheres HCLplga were collected by centrifugation, washed with deionized water, and then resuspended in deionized water; Adding colistin to the HCLplga solution loaded with andrographolide under magnetic stirring for 6 hours to obtain HCLplga microspheres loaded with andrographolide and colistin; (4) adding ambroxol to the chitosan acetic acid solution, the final concentration of ambroxol being 25 mg / mL; mixing the solution with HCLplga loaded with andrographolide, and stirring continuously at room temperature for 4 hours; collecting the microspheres HCLplga-Ab coated with ambroxol chitosan by centrifugation at 3000 rpm, and then washing and freeze-drying; The chitosan acetic acid solution is prepared by dissolving chitosan in a 2% by weight acetic acid solution, wherein the weight percentage content of chitosan is 0.25%.

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