MIL-100(Fe)-based Powdered Inhalant of Imperatorin (PA): Preparation Method and Application

Abstract

This invention belongs to the field of dry powder inhaler technology, and specifically relates to a dry powder inhaler based on MIL-100(Fe) and its preparation method and application. This invention uses a hydrofluoric acid-free hydrothermal method to synthesize MIL-100(Fe) as a drug carrier, and then encapsulates Phenylephrine (PA) as a model drug in inhalable-sized MIL-100(Fe) microparticles to prepare a dry powder inhaler. It can be used to treat bronchial asthma. Compared with aerosols and nebulized inhalers, dry powder inhalers have the advantages of being propellant-free, portable, easy to operate, low equipment cost, and good stability.

Description

Technical Field

[0001] This invention belongs to the field of dry powder inhaler technology, and specifically relates to a dry powder inhaler based on MIL-100(Fe) of angelica dahurica (PA), its preparation method and application. Background Technology

[0002] Asthma is the most common chronic inflammatory lung disease, characterized by airway hyperresponsiveness (AHR), eosinophilic infiltration, reversible airflow obstruction, airway remodeling, mucus hypersecretion, and goblet cell hyperplasia. This disease typically manifests as wheezing, coughing, and dyspnea. Inhaled therapy is crucial for the treatment of acute and chronic asthma, as well as chronic obstructive pulmonary disease (COPD). The dried root of *Peucedanum praeruptorum* has long been used in Traditional Chinese Medicine to treat certain lung diseases, such as asthma, chronic bronchitis, and pulmonary hypertension. Studies have shown that *Peucedanum praeruptorum* A (PA) can inhibit airway inflammation and airway remodeling in a mouse model of allergic asthma, and inhibit the production of transforming growth factor-β-1 and airway remodeling. However, pharmacokinetic studies of PA in rats show that PA has a short half-life in rats, and it is difficult to detect PA in the blood after oral administration. This may be related to its absorption or metabolism by certain digestive enzymes in the gastrointestinal tract, or it may be related to its metabolism by hepatic enzymes shortly after absorption, resulting in low oral bioavailability. Therefore, it is necessary to choose alternative delivery systems.

[0003] Lung drug delivery has become a research hotspot for treating local lung diseases, maximizing drug concentration in the lungs and representing the most effective drug delivery method for diseases such as COPD, bronchial asthma, lung cancer, and pulmonary interstitial fibrosis. However, the clearance function of pulmonary phagocytes and mucociliary cells against foreign bodies exposed in the airways is the biggest obstacle to pulmonary drug delivery. Therefore, to achieve effective nebulization, maximize lung deposition, and minimize macrophage clearance, complex processing and modification of particles are usually required. Metal-organic frameworks (MOFs) have attracted increasing attention in the biomedical field. Due to the presence of organic linkers, Fe-MOFs (MIL-100) can interact with other trivalent metal units such as Fe, Sc, Al, and V. The most widely studied MIL is mesoporous iron(III) carboxylate MIL-100(Fe), with the molecular formula Fe3O(H2O)2OH(BTC)2. MIL-100(Fe) is used as a drug carrier for bioimaging, biosensing, antibacterial applications, and drug release due to its hydrothermal stability, high specific surface area, and high porosity.

[0004] Therefore, how to select a suitable carrier to load PA with drugs in order to solve the problems of PA's first-pass effect and short half-life in vivo is a key issue. Summary of the Invention

[0005] To address the above problems, this invention proposes a dry powder inhaler based on MIL-100(Fe) of angelica dahurica (PA).

[0006] The dry powder inhaler uses MIL-100(Fe) as a carrier and PA as the drug component.

[0007] Furthermore, the powder particle size of the dry powder inhaler and the crystal particle size of MIL-100(Fe) are both 1-5 μm.

[0008] Secondly, the present invention provides a method for preparing the MIL-100(Fe)-based angelica dahurica (PA) dry powder inhaler, the preparation method comprising the following steps:

[0009] MIL-100(Fe) powder was synthesized by feeding FeSO4·7H2O and H3BTC at a molar ratio of 0.64:0.33.

[0010] The drug PA was loaded onto the MIL-100(Fe) powder as a carrier to obtain MIL-100(Fe)-PA powder.

[0011] Furthermore, the synthesis of MIL-100(Fe) powder specifically includes the following steps:

[0012] Add 0.64 mmol FeSO4·7H2O, 0.33 mmol H3BTC and 10 mL H2O to the reaction vessel, stir evenly and sonicate until the reaction is complete, then cool to room temperature;

[0013] After cooling, the product was filtered and washed with water and ethanol more than three times. The product was then soaked in water at 80°C for 6 hours and in ethanol at 60°C for 6 hours. After filtration, the product was activated in a vacuum oven at 120°C for 3 hours to obtain MIL-100(Fe) powder.

[0014] Further, the PA is loaded with drug using the MIL-100(Fe) powder as a carrier to obtain MIL-100(Fe)-PA powder, specifically including the following steps:

[0015] MIL-100(Fe) powder and PA are mixed in a fixed mass ratio and placed in a reactor;

[0016] The reactor was connected to a condenser in a magnetically stirred heater for drug loading. After drug loading was completed, the crystals were collected by centrifugation.

[0017] The crystals were washed three times with ethanol and dried under vacuum at 60°C for 6 hours to obtain MIL-100(Fe)-PA powder.

[0018] Furthermore, the mass ratio of MIL-100(Fe) powder to PA is 1:9.

[0019] Furthermore, the drug loading temperature was 35℃, and the drug loading time was 12h.

[0020] Thirdly, the present invention also proposes a method for evaluating the aforementioned MIL-100(Fe)-based angelica dahurica (PA) dry powder inhaler.

[0021] The method described above evaluates the crystal structure, crystal morphology, powder properties, in vitro lung deposition, in vitro release, and cell activity of the MIL-100(Fe) angelica dahurica (PA) dry powder inhaler.

[0022] The present invention relates to the application of MIL-100(Fe)-based angelica dahurica (PA) dry powder inhaler in the preparation of drugs for treating lung diseases.

[0023] Furthermore, the lung diseases include COPD, bronchial asthma, lung cancer, and pulmonary interstitial fibrosis.

[0024] The beneficial effects of this invention are:

[0025] This invention uses hydrofluoric acid-free hydrothermal synthesis of MIL-100(Fe) as a drug carrier, and for the first time uses angelica dahurica extract (PA) as a model drug, which is embedded in 1-5μm MIL-100(Fe) microparticles to prepare a dry powder inhaler for the treatment of lung diseases. Compared with aerosols and nebulized inhalers, dry powder inhalers have the advantages of being propellant-free, easy to carry, simple to operate, low equipment cost, and good stability.

[0026] This invention provides a new approach to the research and development of pulmonary drug delivery systems by evaluating the absorption of MIL-100(Fe)-PA in the lungs through quality assessment and in vitro evaluation, in order to avoid the first-pass effect of oral administration of angelica dahurica extract and prolong the duration of drug action.

[0027] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The X-ray diffraction patterns of simulated MIL-100(Fe), MIL-100(Fe)-Ⅰ, MIL-100(Fe)-Ⅱ and MIL-100(Fe)-Ⅲ in Embodiment 1 of the present invention are shown.

[0030] Figure 2 SEM images of MIL-100(Fe)-Ⅰ, MIL-100(Fe)-Ⅱ and MIL-100(Fe)-Ⅲ in Embodiment 1 of the present invention are shown;

[0031] Figure 3 The PA standard curve established in Embodiment 2 of the present invention is shown;

[0032] Figure 4 The following graph shows the effect of time on drug loading (n=3) in Embodiment 2 of the present invention;

[0033] Figure 5 The following graph shows the effect of temperature on drug loading (n=3) in Example 2 of the present invention;

[0034] Figure 6 The following graph shows the effect of drug mass ratio on drug loading (n=3) in Example 2 of the present invention;

[0035] Figure 7 The XPRD spectra of simulated MIL-100(Fe), MIL-100(Fe), and MIL-100(Fe)-PA in Example 3 of the present invention are shown.

[0036] Figure 8 The SEM images of MIL-100(Fe) and MIL-100(Fe)-PA in Embodiment 3 of the present invention are shown;

[0037] Figure 9 The DSC diagrams of MIL-100(Fe), MIL-100(Fe)-PA, PA and physical mixtures in Example 3 of the present invention are shown.

[0038] Figure 10 The TGA diagrams of MIL-100(Fe) and MIL-100(Fe)-PA in Embodiment 3 of the present invention are shown;

[0039] Figure 11 The infrared spectra of MIL-100(Fe), MIL-100(Fe)-PA, PA and MIL-100(Fe) physical mixture and PA in Example 3 of the present invention are shown.

[0040] Figure 12 The particle size and distribution diagram of the MIL-100(Fe)-PA dry powder inhaler in Example 4 of the present invention are shown.

[0041] Figure 13 The following graph shows the NGI deposition percentage results for each site in Example 5 of the present invention;

[0042] Figure 14 The following graph shows the release rate of MIL-100(Fe)-PA in simulated lung fluid in Example 6 of the present invention;

[0043] Figure 15 This image shows a rat lung deposition map of MIL-100(Fe)-RhoB after lung administration using an in vivo optical imaging system, as described in Example 7 of the present invention.

[0044] Figure 16A The figure shows the effect of PA, MIL-100(Fe) and MIL-100(Fe)-PA on the survival rate of 16HBE cells in Example 8 of the present invention;

[0045] Figure 16B The figure shows the effect of PA, MIL-100(Fe) and MIL-100(Fe)-PA on the survival rate of Caco-2 cells in Example 8 of the present invention;

[0046] Figure 17A A microscope image of the 16HBE cell model in Example 9 of the present invention is shown;

[0047] Figure 17B A microscope image of the Caco-2 cell model in Example 9 of the present invention is shown;

[0048] Figure 18A A line graph showing the transmembrane resistance of the 16HBE cell layer in Embodiment 9 of the present invention is shown;

[0049] Figure 18B The TEER values ​​of Caco-2 cells measured at different times in the transwell chamber in Example 9 of this invention are shown.

[0050] Figure 19A The figure shows the apparent permeation results of different concentrations of PA and MIL-100(Fe)-PA in the 16HBE cell model in Example 9 of the present invention;

[0051] Figure 19B The diagram shows the apparent permeation results of different concentrations of PA and MIL-100(Fe)-PA in the Caco-2 cell model in Example 9 of the present invention. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Example 1: Selection of the preparation process for MIL-100(Fe)

[0054] 1.1 Preparation of MIL-100(Fe) by magnetic stirring method

[0055] H3BTC (1.67 g) was dissolved in 23.72 g of 1 mol / L NaOH solution to obtain solution 1. FeCl2·4H2O (2.26 g) was dissolved in 97.21 g of H2O water to obtain solution 2. Solution 1 was added dropwise to solution 2, and the mixture was stirred magnetically at room temperature (25 °C) for 24 h. After the reaction was completed, the product was filtered, washed with pure water until neutral, washed three times with ethanol, and then soaked in 80 °C water for 6 h and 60 °C ethanol for 6 h. The filtered product MIL-100(Fe)-Ⅰ was activated in a vacuum oven at 120 °C for 3 h to obtain MIL-100(Fe)-Ⅰ powder (i.e., magnetically stirred MIL-100(Fe)).

[0056] 1.2 Preparation of MIL-100(Fe) by Hydrothermal Synthesis

[0057] FeSO4·7H2O (0.56 g, 2 mmol), H3BTC (0.28 g, 1.34 mmol), and 10 ml of H2O were placed in a reaction vessel, stirred until homogeneous, sonicated for 20 min, and reacted at 150℃ for 24 h. After natural cooling to room temperature, the product was filtered and washed with water and ethanol at least three times each. The product was then soaked in 80℃ water for 6 h and in 60℃ ethanol for 6 h. The filtered product MIL-100(Fe)-II was obtained and activated in a vacuum oven at 120℃ for 3 h to obtain MIL-100(Fe)-II powder (i.e., hydrothermal reaction MIL-100(Fe)).

[0058] 1.3 Modified hydrothermal synthesis method for preparing MIL-100(Fe) with adjusted particle size

[0059] To meet the particle size requirements of dry powder inhalers, the particle size of MIL-100(Fe) crystals is controlled by adjusting the ratio of organic ligands to metal centers. The specific adjustments are as follows:

[0060] In step 1.2, FeSO4·7H2O was adjusted to 0.18 g and 0.64 mmol, and H3BTC was adjusted to 0.07 g and 0.33 mmol, while keeping other conditions unchanged, to prepare MIL-100(Fe)-Ⅲ powder (i.e., particle size-adjusted MIL-100(Fe)).

[0061] 1.4 Morphological Characterization

[0062] The morphology and particle size range of simulated MIL-100(Fe), MIL-100(Fe)-Ⅰ, MIL-100(Fe)-Ⅱ, and MIL-100(Fe)-Ⅲ powders were observed by X-ray diffraction (XRD) and scanning electron microscopy (SEM) to screen for the optimal MOF. The XRD and SEM images of the four MIL-100(Fe) powders are shown below. Figure 1 and Figure 2 As shown.

[0063] Figure 1 The XRD patterns from top to bottom are the simulated MIL-100(Fe), MIL-100(Fe)-Ⅰ, MIL-100(Fe)-Ⅱ, and MIL-100(Fe)-Ⅲ powders. It can be seen that the peak positions of the X-ray diffraction patterns of MIL-100(Fe)-Ⅰ, MIL-100(Fe)-Ⅱ, and MIL-100(Fe)-Ⅲ powders are consistent with the peak positions of the simulated MIL-100(Fe).

[0064] Figure 2 In the image, A, B, and C are scanning electron microscope images of MIL-100(Fe)-Ⅰ, MIL-100(Fe)-Ⅱ, and MIL-100(Fe)-Ⅲ, respectively. It can be seen that the morphology of MIL-100(Fe)-Ⅰ crystals is irregular and aggregated, indicating that MIL-100(Fe)-Ⅰ prepared by magnetic stirring has low crystallinity and small particle size. MIL-100(Fe)-Ⅱ has a cubic structure with different crystal sizes and more clusters, which may be related to the metal-to-ligand ratio and reaction time. Compared with MIL-100(Fe)-Ⅱ, MIL-100(Fe)-Ⅲ has a cubic crystal structure, fewer clusters, and larger particles, meeting the 1-5 μm requirement.

[0065] Based on the above results, the optimal MIL-100(Fe)-Ⅲ was selected to encapsulate PA. According to the literature, there are two types of mesoporous cages in the MIL-100(Fe) structure, with sizes of 2.5 and 2.9 nm and windows of 0.55 and 0.86 nm, respectively. Since the molecular size of PA is approximately 0.9 nm, it is believed that PA can pass through the mesoporous cages and remain in the cavity.

[0066] It should be noted that in the following examples, MIL-100(Fe) refers to the prepared MIL-100(Fe)-Ⅲ powder.

[0067] Example 2: Preparation of MIL-100(Fe)-PA dry powder inhaler

[0068] 2.1 Screening reaction conditions based on the amount of drug loaded (PA) under different conditions. The determination of drug loading includes the following steps:

[0069] (1) Establish PA standard curve

[0070] Using Agilent HC-C 18 (2) A chromatographic column (150×4.6mm, 5μm) was used with methanol-water (75:25, v / v) as the mobile phase, a flow rate of 1.0 mL / min, a detection wavelength of 321 nm, a column temperature of 30℃, and an injection volume of 10 μL. PA stock solution was taken and repeatedly diluted 4-fold with the mobile phase to obtain PA series solutions with concentrations of 63, 15.75, 3.937, 0.984, 0.246, 0.061, and 0.015. These solutions were filtered through a 0.22 μm organic filter membrane. 10 μL of each of the above solutions was precisely injected into the liquid chromatograph, and the peak area was measured. A standard curve was plotted with PA concentration as the abscissa (x) and peak area as the ordinate (y). Figure 3 As shown, the curve equation is: y = 0.3916x + 0.0152, r = 1. The results indicate that the sample concentration is within the range of 0.06–63 μg·mL. -1 The linear relationship between them is good.

[0071] (2) Determine drug loading based on PA standard curve

[0072] Take approximately 2 mg of drug-loaded MIL-100(Fe)-PA powder into a 2 mL EP tube, add 200 μL of a mixed solution of 1 mol / L HCl and 500 μL of dimethyl sulfoxide, sonicate for 30 min to dissolve, centrifuge (6000 rpm, 10 min) to obtain a clear solution, take 200 μL of the supernatant, dilute to 10 mL with anhydrous ethanol, shake well, and filter through a 0.22 μm microporous membrane. Measure the peak area A of each sample at 321 nm using HPLC, substitute into the standard curve equation, and repeat the measurement three times for each sample, calculating the average value. The formula for calculating the drug loading is as follows:

[0073]

[0074] 2.2 Screening of drug loading conditions

[0075] 2.2.1 Investigation of drug loading time

[0076] Weigh 20 mg of MIL-100(Fe) powder into a round-bottom flask, add 20 mL of ethanol solution, add PA with a mass ratio of MIL-100(Fe) powder to drug of 1:9, connect a condenser to a magnetic stirrer heater at 35 °C and 200 rpm for drug loading, and stop drug loading at 4, 6, 8, 12, 16 and 24 h respectively. Centrifuge to collect crystals (6000 rpm, 10 min), wash three times with ethanol and dry under vacuum at 60 °C for 4 h.

[0077] Drug loading results as follows Figure 4 As shown, the drug loading increased with the extension of drug loading time. The drug loading increased significantly and gradually in the first 12 hours. After 12 hours, the drug loading of MIL-100(Fe) decreased, indicating that most PA molecules can enter MIL-100(Fe) within 12 hours.

[0078] 2.2.2 Investigation of drug loading temperature

[0079] Weigh 20 mg of MIL-100(Fe) powder and place 4 portions in round-bottom flasks. Add ethanol and PA according to the drug loading time investigation conditions. Select the optimal drug loading time point and load the drug at 200 rpm in a magnetic stirrer at 35℃, 45℃, 55℃ and 65℃ respectively. After the drug loading is completed, centrifuge at 6000 rpm for 10 min to collect the precipitate. Wash with ethanol 3 times and then vacuum dry at 60℃ for 6 h.

[0080] The results of the investigation on drug loading temperature are as follows: Figure 5 As shown, the drug loading increases significantly when the temperature rises to 35℃. However, the drug loading gradually decreases with increasing temperature, indicating that excessively high temperatures are not conducive to PA loading onto MIL-100(Fe), possibly due to the solvent's boiling point.

[0081] 2.2.3 Evaluation of drug dosage quality

[0082] Weigh out 6 portions of 20 mg MIL-100 (Fe) powder and place them in a round-bottom flask. Add 20 mL of ethanol solution and add the corresponding mass of PA according to the drug loading ratio of 1:3, 1:5, 1:7, 1:9, 1:11, and 1:13. Select the optimal drug loading time and optimal drug loading temperature and carry out drug loading under the same conditions as the above investigation. After drug loading, centrifuge to collect the precipitate (6000 rpm, 10 min), wash with ethanol 3 times, and then vacuum dry at 60℃ for 6 h.

[0083] Results of the drug administration quality ratio study: Figure 6 As shown, the dosage mass ratio (m) can be seen. Mil-100(Fe) / m PA When the molar ratio of Fe to MIL-100 is 1:9, the maximum drug loading is achieved. After the drug loading reaches saturation, increasing the molar ratio actually reduces the drug loading.

[0084] In summary, based on the drug loading results of various samples under different drug loading conditions, it can be seen that the drug loading temperature at 35℃ and the m MIL-100(Fe) / m PA The optimal drug loading conditions for MIL-100(Fe) were 1:9 and a reaction time of 12 h, with a maximum drug loading of 9.01 ± 0.16%. Drug loading was carried out under the above optimal conditions to obtain MIL-100(Fe)-PA.

[0085] Example 3 Characterization of MIL-100(Fe) and MIL-100(Fe)-PA

[0086] 3.1 Powder X-ray Diffraction (PXRD)

[0087] The crystal structures of MIL-100(Fe), MIL-100(Fe)-PA, and simulated MIL-100(Fe) were analyzed using PXRD to examine whether the crystallinity of the drug-loaded powder was compromised. CuKα radiation was used. The sample was scanned with the 2θ angle set in the range of 3-50°, and the tube voltage was 40kV and the tube current was 40mA.

[0088] The result is as follows Figure 7 As shown in the figure, from top to bottom, are the PXRD results of MIL-100(Fe), MIL-100(Fe)-PA, and simulated MIL-100(Fe). It can be seen that the characteristic diffraction peaks of MIL-100(Fe)-PA and MIL-100(Fe) are not significantly different. In the MIL-100(Fe)-PA structure, the reflection intensity of MIL-100(Fe) decreases slightly after PA encapsulation, mostly within the range of 3-10°2θ, with the relative position shifting only slightly towards a lower 2θ position. This confirms the stability of the MIL-100(Fe) matrix during drug incorporation. This result indicates that PA incorporation does not affect the crystal structure of MIL-100(Fe) and may even incorporate PA into the MIL-100(Fe) structure.

[0089] 3.2 Scanning Electron Microscope

[0090] MIL-100(Fe) and MIL-100(Fe)-PA were morphologically characterized using SEM to observe the surface morphology of the powder particles before and after drug loading. Before imaging, the samples were fixed on a metal plate with conductive tape and then gold-plated.

[0091] The result is as follows Figure 8As shown in the figure, A is MIL-100(Fe) and B is MIL-100(Fe)-PA. It can be seen that there is no significant difference in the crystal shape and particle size of MIL-100(Fe) before and after drug loading. Both have the same crystal structure and the particle size is about 1-5μm. This confirms that the loading of PA did not destroy the structure of MIL-100(Fe).

[0092] 3.3 Differential Scanning Calorimetry (DSC)

[0093] The thermal behavior of PA before and after loading was studied using DSC. Samples (5–15 mg) were weighed in aluminum crucibles at a temperature range of 25–200 °C at a heating rate of 10 °C / min. -1 The constant nitrogen purging rate was 30 mL / min. -1 The temperature and enthalpy were calibrated by measuring the melting temperature and enthalpy of indium (T). m =156.6℃, ΔH m =28.5 J·g -1 ).

[0094] The results are as follows Figure 9 As shown in the figure, from top to bottom, are the DSC curves of MIL-100(Fe)-PA, MIL-100(Fe)-PA(PM) (i.e., a physical mixture of MIL-100(Fe) and PA), and MIL-100(Fe) and PA. It can be seen that PA has an endothermic peak at 137℃, but the endothermic peak disappears on the MIL-100(Fe)-PA DSC curve, indicating that PA may have entered the MIL-100(Fe) framework. In the physical mixture of PA and MIL-100(Fe), the same endothermic peak is clearly observed at 137℃, which may be due to the melting of PA. The endothermic peak of the support shifts, indicating that there may be a weak interaction between the two substances after physical mixing, and no new substances are generated.

[0095] 3.4 Thermogravimetric Analysis (TGA)

[0096] The thermal stability of MIL-100(Fe) and MIL-100(Fe)-PA was studied using a TG 209F1 Libra thermogravimetric analyzer manufactured by Netzsch GmbH, Germany, in a nitrogen atmosphere with a heating rate of 10℃ / min and a heating range of 30℃ to 680℃.

[0097] The result is as follows Figure 10As shown, the first weight loss below 100℃ corresponds to the departure of free water molecules, and the weight loss between 100℃ and 420℃ may be due to the decomposition of water molecules and ligands bound in the framework. The MIL-100(Fe) framework collapses and decomposes between 400℃ and 605℃, indicating that the synthesized MIL-100(Fe) has good thermal stability. At 600℃, the weight loss rate of MIL-100(Fe)-PA is lower than that of MIL-100(Fe), indicating that PA is successfully encapsulated within MIL-100(Fe).

[0098] 3.5 Fourier Transform Infrared Spectroscopy (FTIR)

[0099] The sample to be tested was ground and mixed with potassium bromide at a ratio of 1:10, then compressed into tablets. FTIR was used to determine the wavelengths of each sample in the range of 400–4000 cm⁻¹. -1 Infrared absorption spectrum within the wavelength range.

[0100] The result is as follows Figure 11 As shown in the figure, from top to bottom, are the FTIR spectra of physical mixtures of MIL-100(Fe)-PA, MIL-100(Fe), and MIL-100(Fe)-PA(PM), and PA. The FTIR spectrum of MIL-100(Fe)-PA shows that MIL-100(Fe) has peak values ​​at 1617, 1571, 1448, and 1382 cm⁻¹. -1 The nearby vibrational bands confirmed the stability of MIL-100(Fe) during loading, indicating no chemical reaction with PA molecules. PA exhibited vibrations at 3079, 1735, 1602, 1232, and 1006 cm⁻¹. -1 Peaks were observed. PA was observed at 1602, 1232, and 854 cm⁻¹ in the MIL-100 (Fe) spectrum. -1 The absorption peaks disappear at 1735 and 1006 cm⁻¹, and then reappear at 1735 and 1006 cm⁻¹. -1 The absorption peak at that location was significantly weakened. In summary, this confirms the loading of PA. Furthermore, for MIL-100(Fe)-PA, the vibrational bands of νAs(C=O) and νAs(CO) are 1735 and 1006 cm⁻¹, respectively. -1 ) and free PA (1710 and 1058 cm⁻¹, respectively) -1 The displacement is slightly different from that of the PA. This displacement reveals the confinement effect of PA in porous solids.

[0101] Example 4: Investigation of the powder properties of MIL-100(Fe)-PA

[0102] 4.1 Particle size and distribution

[0103] The SEM images of MIL-100(Fe)-PA from Example 3 were analyzed and calculated using ImageJ software. The distribution of particles of different sizes was represented by frequency and cumulative distributions, and a particle size distribution map was plotted. The results are as follows: Figure 12 As shown, the particle size of MIL-100(Fe)-PA powder is in the range of 1 to 5 μm, with about 60% of the particles having a particle size between 1.6 and 2.4 μm, which meets the particle size requirements for dry powder inhalers.

[0104] 4.2 Powder Flowability

[0105] Take a 5mL graduated cylinder and record its mass as m0. Fill the graduated cylinder with the MIL-100(Fe)-PA powder sample to the 5mL mark and record the mass of the cylinder and powder as m1, and the powder volume as v0. Tap the graduated cylinder vertically onto the experimental platform from a uniform height (2cm) until the powder maintains a constant volume; record this volume as v1. Perform three parallel measurements and calculate the powder bulk density, tap density, and Carr's index to evaluate powder flowability. Using appropriate equipment, flow the powder through a funnel onto a flat surface and measure the height h and radius r of the cone formed by the powder. Repeat this operation three times, using the ratio of cone height to radius as the tangent to calculate the angle of repose θ. The formulas for calculating powder flowability parameters are as follows:

[0106] Bulk density (ρ0) = (m1 - m0) / v0 × 100%

[0107] Tap density (ρ) f )=(m1-m0) / v1×100%

[0108] Carr's exponent = (ρ f -ρ0) / ρ f ×100%

[0109] tanθ = h / r × 100%

[0110] The calculation results are shown in Table 1:

[0111] Table 1. Powder flowability parameters of MIL-100(Fe)-PA (n=3)

[0112] factor MIL-100(Fe)-PA Angle of repose (°) 38.91±0.82 <![CDATA[Bulk density (g·mL -1 )]]> 0.37±0.01 <![CDATA[Tap density (g·mL -1 )]]> 0.49±0.01 Carr's Index (%) 24.48±2.18

[0113] 4.3 Hygroscopicity Investigation

[0114] The hygroscopicity of MIL-100(Fe)-PA powder was determined according to the method for determining the hygroscopicity of powders in Part IV of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 2.

[0115] Table 2. Hygroscopicity of MIL-100(Fe)-PA powder (n=3)

[0116]

[0117] As can be seen from the table above, after 24 hours, some powder of MIL-100(Fe)-PA showed slight aggregation and agglomeration, with a moisture-induced weight gain of 9.81±0.20%. According to the 2020 edition of the Pharmacopoeia, the moisture-induced weight gain is between 2% and 15%, indicating that the powder is hygroscopic. This may be due to the large porosity and specific surface area of ​​MIL-100(Fe)-PA.

[0118] Example 5: In vitro lung deposition of MIL-100(Fe)-PA

[0119] The in vitro lung deposition rate of dry powder inhalers was determined using a new-generation pharmaceutical impactor (NGI). Approximately 10 mg of MIL-100(Fe)-PA powder was filled into hydroxypropyl methylcellulose capsules, yielding 10 capsules. The NGI instrument was operated according to the prescribed procedure. Prior to operation, the eight collection trays were coated with a 1% w / v solution of methyl silicone oil and n-hexane to capture particles. The airflow rate was adjusted to 60 L / min. -1 Repeat the procedure until all 10 capsules have been inhaled. Based on the European Pharmacopoeia, the airflow rate is 60 L / min. -1 At that time, the cutoff diameters of particles in the collection cups of stages 1–7 were 8.06, 4.46, 2.82, 1.66, 0.94, 0.55, and 0.34 μm, respectively. Particles deposited in the inhalation device, throat (including adapter), pre-separator, collection cups of stages S1–S7, and microporous collector (MOC) were collected and dissolved in a mobile phase of methanol-water (75:25). The PA content in the solution was determined by chromatography.

[0120] The delivery rate (EF) and the fraction of fine particles (FPF) are calculated using the following formulas:

[0121]

[0122]

[0123] Wherein, ED represents the discharge volume, which is the amount of powder stored in the adapter, simulated throat, pre-separator, and all impact stages; TD represents the total dose, which is the amount of powder filled in the capsule; FPD represents the fine particle dose, which is the sum of the powder amounts in the S2 to S7 stage collection cups and MOC section, i.e., powder with an aerodynamic diameter of less than 4.46 μm.

[0124] Plot the logarithm of the cumulative distribution percentage against the logarithm of the cutoff diameter. The particle size at which the cumulative distribution percentage reaches 50% is the mass median aerodynamic diameter (MMAD). The square root of the ratio of the aerodynamic diameters at cumulative distribution percentages of 84.13% and 15.87% is the geometric standard deviation (GSD).

[0125] NGI determination of deposition percentage in various locations is as follows: Figure 13 As shown, less than 10% of the powder remained in the capsule, indicating that the MIL-100(Fe)-PA dry powder formulation is easily atomized and passed through the device.

[0126] The aerodynamic parameters of MIL-100(Fe) dry powder inhaler are shown in Table 3:

[0127] Table 3 Aerodynamic parameters of MIL-100(Fe) dry powder inhaler (n=3)

[0128]

[0129]

[0130] As shown in the table above, the MMAD of MIL-100(Fe)-PA dry powder inhaler is 4.16±0.07μm, and the GSD is 1.52±0.02. GSD describes the shape of the powder particle size distribution curve. Generally, the closer this parameter value is to 1, the narrower the particle size distribution of the dry powder inhaler.

[0131] The above results indicate that MIL-100(Fe)-PA dry powder inhaler basically meets the requirements for pulmonary inhalation administration and can be used as a pulmonary delivery carrier to deliver PA to the lungs.

[0132] Example 6: In vitro release study of MIL-100(Fe)-PA

[0133] Accurately weigh three 10.0 mg portions of MIL-100(Fe)-PA powder and place them in three 50 mL round-bottom flasks. Add 10 mL of freshly prepared simulated lung fluid to each flask, and then add 2% Tween 80 to aid suspension. Place the flasks in a constant temperature water bath at 37°C and 100 rpm. Collect 200 μL of the release medium at 0, 2, 4, 6, 8, 10, 16, and 24 h, respectively. Then, fill the original release medium with an equal volume of fresh simulated lung fluid. Centrifuge the collected release solution (6000 rpm, 10 min), and filter the supernatant through a 0.22 μm organic filter membrane. Determine the PA content in each sample solution according to the method in 2.1 of Example 2.

[0134] Preparation of simulated alveolar fluid: Add the solutes of each component in Table 4 to ultrapure water in sequence, and after dissolution, dilute to a 1L volumetric flask. Add the next solute only after each solute has completely dissolved to avoid salt precipitation. All chemical reagents are analytical grade.

[0135] Table 4. Composition of simulated lung fluid (SLF, pH = 7.4)

[0136]

[0137]

[0138] The release rate of MIL-100(Fe)-PA in simulated lung fluid is as follows: Figure 14 As shown, MIL-100(Fe)-PA powder tends to stabilize in SLF after 24 hours with a release rate of approximately 73%. This may be because PA adsorbed within the pores of the MOF can slowly dissolve in the release medium, potentially exhibiting a sustained-release effect. These properties of PA-loaded MIL-100(Fe) highlight its potential for sustained treatment in lung tissue and PA absorption.

[0139] Example 7: In vivo fluorescence imaging of small animals

[0140] Since MIL-100(Fe) does not change its crystal morphology after drug loading, RhoB was used as a fluorescent agent to synthesize MIL-100(Fe)-RhoB using a magnetic stirring heating method, replacing the distribution of MIL-100(Fe)-PA dry powder inhaler in the lungs. Specifically, the IVIS Spectrum small animal in vivo optical imaging system was used to record the fluorescence imaging of lung tissue, with RhoB excitation wavelength at 550 nm and emission wavelength at 580 nm.

[0141] The results are as follows Figure 15 As shown, a strong signal was observed in the respiratory tract 1 hour after administration, and it slowly diffused into the lungs after 4–16 hours. The results indicate that the MIL-100(Fe)-Rho-B dry powder inhaler was successfully retained in the lungs, and the fluorescence intensity in lung tissue remained stable over 24 hours during the MIL-100(Fe)-mediated RhoB retention. Overall, the results demonstrate that MIL-100(Fe) particles carrying RhoB can be effectively delivered to the lungs, rather than being cleared directly from the respiratory tract.

[0142] Example 8 Cell viability experiment

[0143] Culture of 16HBE cells

[0144] Culture flasks containing 16HBE cells were placed in a cell culture incubator at 37°C and 5% CO2. After 24 hours, cell growth was observed under an inverted microscope, and the medium was replaced with DMEM containing 10% fetal bovine serum. Subsequently, the medium was changed according to cell growth. When the cells reached 80-90% confluence in the culture flasks, they were digested with 0.25% trypsin for 2-3 minutes, centrifuged to collect the cells, and passaged. After passage, the cells were continued to be cultured in DMEM medium containing 10% fetal bovine serum for approximately three passages before being used in experiments.

[0145] Caco-2 cell culture

[0146] Human colon cancer Caco-2 cells from cryopreservation tubes were revived and transferred to culture flasks containing DMEM medium with 20% fetal bovine serum. The flasks containing Caco-2 cells were placed in a cell culture incubator at 37°C and 5% CO2. After 24 hours, cell growth was observed under an inverted microscope, and the medium was replaced with DMEM medium containing 10% fetal bovine serum. Subsequently, the medium was changed according to cell growth. When the cells reached 80-90% confluence in the culture flasks, they were digested with 0.25% trypsin for 3-5 minutes, centrifuged to collect the cells, and passaged. After passage, the cells were continued to be cultured in DMEM medium containing 10% fetal bovine serum.

[0147] The cells used in this invention were purchased from Shanghai Zishi Biotechnology Co., Ltd.

[0148] Cell viability assay

[0149] 16HBE cells and Caco-2 cells in the logarithmic growth phase were counted using a cell counting chamber, and then analyzed at a concentration of 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates, with 100 μL of sterile PBS added to the periphery of each well. Three experimental groups were set up: a zeroing group, a control group, and an experimental group. No cells were seeded in the zeroing group. Cells were cultured at 37°C and 5% CO2 in DMEM medium containing 10% fetal bovine serum. When cell confluence reached approximately 70%, the medium was replaced with fresh serum-free DMEM for maintenance. After 24 hours of medium replacement, 100 μL of different concentrations of PA, MIL-100(Fe), and MIL-100(Fe)-PA solutions were added to the experimental groups, while serum-free DMEM was added to the control group. After 24 hours of incubation, 10 μL of CCK-8 solution was added to each well except for the peripheral wells, and incubation continued for another 8 hours. After termination of culture, the OD value at 450 nm was measured using a microplate reader. The cytotoxicity of MIL-100(Fe) was assessed by calculating the cell viability of each group.

[0150] Cell viability (%) = [(OD) 实验组 -OD 调零组) / (OD 对照组 -OD 调零组 )]×100%.

[0151] Preparation of cell drug delivery solution: Dissolve 8.2 mg of PA in 50 μL LDMSO, then bring the volume to 8 mL with DMEM medium to prepare a solution with a concentration of 102.5 μg / mL. -1 The stock solution (3200 μM) was filtered through a 0.22 μm sterile filter membrane, diluted 10-fold with culture medium, and then gradually diluted 2-fold to achieve a PA concentration range of 0.104–53.75 μg·mL. -1 MIL-100(Fe) and MIL-100(Fe)-PA powder were suspended in DMEM medium at a concentration equivalent to the PA concentration. The solution was prepared in a clean bench.

[0152] The effects on the survival rates of 16HBE cells and Caco-2 cells are as follows: Figure 16A and 16B As shown, the results indicate that PA, MIL-100(Fe), and MIL-100(Fe)-PA had no significant inhibitory effect on the growth of 16HBE and Caco-2 cells within the PA concentration range of 0.104–26.875 μg / mL, and the cell survival rate reached over 80%. This indicates that MIL-100(Fe) has good biocompatibility and can be used as a novel dry powder inhaler carrier to safely deliver PA into the lungs.

[0153] Example 9: Permeability Study of MIL-100(Fe)-PA Dry Powder Inhaler in 16HBE and Caco-2 Cell Models

[0154] Establishment of the 16HBE cell model

[0155] Mycoplasma-free 16HBE cells were used at 2.5 × 10⁻⁶. 5 cells / cm 2 The density in a 12-pore polycarbonate membrane Transwell chamber (0.4 μm pore size, 1.12 cm⁻¹) 2 Seeds were placed on the transwell chamber (specific surface area), and 0.5 mL of cell suspension was added. 1.5 mL of DMEM medium containing 10% fetal bovine serum was added to the transwell chamber, and the cells were incubated at 37°C in a 5% CO2 incubator. After 2 days of culture, the culture medium from the apical chamber and the transwell chamber was aspirated, and another 1.5 mL of medium containing 10% fetal bovine serum was added to the transwell chamber to maintain the cells at the gas-liquid interface. The culture medium in the transwell chamber was changed every 24 hours (1.5 mL), and the cells were cultured at the gas-liquid interface for 6 days. When changing the culture medium, care must be taken to ensure that there are no air bubbles between the culture medium and the Transwell chamber. The cell morphology is as follows: Figure 17AAs shown, the cells form a dense cell layer on the polycarbonate membrane, with tight junctions between the cells.

[0156] Measurement of transmembrane resistance of 16HBE cells

[0157] The number of culture days required for cell layers to reach confluence was determined by measuring transmembrane resistance (TEER), and the growth of confluent cell layers with tight junctions was monitored. TEER of epithelial cell layers was measured using an EVOM transmembrane resistance meter and an STX2 electrode. Before measurement, 0.5 mL and 1.5 mL of fresh culture medium preheated to 37°C were added to the top chamber and the basal outer chamber, respectively, and equilibrated at 37°C for 30 min before measurement. The TEER of each cell layer was subtracted from the resistance of a blank permeable membrane to obtain the TEER of the epithelial cell layers (n=3).

[0158] The result is as follows Figure 18A As shown, TEER reached its maximum value (225±2.08Ω·cm) after 6 days of culture. -2 The cell layer has the best barrier performance.

[0159] Permeability study of PA and MIL-100(Fe)-PA dry powder inhalers in a 16HBE cell model

[0160] Based on the results of cytotoxicity assays, low, medium, and high concentrations were selected for 16HBE cell model transport experiments. The amount of PA and MIL-100(Fe)-PA dry powder inhalers from the apex to the basal side of the cell layer was measured, and Papp was calculated to evaluate their absorption in the lungs. The results are as follows: Figure 19A As shown, the permeability values ​​of PA and MIL-100(Fe)-PA at low, medium, and high concentrations are all greater than 1×10⁻⁶. -6 cm·s -1 This indicates that PA is well absorbed in the lungs and can be used for pulmonary administration. Furthermore, the permeability of PA and MIL-100(Fe)-PA dry powder inhaler increases with increasing concentration, showing a concentration-dependent relationship. After treatment with MIL-100(Fe), the permeability of PA in the 16HBE cell model increased threefold compared to monotherapy, especially at 16 μg / mL. -1 The effect is more pronounced at higher concentrations.

[0161] Establishment of the Caco-2 cell model

[0162] Cells were loaded at 2.5 × 10 5 cells / cm 2 The density in a 12-pore polycarbonate membrane Transwell chamber (0.4 μm pore size, 1.12 cm⁻¹) 2Cells were seeded on substrates with a specific surface area of ​​[missing information]. 0.5 mL of cell suspension was added to the apical side (AP), and 1.5 mL of DMEM medium containing 10% fetal bovine serum was added to the basal side (BL). The cells were incubated at 37°C in a 5% CO2 cell culture incubator. The medium was changed every two days for the first week after seeding, and daily thereafter, for a total of 21 days. The cell morphology was as follows: Figure 17B As shown, after day 21, the cell monolayer structure was clearly intact, and the cells were tightly connected.

[0163] Measurement of transmembrane resistance in Caco-2 cells

[0164] Before conducting the transport experiment, the transepithelial electrical resistance (TEER) of Caco-2 cells needs to be measured using a transepithelial electrical resistance meter to select dense and intact cell monolayers. After seeding Caco-2 cells in Transwell chambers, the TEER of the epithelial cell layer was measured using an EVOM transepithelial electrical resistance meter and an STX2 electrode on days 1, 3, 7, 12, 16, and 21 of cell growth. Caco-2 cell monolayers with intact growth and a resistance value greater than 300 Ω·cm were selected. -2 The membrane was used for transport experiments. Before measurement, 0.5 mL and 1.5 mL of fresh culture medium preheated to 37°C were added to the top chamber and basal side chamber, respectively, and equilibrated at 37°C for 30 min before measurement. The TEER of the epithelial cell layer (n=3) was obtained by subtracting the resistance of the blank permeable filter membrane from the measured TEER of each cell layer.

[0165] Test results as follows Figure 18B As shown, after Caco-2 cells were seeded into Transwell chambers, the compactness of the Caco-2 cell monolayer gradually increased with the extension of culture time, reaching 331±3.05 Ω·cm on day 12. 2 Subsequently, the resistance value increases more slowly, almost reaching saturation, and then stops increasing.

[0166] Permeability study of PA and MIL-100(Fe)-PA in Caco-2 cell model

[0167] Based on the results of the cytotoxicity assay, low, medium, and high concentrations were selected for transport experiments in the Caco-2 cell model. The amounts of PA and MIL-100(Fe)-PA from the apex to the basal side of the cell layer were measured, and Papp was calculated to evaluate its absorption in the gastrointestinal tract. The results are as follows: Figure 19B As shown, the permeability values ​​of PA and MIL-100(Fe)-PA at low, medium, and high concentrations are all greater than 1×10⁻⁶. -6 cm·s -1This indicates that PA can be absorbed in the gastrointestinal tract. Furthermore, the permeability of PA and MIL-100(Fe)-PA increases in a concentration-dependent manner. However, after treatment with MIL-100(Fe), the permeability of MIL-100(Fe)-PA only slightly improves compared to when used alone.

[0168] The above results show that, by comparing the permeability of PA and MIL-100(Fe)-PA dry powder inhaler in 16HBE and Caco-2 cell models, PA as MIL-100(Fe)-PA is absorbed more when administered via pulmonary inhalation than when administered orally. It can be predicted that MIL-100(Fe) may promote the absorption of PA in the lungs.

[0169] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dry powder inhaler based on MIL-100(Fe) of angelica dahurica (PA), characterized in that, The dry powder inhaler uses MIL-100(Fe) as a carrier and PA as the drug component; the dry powder inhaler is prepared according to the following method: Add 0.64 mmol FeSO4·7H2O, 0.33 mmol H3BTC and 10 mL H2O to the reaction vessel, stir evenly and sonicate until the reaction is complete, then cool to room temperature; After cooling, the product was filtered and washed with water and ethanol more than three times. The product was then soaked in water at 80°C for 6 hours and in ethanol at 60°C for 6 hours. After filtration, the product was activated in a vacuum oven at 120°C for 3 hours to obtain MIL-100(Fe) powder. MIL-100(Fe) powder and PA are mixed in a mass ratio of 1:9 and placed in a reactor; The reactor was connected to a condenser and a magnetically stirred heater for drug loading. After drug loading was completed, the crystals were collected by centrifugation. The drug loading temperature was 35˚C and the drug loading time was 12 h. The crystals were washed three times with ethanol and dried under vacuum at 60˚C for 6 h to obtain MIL-100(Fe)-PA powder.

2. The MIL-100(Fe)-based dry powder inhaler of angelica dahurica (PA) according to claim 1, characterized in that, The powder particle size of the dry powder inhaler and the crystal particle size of MIL-100(Fe) are both 1-5 μm.

3. A method for evaluating the MIL-100(Fe)-based angelica dahurica (PA) dry powder inhaler according to claim 1, characterized in that: The method described above evaluates the crystal structure, crystal morphology, powder properties, in vitro lung deposition, in vitro release, and cell activity of MIL-100(Fe) angelica dahurica (PA) dry powder inhaler.

4. The use of the MIL-100(Fe)-based angelica dahurica (PA) dry powder inhaler as described in claim 1 or 2 in the preparation of a medicament for treating lung diseases.

5. The application according to claim 4, characterized in that, The lung diseases mentioned include one or more of COPD, bronchial asthma, lung cancer, and pulmonary interstitial fibrosis.

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