Quercetin and ketotifen co-assembled nanodrug and construction method and application thereof

Abstract

The present application relates to a kind of quercetin and ketotifen co-assembly nanomedicine and its construction method and application, belong to nanomedicine technical field;The construction method includes the following steps: S1, quercetin and ketotifen are dissolved in organic solvent, and mixed uniformly;S2, remove organic solvent, so that quercetin and ketotifen co-assembly occurs, and forms co-assembly solid;S3, to co-assembly solid is added dispersion medium and is hydrated, and is treated by ultrasonic dispersion, and the dispersion liquid of quercetin and ketotifen co-assembly nanomedicine is obtained.The present application is creatively constructed as co-assembly nanomedicine without any exogenous carrier, not only simultaneously solves the technical problems of poor water-solubility of quercetin and obvious central inhibition side effects of ketotifen, but also realizes the treatment goal of high efficiency, low toxicity, synergistic anti-asthma, with significant clinical conversion prospect and industrial application value.

Description

Technical Field

[0001] This invention relates to a quercetin and ketotifen co-assembled nanomedicine, its construction method and application, belonging to the field of nanomedicine technology. Background Technology

[0002] Asthma is a common chronic inflammatory airway disease, and anti-inflammatory and anti-allergic treatments are central to its long-term management. Ketotifen (KET), a drug with both antihistamine and anti-allergic effects, is widely used clinically for the preventive treatment of asthma. However, oral ketotifen is prone to causing significant central nervous system depressant adverse reactions in the early stages of treatment, such as drowsiness, slowed reaction, and decreased psychomotor function. These side effects seriously affect patient tolerance and long-term medication adherence, thus limiting its clinical application value.

[0003] On the other hand, the natural flavonoid quercetin (QUE) possesses multi-target anti-inflammatory, antioxidant, and immunomodulatory activities, showing promising intervention potential for asthma-related allergic airway inflammation. However, quercetin has extremely poor drug-like properties. Existing studies clearly indicate that quercetin has extremely low water solubility, with a solubility in water of only 0.166–7.7 µg / mL. It also exhibits a strong first-pass metabolism in the liver, resulting in an oral bioavailability of less than 2% [References: Quercetin as a Therapeutic Product: Evaluation of Its Pharmacological Action and Clinical Applications—A Review (PMC, 2024) and Materials, Syntheses and Biomedical Applications of Nano-Quercetin Formulations: A Comprehensive Literature Review (Int J Nanomed, 2024 / 2025)]. This extremely poor solubility and absorption characteristics mean that free quercetin cannot effectively dissolve and penetrate intestinal epithelial cells in the gastrointestinal tract, severely limiting its in vivo delivery efficiency and clinical translation.

[0004] To overcome the shortcomings of single-drug therapy, those skilled in the art have attempted to combine ketotifen with quercetin. The simplest strategy is a simple physical mixture or oral administration of both. While this approach can exert their respective pharmacological effects to some extent, it fails to address the fundamental problems: Firstly, physical mixing cannot alter the physicochemical nature of quercetin; its solubility and absorption characteristics remain completely identical to those of free quercetin, offering no improvement [Reference: Improved Oral Absorption of Quercetin from Quercetin Phytosome®, a New Delivery System (Eur J Drug Metab Pharmacokinet)]. Secondly, physical mixing cannot modulate the pharmacokinetic behavior of ketotifen; it can still freely cross the blood-brain barrier, and central nervous system depressant side effects (drowsiness, decreased activity, etc.) are not reduced, failing to achieve the therapeutic goal of "enhanced efficacy and reduced toxicity."

[0005] To further improve drug delivery performance, researchers have attempted to co-encapsulate ketotifen and quercetin using traditional nanoparticle drug delivery systems (such as liposomes, polymer nanoparticles, and micelles). These methods can improve the apparent solubility of poorly soluble drugs to some extent and may enable sustained-release or targeted delivery. However, traditional nanocarriers still have significant technical limitations: First, the introduction of a large number of non-pharmacologically active carrier materials (such as polymeric excipients or lipid materials) into the system results in generally low drug loading of the effective active ingredient and an excessively high proportion of excipients in the formulation; second, ketotifen and quercetin have significantly different physicochemical properties, making it difficult for traditional carriers to ensure that both are delivered synchronously and quantitatively to the same lesion site; more importantly, the degradation, metabolism, and long-term accumulation of exogenous carrier materials in vivo may cause additional metabolic burden, cytotoxicity, or immunogenicity problems, increasing the potential risk of toxic side effects [References: Carrier-Free Nanodrugs: From Benchto Bedside (ACS Nano, 2024, DOI: 10.1021 / acsnano.4c09027) and Advance Progress in Assembly Mechanisms of Carrier-Free Nanodrugs for Cancer Treatment (Molecules, 2023)]. These shortcomings severely restrict the clinical translation and application prospects of traditional nanocarrier drug delivery systems.

[0006] In summary, existing technologies still lack a strategy for effectively integrating ketotifen and quercetin without introducing additional excipients or carriers. How to simultaneously improve the water solubility and bioavailability of quercetin, reduce the central nervous system depressant side effects of ketotifen, and achieve a synergistic anti-asthmatic effect of both is a pressing technical challenge in this field. Summary of the Invention

[0007] Based on the technical problems existing in the prior art, the present invention proposes a carrier-free co-assembled nanomedicine based on quercetin and ketotifen, its construction method and application. The construction method does not require any exogenous carrier material and relies entirely on the non-covalent interactions (hydrogen bonds, hydrophobic interactions and van der Waals forces, etc.) between the two drug molecules for co-assembly.

[0008] The technical solution provided by this invention is as follows: One objective of this invention is to provide a method for constructing a nanomedicine co-assembled with quercetin and ketotifen, comprising the following steps: S1. Co-solution: Weigh quercetin and ketotifen, dissolve them together in an organic solvent, and mix thoroughly with magnetic stirring at room temperature; S2, Evaporation: Organic solvents are removed by stirring and rotating to allow quercetin and ketotifen to co-assemble and form co-assembled nanoparticles; S3. Hydration: A dispersion medium is added to the co-assembled nanoparticles for hydration. After ultrasonic dispersion, a dispersion of quercetin and ketotifen co-assembled nanomedicine is obtained.

[0009] Based on the above technical solution, the present invention can be further improved as follows: Further, in step S1, the ratio of quercetin, ketotifen, and organic solvent is 0.5~2g:1g:1~1.5L.

[0010] Further, in step S1, the organic solvent is methanol or anhydrous ethanol; the magnetic stirring speed is 500~800 r / min, and the time is 1~2 h.

[0011] Furthermore, in step S2, the temperature for stirring and evaporation is 37~42℃, the stirring speed is 350~500rpm, and the time is 0.5~2h.

[0012] Furthermore, in step S2, the stirring and rotational evaporation is carried out using a magnetic stirrer.

[0013] Further, in step S3, the dispersion medium is ultrapure water, physiological saline, or PBS buffer. Further, in step S3, the ratio of the co-assembled nanoparticles to the dispersion medium is 2.5~4.0 mg: 5~8 mL. Furthermore, in step S3, the ultrasonic dispersion treatment time is 3-5 minutes and the power is 200-300W.

[0014] The second objective of this invention is to provide a quercetin and ketotifen co-assembled nanomedicine, which is constructed using the method described above.

[0015] A third objective of this invention is to provide the application of the quercetin and ketotifen co-assembled nanomedicine described above in the preparation of drugs for treating respiratory diseases.

[0016] Furthermore, the respiratory diseases include asthma.

[0017] The technical solution provided by this invention has the following advantages compared with the prior art: 1. This invention does not rely on exogenous carrier materials such as liposomes and polymers. It drives co-assembly solely through non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and van der Waals forces between the two small drug molecules, ketotifen and quercetin. The drug loading capacity is much higher than that of traditional nanocarriers (usually less than 20%). This completely avoids the metabolic burden, cytotoxicity, and immunogenicity problems caused by the long-term accumulation of polymeric excipients or inorganic carriers in vivo, and significantly improves the safety of the formulation.

[0018] 2. This invention utilizes the molecular co-assembly of ketotifen and quercetin, taking advantage of the polar groups (such as piperidine rings and carbonyl groups) in the ketotifen structure to form intermolecular hydrogen bonds with the phenolic hydroxyl groups of quercetin. At the same time, the hydrophobic regions are synergistically stacked, effectively improving the apparent solubility of quercetin. The nanoparticles formed by co-assembly have a large specific surface area, which can promote the dissolution and transmembrane transport of quercetin in the gastrointestinal tract, thereby significantly improving its oral absorption efficiency and in vivo exposure level, overcoming the long-standing drug-like bottleneck of quercetin.

[0019] 3. In this invention, after co-assembling ketotifen and quercetin into nanomedicines, quercetin has antioxidant and blood-brain barrier permeability regulation effects, and the co-assembled structure may delay the release of ketotifen from the nanoparticles. Behavioral experiments have confirmed that mice in the ketotifen monotherapy group showed significant central inhibitory effects such as drowsiness and decreased total activity distance. Mice that received co-assembled nanomedicine intervention showed significant recovery in spontaneous exploration behavior in the open field test and voluntary movement level in the wheel test compared to the monotherapy group, and their water intake, food intake and weight changes were more likely to be in a normal and stable state.

[0020] 4. In this invention, ketotifen and quercetin are co-assembled and simultaneously delivered to the site of airway inflammation, achieving co-release and co-deposition at the pharmacokinetic level, thereby exerting complementary and synergistic effects. Experiments have demonstrated that the nanomedicine of this invention is significantly superior to the simple physical mixture of two drugs and the single drug group in inhibiting airway hyperresponsiveness, reducing the levels of eosinophils and inflammatory factors in bronchoalveolar lavage fluid, and alleviating pathological damage to lung tissue, exhibiting a significant synergistic effect. Attached Figure Description

[0021] Figure 1 The diagram shows the interaction energy heatmap of the ketotifen single-component system in Test Example 1. In the diagram, A represents the total interaction energy, B represents the coulombic short-range interaction energy, and C represents the Leonard-Jones short-range interaction energy.

[0022] Figure 2 This is a heatmap of the interaction energies of the quercetin single-component system in Test Example 1. In the diagram, A represents the total interaction energy, B represents the Coulomb short-range interaction energy, and C represents the Leonard-Jones short-range interaction energy.

[0023] Figure 3 This is a heatmap of the interaction energy of the mixed system in Test Example 1. In the diagram, A represents the total interaction energy, B represents the Coulomb short-range interaction energy, and C represents the Leonard-Jones short-range interaction energy.

[0024] Figure 4 The graph shows the total interaction energy of the mixed system in Test Example 1 as a function of time.

[0025] Figure 5 The graph shows the dynamic change of the interaction energy of the mixed system in Test Example 1.

[0026] Figure 6 The diagram shows the cluster analysis results for different systems in Test Example 1.

[0027] Figure 7 The graph shows the change in the radius of gyration of the mixed system in Test Example 1.

[0028] Figure 8 The plot shows the trajectory changes of the ketotifen single-component system in Test Example 2 at 0, 200, 400, 600, 800 and 1000 ps.

[0029] Figure 9 The plot shows the trajectory changes of the quercetin single-component system in Test Example 2 at 0, 200, 400, 600, 800 and 1000 ps.

[0030] Figure 10The molecular dynamics trajectory diagrams for the mixed system of ketotifen and quercetin at different feed ratios in Example 2 are shown at 0, 500, 1000, 1500, and 2000 ps. A is the molecular dynamics trajectory diagram of the nano-drug dispersion prepared in Example 1; B is the molecular dynamics trajectory diagram of the nano-drug dispersion prepared in Example 2; and C is the molecular dynamics trajectory diagram of the nano-drug dispersion prepared in Example 3.

[0031] Figure 11 The diagram shows the zeta potential distribution of the QUE single-component system in Test Example 3.

[0032] Figure 12 The diagram shows the zeta potential distribution of the KET single-component system in Test Example 3.

[0033] Figure 13 The diagram shows the zeta potential distribution of the QUE / KET co-incubation system in Test Example 3.

[0034] Figure 14 The statistical results of the Zeta potentials of the QUE, KET, and QUE / KET systems in Test Example 3 are presented.

[0035] Figure 15 The particle size distribution curve of the quercetin:ketotifen = 0.5:1 group in test example 4 is shown.

[0036] Figure 16 The particle size distribution curve of the quercetin:ketotifen = 1:1 group in test example 4.

[0037] Figure 17 The particle size distribution curves for the quercetin:ketotifen = 2:1 group in Test Example 4 are shown.

[0038] Figure 18 The average hydrated particle size of the co-assembled nanoparticles under different feed ratios in Test Example 4 is statistically analyzed.

[0039] Figure 19 The results of the elevated cross maze experiment in Test Example 6 are shown in the figure. Among them, (A) is the representative movement trajectory of mice in each group; (B) is the percentage of time mice spend with their open arms in each group; and (C) is the statistical result of the movement distance of mice with their open arms in each group.

[0040] Figure 20 The results of the open field experiment in Test Example 6 are shown in the figure. Among them, (A) is the exploration map of the central area of ​​mice in each group; (B) is the comparison map of the total movement distance of mice in each group; (C) is the comparison map of the dwell time in the central area of ​​mice in each group; and (D) is the comparison map of the movement distance in the central area of ​​mice in each group.

[0041] Figure 21 This is a graph showing the changes in airway hyperresponsiveness in mice under gradient concentrations of methacholine in test example 5.

[0042] Figure 22 The graph shows the expression levels of p-p65 and p65 proteins in the lung tissue of mice in each group as detected by Western blotting in Test Example 5.

[0043] Figure 23 The graph shows the statistical results of the total inflammatory cell count in the bronchoalveolar lavage fluid (BALF) of mice in each group in Test Example 5.

[0044] Figure 24 This is a graph showing the changes in airway resistance in mice under gradient concentrations of methacholine in test example 5.

[0045] Figure 25 The figure shows the results of ELISA detection of IgE inflammatory factor expression levels in the BALF of mice in each group in Test Example 5.

[0046] Figure 26 The figure shows the results of ELISA detection of IL-4 inflammatory factor expression levels in the BALF of mice in each group in Test Example 5.

[0047] Figure 27 The figure shows the results of ELISA detection of IL-5 inflammatory factor expression levels in the BALF of mice in each group in Test Example 5.

[0048] Figure 28 The figure shows the results of ELISA detection of IL-13 inflammatory factor expression levels in the BALF of mice in each group in Test Example 5. Detailed Implementation

[0049] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0050] Example 1 A method for constructing a quercetin and ketotifen co-assembled nanomedicine includes the following steps: S1. Co-dissolving: Weigh 0.005g of quercetin and 0.01g of ketotifen (the mass ratio of quercetin to ketotifen is 0.5:1), dissolve them together in 10mL of anhydrous ethanol, and stir magnetically at 800rpm for 1h at room temperature to mix thoroughly. S2. Evaporation: The organic solvent is completely removed by stirring and rotating the mixture for 1.5 hours at a temperature of 40℃ and a stirring speed of 400rpm using a magnetic stirrer, so that quercetin and ketotifen can co-assemble to form co-assembled nanoparticles. S3. Hydration: Add 5 mL of ultrapure water to 2.5 mg of co-assembled nanoparticles for hydration. After ultrasonic dispersion treatment at 200 W for 5 min, a dispersion of quercetin and ketotifen co-assembled nanomedicine is obtained.

[0051] Example 2 A method for constructing a quercetin and ketotifen co-assembled nanomedicine includes the following steps: S1. Co-dissolving: Weigh 0.01g of quercetin and 0.01g of ketotifen (the mass ratio of quercetin to ketotifen is 1:1), dissolve them together in 15mL of anhydrous ethanol, and stir magnetically at 600rpm for 1.5h at room temperature until fully mixed. S2. Evaporation: The organic solvent is completely removed by stirring and rotating the mixture for 2 hours at a temperature of 37°C and a stirring speed of 350 rpm using a magnetic stirrer, so that quercetin and ketotifen can co-assemble to form co-assembled nanoparticles. S3. Hydration: 7 mL of physiological saline was added to 3.5 mg of co-assembled nanoparticles for hydration. After ultrasonic dispersion treatment at 250 W for 4 min, a dispersion of quercetin and ketotifen co-assembled nanomedicine was obtained.

[0052] Example 3 A method for constructing a quercetin and ketotifen co-assembled nanomedicine includes the following steps: S1. Co-dissolving: Weigh 0.01g of quercetin and 0.005g of ketotifen (the mass ratio of quercetin to ketotifen is 2:1), dissolve them together in 12mL of anhydrous ethanol, and stir magnetically at 500rpm for 2 hours at room temperature to ensure thorough mixing. S2. Evaporation: The organic solvent is completely removed by stirring and rotating the mixture at 42℃ and 500rpm using a magnetic stirrer for 0.5h, allowing quercetin and ketotifen to co-assemble and form co-assembled nanoparticles. S3. Hydration: Add 8 mL of ultrapure water to 4 mg of co-assembled nanoparticles for hydration. After ultrasonic dispersion treatment at 300 W for 3 min, a dispersion of quercetin and ketotifen co-assembled nanomedicine is obtained.

[0053] Comparative Example 1 The difference from Example 1 is that quercetin is not added in step S1; instead, an equal amount of ketotifen is used. A dispersion of the ketotifen single-component system is obtained.

[0054] Comparative Example 2 The difference from Example 1 is that ketotifen is not added in step S1, but instead an equal amount of quercetin is added. A dispersion of the quercetin single-component system is obtained.

[0055] test: The dispersions of nanomedicines obtained in Examples 1-3 and Comparative Examples 1-2 were tested: Test Example 1: Molecular dynamics analysis of the stability of the interaction between quercetin and ketotifen Molecular dynamics simulations were primarily performed using the GROMACS software package. First, the three-dimensional molecular structures of quercetin and ketotifen were obtained, and suitable topology files and small molecule force field parameters were generated. The constructed single-component or mixed systems with different feed ratios were placed in a cubic periodic boundary box, and thorough solvation was performed using the TIP3P water model. Appropriate counterions were added as needed to maintain the electroneutrality of the system. After the system was constructed, the steepest descent method was first used to minimize energy, eliminating unreasonable intermolecular collisions and local steric hindrance. Subsequently, position-restricted equilibrium simulations were performed on the system under NVT (constant number of atoms, volume, and temperature) and NPT (constant number of atoms, pressure, and temperature), stabilizing the system temperature at 300 K and the pressure at 1 bar. After equilibrium was achieved, the position restrictions were removed, and a production molecular dynamics simulation (Production MD) was performed for 2000 ps. After the simulation was completed, the total interaction energy, Coulomb short-range interaction energy, Leonard-Jones short-range interaction energy and radius of gyration (Rg) of the system were extracted using the built-in analysis program of GROMACS. Molecular visualization software such as PyMOL was used to visualize and analyze the co-aggregation behavior and trajectory snapshots between molecules.

[0056] Molecular dynamics simulations indicate a stable intermolecular interaction between quercetin and ketotifen, with the potential to form complex aggregates. Analysis of the total interaction energy, coulombic short-range interaction energy, and Leonard-Jones short-range interaction energy of different systems shows that the co-assembled mixtures in Examples 1-3 exhibit more pronounced low-energy interaction characteristics, suggesting that quercetin and ketotifen can form a stable complex through various non-covalent interactions, with the Leonard-Jones short-range interaction energy contributing more significantly. In contrast, the single-component systems in Examples 1-2, lacking synergistic non-covalent interactions between the heterogeneous molecules of quercetin and ketotifen (such as complementary hydrogen bonds and π-π stacking), show significantly weaker coulombic and Leonard-Jones short-range interaction energies compared to the co-assembled mixtures. Figures 1-3 Further energy dynamics analysis showed that although the system experienced some fluctuations during the simulation, it remained at a low level overall, without any obvious trend of instability. Meanwhile, the radius of gyration stabilized after a brief initial adjustment, indicating that the composite system possesses good spatial compactness and dynamic stability. Figures 4-7 ).

[0057] Test Example 2: Analysis of the self-aggregation and co-aggregation trajectories of quercetin and ketotifen To further evaluate the self-aggregation behavior and co-assembly feasibility of quercetin and ketotifen, a visualization analysis of their molecular dynamics trajectories was performed.

[0058] First, the original molecular dynamics simulation trajectory of 2000 ps was post-processed using the built-in gmx trjconv command in GROMACS to eliminate the influence of periodic boundary conditions (PBCs) and center the complex aggregates within the box. Then, the processed trajectory file and structural topology file were imported into PyMOL software for visualization and rendering. To visually demonstrate the dynamic evolution of single-component self-aggregation and co-assembly of hybrid systems, 3D structural snapshots of the initial state (0 ps) and key simulation nodes were extracted and rendered using uniform shading and ball-and-stick / stick models.

[0059] The results are as follows Figures 8-10 As shown, both the quercetin single-component system (the nano-drug dispersion prepared in Comparative Example 2) and the ketotifen single-component system (the nano-drug dispersion prepared in Comparative Example 1) gradually transformed from an initial dispersed state to an aggregated state during the simulation, suggesting that both have a certain degree of self-aggregation ability. Figure 8 and Figure 9 Further observation of the quercetin and ketotifen mixtures with different feed ratios (nanomedicine dispersions prepared in Examples 1-3) revealed that each system gradually formed complex aggregates of varying degrees during the simulation process. Furthermore, with increasing time, intermolecular contact increased and clusters gradually enlarged. Figure 10 The above results demonstrate that quercetin and ketotifen can not only interact stably, but also achieve synergistic aggregation under dynamic conditions, providing a direct basis for the subsequent construction of carrier-free co-assembled nanosystems.

[0060] Test Example 3: Surface electrical characterization of nanoparticles formed by co-assembling quercetin and ketotifen To verify whether quercetin and ketotifen form a new assembly system, the zeta potentials of the single-component system and the co-assembled system were characterized.

[0061] The specific characterization method was as follows: Appropriate amounts of freshly prepared dispersions from Comparative Examples 1 and 2 (QUE and KET groups) and the co-assembled nanomedicine dispersion from Example 1 (QUE / KET group) were taken and appropriately diluted with ultrapure water to avoid multiple light scattering effects. Approximately 1 mL of the diluted sample was injected into a dedicated Zeta potential testing cell (folded capillary sample cell). Under constant temperature conditions of 25°C, the surface Zeta potential of each system was measured using a laser particle size and Zeta potential analyzer (such as the Malvern Zetasizer Nano ZS series or equivalent equipment). Each sample was measured in triplicate, and the average value was recorded as the potential distribution curve.

[0062] The results are as follows Figures 11-14 As shown, the three groups of samples all exhibit a relatively concentrated single-peak distribution, indicating that the electrophoretic behavior of the system is quite consistent. Figures 11-13Further statistical analysis showed that the Zeta potential of the KET monocomponent system was close to neutral, the QUE monocomponent system was slightly negatively charged, and the Zeta potential of the QUE / KET co-assembled system shifted further towards the negative potential. Figure 14 Compared to the two single components, the surface charge of the co-assembled system changed significantly, indicating that there is an interaction between QUE and KET, and that a new dispersion system was formed.

[0063] Test Example 4: Effect of different feed ratios on the particle size of QUE / KET co-assembled nanoparticles The specific testing procedure is as follows: Take an appropriate amount of the freshly prepared co-assembled nanoparticle dispersions from Examples 1-3, and dilute them appropriately with ultrapure water to avoid multiple light scattering effects caused by excessively high system concentration. Place the diluted sample in a clean sample cell, and under constant temperature of 25°C, acquire the light scattering signal of the sample using a dynamic light scattering (DLS) particle size analyzer (such as the Malvern Zetasizer Nano ZS series or equivalent). Each sample ratio is measured in triplicate, and the average hydrated particle size distribution and polydispersity index (PDI) of the nanoparticles are calculated and output using the instrument's built-in software.

[0064] The results are as follows Figures 15-18 As shown, all three groups of samples exhibited a relatively concentrated single-peak distribution with no obvious abnormal peaks, suggesting that a well-dispersed nanosystem can be formed under different feed ratios. Figures 15-17 Further statistical analysis showed that as the QUE feed ratio increased, the average hydrated particle size of the nanoparticles gradually increased, with the 0.5:1 group being approximately 47 nm, the 1:1 group approximately 73 nm, and the 2:1 group approximately 88 nm. Figure 18 The above results indicate that the feed ratio can affect the particle size of the QUE / KET co-assembled system.

[0065] Test Example 5: Evaluation of the in vivo anti-asthmatic efficacy of QUE / KET co-assembled nanomedicines in OVA-induced allergic asthma model mice. In an OVA-induced mouse model of allergic asthma, the in vivo anti-asthmatic efficacy of the QUE / KET carrier-free co-assembled nanosystem was systematically evaluated.

[0066] Experimental setup: Animal strain: Female BALB / c mice (SPF grade); Age and weight: 6-8 weeks old, weight approximately 18-20g; Sample size: Considering statistical validity and animal ethics, n=6 animals per group.

[0067] Group design (5 groups in total): NC group (Normal Control, blank control group) OVA group (model group: ovalbumin sensitization group) DEX group (positive drug control group: dexamethasone group) KET group (ketotifen group, dispersion prepared in Comparative Example 1) QUE / KET group (ketotifen + quercetin group, dispersion prepared in Example 1) Setting up OVA model groups (applicable to OVA group, DEX group and each drug administration group): Basic sensitization (days 0, 7, and 14): 0.2 mL of a saline suspension containing OVA (20 μg) and aluminum hydroxide adjuvant (2 mg) was administered via intraperitoneal injection (ip) to activate a systemic allergic immune response.

[0068] Airway provocation (days 21-27): For 7 consecutive days, mice were placed in a closed nebulizer and nebulized with 1% OVA saline solution for 30 minutes each time to induce local airway inflammation.

[0069] Setup of the NC group (blank control group): Objective: To eliminate the influence of experimental procedures and environmental stress on mice (especially their behavioral performance).

[0070] Procedure: The entire process was performed in parallel with the OVA model group. However, sensitization on days 0, 7, and 14 involved injection of an equal volume of sterile saline with aluminum hydroxide adjuvant; challenge on days 21-27 involved nebulization of pure saline.

[0071] Drug intervention was uniformly concentrated in the later airway provocation phase (days 21-27).

[0072] DEX group (dexamethasone positive control): Administration: Dexamethasone (1-2 mg / kg) is administered via intraperitoneal injection (ip) 1 hour before OVA nebulization challenge on days 21-27.

[0073] QUE / KET group (co-assembled nano-dispersion group) and KET group (ketotifen group): Administration time: Also on days 21-27, administer 1-2 hours before each OVA nebulization stimulation.

[0074] Route of administration (intraperitoneal injection).

[0075] The specific methods and procedures for evaluating drug efficacy are as follows: (1) Measurement of airway hyperresponsiveness (AHR) and airway resistance: 24 h after the last challenge, the lung function of mice in each group was assessed using an animal lung function testing system (such as a non-invasive / invasive plethysmography system). Mice were placed in a plethysmography chamber and airway challenge was performed by sequentially administering a basal dose (physiological saline) and gradient concentrations of methacholine (Mch, e.g., 0, 6.25, 12.5, 25, 50 mg / mL) via ultrasonic nebulization. The changes in airway resistance and airway hyperresponsiveness were continuously recorded and calculated.

[0076] (2) Bronchoalveolar lavage fluid (BALF) collection and cell counting: After the pulmonary function test, mice were anesthetized and their trachea exposed. Endotracheal intubation was performed, and pre-cooled PBS buffer (approximately 0.8–1.0 mL) was slowly injected to lavage the lungs. The lavage fluid was collected after gentle massage of the lungs, and this process was repeated three times to ensure thorough collection. The collected BALF was centrifuged at 1500 rpm for 10 min at 4°C. The supernatant was collected and stored at -80°C for later use. The precipitated cell clusters were resuspended in an appropriate amount of PBS, and a total inflammatory cell count was performed using a cell counter or hemocytometer.

[0077] (3) Detection of inflammatory factors and IgE levels (ELISA): Take the BALF supernatant and mouse peripheral serum that have been centrifuged and preserved above, and strictly follow the operation steps of the corresponding enzyme-linked immunosorbent assay (ELISA) kit instructions to quantitatively detect the expression concentration of key inflammatory factors and antibodies such as IgE, IL-4, IL-5, and IL-13 in the samples.

[0078] (4) Lung tissue protein expression analysis (Western Blot): Right lung tissue from mice was extracted and homogenized using RIPA lysis buffer containing protease and phosphatase inhibitors. Total protein was extracted by centrifugation, and protein concentration was quantified using the BCA method. Equal volumes of protein samples were separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk, primary antibodies against p-p65, p65, and the internal control protein β-actin were added and incubated overnight at 4°C. After washing, horseradish peroxidase (HRP)-labeled secondary antibodies were added and incubated at room temperature. Finally, chemiluminescence (ECL) imaging was performed, and the gray values ​​of the protein bands were analyzed using ImageJ software.

[0079] Animal models were constructed using the classic sensitization-challenge method: On days 0, 7, and 14, except for the NC group which received an equal volume of blank saline, mice in all other groups were systematically sensitized via intraperitoneal injection of a mixture of OVA and aluminum hydroxide adjuvant; from days 21 to 27, except for the NC group which received nebulized saline, all other groups received nebulized inhalation of 1% OVA solution for 30 minutes daily to induce local airway inflammation. Drug intervention was concentrated in the airway challenge phase (days 21-27). Each treatment group received treatment one hour before daily nebulization. The DEX group received intraperitoneal injection of dexamethasone at the standard dose, while the comparative formulation group and the QUE / KET group received the corresponding drug dispersion via intraperitoneal injection at the set concentrations.

[0080] Methacholine (Mch) challenge and airway hyperresponsiveness (AHR) assessment: 24 hours after the last airway challenge (e.g., day 27), airway hyperresponsiveness in mice was assessed using a non-invasive / invasive mouse pulmonary function system (e.g., Buxco plethysmography). Mice were properly secured or placed in the testing chamber, and after 5 minutes of stable breathing adaptation, physiological saline (0 mg / mL) was first nebulized to measure baseline airway resistance. Subsequently, a gradient of acetylcholine (Mch) solution with increasing concentrations was nebulized sequentially (conventional concentration gradients were set at 3.125, 6.25, 12.5, 25, and 50 mg / mL), with each concentration challenge lasting 2–3 minutes. The system automatically recorded the peak changes in pulmonary function parameters such as airway resistance (Rl) within 3–5 minutes after each Mch challenge, thereby quantitatively assessing the degree of airway hyperresponsiveness in each experimental group to acetylcholine challenge.

[0081] The results showed that administration of QUE / KET significantly improved the adverse physical signs in mice stimulated by methacholine. Figure 21 The airway hyperresponsiveness and airway resistance were significantly better than those in the KET group. Figure 24 ), can significantly reduce the total number of inflammatory cells in BALF ( Figure 23 ), downregulates the levels of key pro-inflammatory factors IgE, IL-4, IL-5, and IL-13 ( Figures 25-28 Simultaneously, it can significantly inhibit excessive activation of the NF-κB signaling pathway in lung tissue and downregulate p65 phosphorylation levels. Figure 22 The above results confirm that the QUE / KET co-assembly system can exert significantly better anti-asthmatic efficacy than single drugs through multi-target synergistic effects.

[0082] Test Example 6: QUE / KET co-assembled nanomedicines improve allergic asthma phenotype and alleviate behavioral abnormalities. All mice in Test Example 5 underwent behavioral assessments, including elevated cross maze and open field tests, and subsequent sample collection within 24 hours after the last nebulization challenge.

[0083] The specific behavioral experimental procedures and methods are as follows: (1) Elevated Plus Maze Test: This test was used to assess anxiety-like behavior in mice after OVA induction and drug intervention. The maze consisted of two opposing open arms, two opposing closed arms, and a central platform, all at a certain height above the ground. At the start of the experiment, the mice were gently placed on the central platform with their heads facing one of the open arms. In a quiet and evenly lit environment, a camera suspended above the maze, combined with an animal behavior video tracking system, continuously recorded the mice's free movement trajectory for 5 minutes. After the experiment, the software automatically analyzed and statistically analyzed the percentage of time the mice spent in the open arms and the distance they moved in those arms. After each mouse was tested, the maze was thoroughly wiped with 75% alcohol to completely eliminate odor interference. (2) Open Field Test: This test assesses the spontaneous exploration ability and overall activity level of mice. The experimental setup is a standard square open box, the bottom of which is divided into a central area and a peripheral area. During the test, the mouse is placed stably in the center of the bottom of the open box. In an environment free from external noise interference, the mouse's movement trajectory is continuously recorded over 5 minutes using a video tracking system. Subsequently, the total movement distance, the time spent in the central area, and the distance moved in the central area are extracted and statistically analyzed. Between each mouse's test intervals, the open box is cleaned with 75% alcohol.

[0084] Representative trajectories of elevated cross mazes include: Figure 19 The results showed that the OVA and KET groups explored the open arm region less, while the QUE / KET and DEX groups showed significantly increased open arm activity, more similar to the NC group. Figure 19 (A); Corresponding quantitative results showed that the QUE / KET group had higher open arm dwell time and open arm movement distance than the OVA and KET groups ( Figure 19 (B and C in the middle). Representative trajectories of the open field experiment are as follows: Figure 20 The results showed that central region exploration decreased in the OVA and KET groups, while central region activity significantly improved in the QUE / KET group. Figure 20 Quantitative analysis further showed that there was no significant difference in total movement distance among the groups, but the QUE / KET group had higher central region dwell time and central region movement distance than the OVA and KET groups, and was closer to the NC and DEX groups. Figure 20 (B~D). The above results indicate that QUE / KET can alleviate asthma phenotypes and improve exploratory behavior and overall condition in mice, demonstrating potential for synergistic effects and toxicity reduction.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a nanomedicine co-assembled with quercetin and ketotifen, characterized in that, Includes the following steps: S1. Co-solution: Weigh quercetin and ketotifen, dissolve them together in an organic solvent, and mix thoroughly with magnetic stirring at room temperature; S2, Evaporation: Organic solvents are removed by stirring and rotating to allow quercetin and ketotifen to co-assemble and form co-assembled nanoparticles; S3. Hydration: A dispersion medium is added to the co-assembled nanoparticles for hydration. After ultrasonic dispersion, a dispersion of quercetin and ketotifen co-assembled nanomedicine is obtained.

2. The method for constructing nanomedicine co-assembled with quercetin and ketotifen according to claim 1, characterized in that, In step S1, the ratio of quercetin, ketotifen, and organic solvent is 0.5~2g:1g:1~1.5L.

3. The method for constructing nanomedicine co-assembled with quercetin and ketotifen according to claim 1, characterized in that, In step S1, the organic solvent is methanol or anhydrous ethanol; the magnetic stirring speed is 500~800 r / min, and the time is 1~2 h.

4. The method for constructing nanomedicine co-assembled with quercetin and ketotifen according to claim 1, characterized in that, In step S2, the temperature for stirring and evaporation is 37~42℃, the stirring speed is 350~500rpm, and the time is 0.5~2h.

5. The method for constructing quercetin and ketotifen co-assembled nanomedicines according to claim 1 or 4, characterized in that, In step S2, the stirring and evaporation is carried out using a magnetic stirrer.

6. The method for constructing nanomedicine co-assembled with quercetin and ketotifen according to claim 1, characterized in that, In step S3, the dispersion medium is ultrapure water, physiological saline, or PBS buffer.

7. The method for constructing nanomedicine co-assembled with quercetin and ketotifen according to claim 1, characterized in that, In step S3, the ratio of the amount of co-assembled nanoparticles to the dispersion medium is 2.5~4.0 mg: 5~8 mL.

8. The method for constructing nanomedicine co-assembled with quercetin and ketotifen according to claim 1, characterized in that, In step S3, the ultrasonic dispersion treatment takes 3-5 minutes and the power is 200-300W.

9. A nanomedicine co-assembled with quercetin and ketotifen, characterized in that, It is constructed using the construction method described in any one of claims 1 to 8.

10. The application of the quercetin and ketotifen co-assembled nanomedicine as described in claim 9 in the preparation of drugs for treating respiratory diseases.

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