Cascade targeted drug delivery system and preparation method and application thereof

Abstract

The invention discloses a cascade targeted drug delivery system as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention designs a drug delivery system with a cascade targeting function, and the drug delivery system is used for delivering a phosphodiesterase 4 inhibitor A5. Through polypeptide modification and exosome fusion, phagocytosis and ingestion of cells to a drug delivery system are promoted, and the bioavailability of the drug is significantly improved. The drug delivery system shows excellent in-vivo lung targeting, can effectively inhibit the aggravation of inflammatory infiltration in the lung, and plays a role in treating acute lung injury. The invention provides a new strategy for improving the targeting property and the utilization rate of the medicine, provides a new medicine for treating the acute lung injury, and has a wide application prospect and an extremely high application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a cascade targeted drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Acute lung injury (ALI) is an acute diffuse infiltrative lesion and pulmonary edema caused by damage to pulmonary capillary endothelial cells and alveolar epithelial cells in non-cardiogenic diseases such as severe infection, trauma and burn, resulting in acute hypoxia respiratory dysfunction or respiratory failure. Damage to a certain extent can cause acute respiratory distress syndrome (ARDS), that is, the final severe stage of ALI or ALI. Various clinical diseases can cause acute lung injury, the incidence of severe infection can be as high as 25%-50%, and the mortality can be more than 40%. Meta-analysis suggests that there is no difference in mortality between acute lung injury caused by internal and external factors.

[0003] Although there are a large number of studies on acute lung injury at present, there is still no specific treatment for this disease in clinic. The usual treatment method first needs to find the cause and effectively treat the primary disease. The conventional treatment method of ALI mainly includes mechanical ventilation therapy, surfactant, antioxidant, glucocorticoid, anti-inflammatory drug and other drug treatment method, stem cell treatment method, nano drug delivery system auxiliary treatment method, etc. However, when acute lung injury occurs, pathological changes such as neutrophil inflammation in alveolar part, formation of glass membrane composed of fibrin and matrix protein in alveolar duct and alveolar cavity, etc. These lesions will cause difficulty in drug deposition in the lung, reduce the bioavailability of the drug, and thus affect the treatment effect. Part of the reason for insufficient drug treatment may be that the drug delivery to the damaged alveoli is blocked, the drug is not accumulated enough in the lung, the circulating half-life is low, and it cannot cross the physiological barrier (mucus and alveolar fluid) for systemic delivery. In addition, many small molecule drugs also lack targeting, so there are still many problems to be solved in the treatment of ALI. SUMMARY

[0004] The application aims to provide a cascade targeted drug delivery system and a preparation method and application thereof to solve the problems of the prior art.

[0005] To achieve the above-mentioned object, the application provides the following solutions.

[0006] The application provides a preparation method of a cascade targeted drug delivery system, comprising the following steps.

[0007] (1) pardaxin peptide is co-incubated with DSPE-PEG2000-NHS to obtain polypeptide modified lipids;

[0008] (2) the polypeptide modified lipids, Dlin-MC3-DMA, DSPC, cholesterol, DOTAP and phosphodiesterase 4 inhibitor are mixed and dissolved, and drug-loaded lipid nanoparticles are prepared by a film dispersion-extrusion method;

[0009] (3) the drug-loaded lipid nanoparticles are co-incubated with exosomes by a membrane extrusion method to prepare the cascade targeted drug delivery system.

[0010] Further, in step (1), the mass ratio of the pardaxin peptide to the DSPE-PEG2000-NHS is 1:1.

[0011] Further, in step (1), the co-incubation condition is as follows: in a water-free dimethylformamide solvent, under the protection of N,N-diisopropyl ethylamine and nitrogen, co-incubation is performed at 250 rpm for 2 h.

[0012] Further, in step (2), the mass ratio of the polypeptide modified lipids, Dlin-MC3-DMA, DSPC, cholesterol and DOTAP is 4:15:6:7:25.

[0013] Further, in step (2), the mixing and dissolving condition is as follows: in a mixed solvent of water-free ethanol:chloroform=1:2, ultrasonic treatment is performed in a water bath until dissolution, and then the organic solvent is removed.

[0014] Further, in step (3), the mass ratio of the drug-loaded lipid nanoparticles to the exosomes is 1:0.2.

[0015] Further, in step (3), the co-incubation condition is that: after mixing the drug-loaded lipid nanoparticles and the exosomes, incubate at 37 DEG C for 12 hours.

[0016] Further, the phosphodiesterase 4 inhibitor is A5, and the structural formula is as follows:

[0017] ;

[0018] The exosomes are human fat-derived mesenchymal stem cell exosomes.

[0019] The application further provides a cascade targeted drug delivery system prepared by the preparation method.

[0020] The application further provides application of the cascade targeted drug delivery system in preparation of a drug for treating acute lung injury or an anti-inflammatory drug.

[0021] The application discloses the following technical effects:

[0022] The application is based on a lung organ targeted lipid nanoparticle (LNP), which is modified by a cell penetrating peptide to realize targeting, and is fused with a negatively charged human fat-derived mesenchymal stem cell exosome (Exo), so that the defects of the LNP, such as high surface positive charge and possible side effects of aggravating inflammation, are overcome, a drug delivery system with cascade targeting function is designed, and a phosphodiesterase 4 inhibitor A5 is delivered. The cascade targeted drug delivery system (A5@ELNP) prepared in the application is safe, the modification of the polypeptide and the fusion of the exosome can promote phagocytosis of cells, the uptake is stronger in an inflammatory state, and the bioavailability of the drug is significantly improved. The A5@ELNP has good DPPH and ABTS free radical scavenging performance, can reduce the levels of inflammation-related factors such as NO, IL-6 and TNF-alpha, and can relieve the degree of oxidative stress in cells. Through in-vivo biological activity evaluation experiments on an acute lung injury animal model, it is proved that the A5@ELNP provided in the application has excellent lung targeting in vivo, can effectively inhibit the aggravation of inflammatory infiltration in the lung, can regulate the changes of Ca 2+ and then affect the mitochondrial-related endoplasmic reticulum membrane homeostasis and the change of the mitochondrial membrane potential to play a role in treating acute lung injury.

[0023] The application provides a new strategy for improving the targeting and utilization of drugs, provides a new drug for treating acute lung injury, and has a wide application prospect and high application value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1Transmission electron microscopy (TEM) morphology of exosomes;

[0025] Figure 2 This is a particle size-particle distribution diagram of exosomes;

[0026] Figure 3 Image showing the identification results of exosome surface markers;

[0027] Figure 4 For DSPE-PEG 2000 -PAR's 1H NMR spectrum;

[0028] Figure 5 For DSPE-PEG 2000 Fourier infrared spectrum of -PAR;

[0029] Figure 6 Fluorescence spectrum of a cascaded targeted drug delivery system;

[0030] Figure 7 This is a diagram showing the laser confocal results of a cascaded targeted drug delivery system.

[0031] Figure 8 The graph shows the zeta potential detection results for Exo, A5@LNP, and A5@ELNP.

[0032] Figure 9 Transmission electron microscopy (TEM) morphology characterization of A5@LNP;

[0033] Figure 10 The image shows the negatively stained transmission electron microscopy morphology of A5@ELNP.

[0034] Figure 11 The UV absorption spectra of A5, A5@LNP, and A5@ELNP are shown.

[0035] Figure 12 Drug release curves for A5 and A5@ELNP;

[0036] Figure 13 The graph shows the results of DPPH and ABTS free radical scavenging rate detection.

[0037] Figure 14 This is a graph showing the results of a cytotoxicity assay.

[0038] Figure 15 Fluorescence pattern for cytotoxicity assay;

[0039] Figure 16 This is a graph showing the results of quantitative fluorescence analysis in a cytotoxicity assay.

[0040] Figure 17 The image shows the results of the cell uptake experiment.

[0041] Figure 18 This is a graph showing the results of in vitro anti-inflammatory activity assay.

[0042] Figure 19 The image shows the fluorescence pattern of DNA damage (A) and the results of its quantitative analysis (B).

[0043] Figure 20 This is a graph showing the results of flow cytometry detection of apoptosis.

[0044] Figure 21 Figure 1 shows the results of CAT activity, SOD activity and T-AOC detection in cells;

[0045] Figure 22 The images show the tissue distribution results of the drug at 0.25 h, 2 h, and 6 h.

[0046] Figure 23 This is a biodistribution map of the drug in vivo.

[0047] Figure 24 This image shows the distribution of the drug in different isolated organs and the results of quantitative analysis.

[0048] Figure 25 This is a distribution map of the drug in lung tissue;

[0049] Figure 26 Figure 1 shows the results of total cell count detection in bronchoalveolar lavage fluid of mice in each group.

[0050] Figure 27 The graph shows the detection results of TNF-α in the bronchoalveolar lavage fluid of mice in each group;

[0051] Figure 28 The graph shows the results of T-AOC, SOD activity, CAT activity and ROS production rate detection in each group of mice;

[0052] Figure 29 H&E pathological sections of lung tissue from each group of mice (numerous alveoli at the terminal branches of the bronchi at all levels in the lungs (red arrows); alveolar walls (black arrows); inflammatory cell infiltration (blue arrows) and necrotic cell debris (gray arrows) in the alveolar walls; granulocytes and lymphocytes exudated in the alveolar cavities (green arrows); necrotic bronchiolar epithelial cells (brown arrows); alveolar dilation (yellow arrows); perivascular erythrocytes (orange arrows)).

[0053] Figure 30 Immunohistochemical images of lung tissue from mice in each group;

[0054] Figure 31 Figure 1 shows the results of quantitative analysis of Arg-1, MPO and iNOS in the lung tissue of mice in each group by immunohistochemistry.

[0055] Figure 32The graph shows the results of routine blood tests (hemoglobin, lymphocyte count, neutrophil count, white blood cell count, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, mean corpuscular volume, coefficient of variation of red blood cell distribution width, number of monocytes, percentage of monocytes, mean platelet volume, and number of red blood cells) for each group of mice.

[0056] Figure 33 H&E pathological sections of major organs in mice from each group are shown in the images.

[0057] Figure 34 For the mitochondrial Ca of each group of mice 2+ Content detection results (image);

[0058] Figure 35 Ca2+ in the endoplasmic reticulum of mice in each group 2+ Content change detection results graph;

[0059] Figure 36 Figure 1 shows the results of detecting the rate of mitochondrial ROS production in mouse cells of each group.

[0060] Figure 37 The graph shows the results of mitochondrial membrane potential detection based on the JC-1 method.

[0061] Figure 38 The graph shows the results of mitochondrial membrane potential detection based on the Disc 3 (5) method;

[0062] Where *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant. Detailed Implementation

[0063] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0064] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0065] The technical concept of this invention is as follows:

[0066] Our research group previously independently developed a phosphodiesterase 4 (PDE4) inhibitor A5 (Li M, Li G, Liu Y, et al. Design, Synthesis, and Evaluation of Selective PDE4 Inhibitors for the Therapy of Pulmonary Injury[J]. Journal of Medicinal Chemistry, 2025(3):68.), which inhibits PDE4 at an IC50. 50 The value is 5.9 nM, with selectivity exceeding that of other subtypes by more than 200 times, and the risk of toxicity to hERG is very low (IC50). 50 >30 μM). Animal experiments using LPS-induced acute lung injury confirmed that A5 can inhibit lung inflammation, alleviate acute lung injury symptoms, and reduce pulmonary bronchial inflammatory infiltration and fibrosis. However, the inhibitor A5 faces the problems of insufficient targeting and low bioavailability compared to conventional therapeutic drugs. Based on this, the present invention conducted the following research.

[0067] The structural formula for A5 is as follows:

[0068] .

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0070] Example 1: Isolation, extraction and identification of exosomes

[0071] The main reagent materials involved in this embodiment are shown in Table 1.

[0072] Table 1 Main Reagent Materials

[0073]

[0074] 1. Isolation and extraction of exosomes

[0075] Adult adipose-derived mesenchymal stem cells (ADMSCs) were revived and cultured in complete human adipose-derived mesenchymal stem cell culture medium, with the medium changed every 3 days until the attached cells exhibited a spindle-shaped morphology. When the cell density reached approximately 90%, the cells were digested with trypsin. When approximately 70%-80% of the cells were nearly round, an appropriate amount of complete culture medium was added and the digestion was stopped by gentle agitation. The cell suspension was aspirated, centrifuged at room temperature, and the supernatant was discarded. The cells were resuspended in complete culture medium and gently pipetted, then passaged at a ratio of 1:3. Exosomes were isolated and extracted from ADMSCs after 3-8 passages.

[0076] When the adherence and confluence of passaged ADMSC cells reached 25%-50%, they were continuously cultured in exosome-specific mesenchymal stem cell complete culture medium until the cell confluence reached 80%. The supernatant was then collected. The supernatant was first centrifuged at low temperature for 5 min to remove cells, followed by centrifugation at 3000×g for 10 min to remove cell debris and other impurities, and then filtered through a 0.22 μm filter membrane. A 100 kDa ultrafiltration membrane was moistened with ultrapure water, pre-cooled at 4°C for 5 min, and the filtered supernatant was added to the tube and concentrated by centrifugation at 3000×g for 10 min. Following the instructions of the cell culture supernatant exosome extraction and purification kit, the concentrated cell supernatant was mixed with exosome extraction reagent (ECS reagent) at a ratio of 4:1, vortexed for 1 min, and then incubated at 4°C for 18 h. After standing, the mixture was centrifuged at 10000×g for 60 min at 4°C, the supernatant was discarded, and the mixture was centrifuged again at 10000×g for 2 min to remove any residual liquid. The precipitate was resuspended in 200 μL of 1× PBS, mixed well, and centrifuged at 12000×g for 2 min at 4°C. The supernatant was retained and transferred to the upper chamber of the EPF purification column, centrifuged at 3000×g for 10 min at 4°C, and the liquid at the bottom of the EPF column was collected. This liquid is the purified human adipose-derived mesenchymal stem cell exosomes (Exo).

[0077] 2. Identification of exosomes

[0078] 2.1 Protein concentration determination

[0079] The protein concentration of the exosome suspension was determined using a BCA protein concentration assay kit for later use.

[0080] 2.2 Morphological observation by electron microscopy (TEM)

[0081] Add 10 μL of ultrapure water to the center of the hydrophobic membrane, place the copper mesh face down (reflective side) on the ultrapure water for a few seconds, and remove excess liquid. Dilute the Exo suspension appropriately with PBS, add 10 μL of the diluted exosome suspension to the copper mesh for adsorption for 5 min, perform phosphotungstic acid negative staining, allow it to air dry, and observe and photograph it using a transmission electron microscope (TEM).

[0082] The results are as follows Figure 1 As shown, exosomes exhibit typical saucer-like ultrastructural features, with virtually no other impurities in the background, indicating high purity of the extracted exosomes. Because exosomes contain membrane proteins, lipids, or intraluminal substances (such as RNA) that tend to be distributed to one side, staining leads to differences in electron density, resulting in one side appearing brighter during TEM imaging.

[0083] 2.3 Particle size determination

[0084] Exo was diluted to a suitable concentration with PBS. Using a Nanosight NS300 instrument, a 60-second video recording was acquired at a frame rate of 30 frames per second under 405 nm excitation light. Finally, the particle motion trajectory was analyzed using NTA software to evaluate the size and concentration of exosome particles.

[0085] The results are as follows Figure 2 As shown, the extracted exosomes were mainly concentrated at a size of 139.5 nm, which is consistent with the typical exosome size range (50-150 nm). Preliminary TEM and NTA results indicate that the extracted exosomes were secreted by cells.

[0086] 2.4 Surface Marker Detection

[0087] Western blotting was used to detect the positive surface protein markers TSG101 and CD63, transmembrane protein, and the negative surface protein marker Calnexin.

[0088] The results are as follows Figure 3 As shown, two positive protein markers were successfully detected on the extracted exosomes: the 44 kDa solute protein TSG101 and the 26 kDa transmembrane protein CD63. Meanwhile, the negative protein marker, endoplasmic reticulum protein Calnexin, was not detected. This result confirms that the extracted component is indeed an exosome and further demonstrates its high purity, indicating that it was not contaminated by other proteins.

[0089] Example 2: Preparation of a cascaded targeted drug delivery system

[0090] 1. Peptide-modified lipids (DSPE-PEG) 2000 Synthesis of -PAR)

[0091] Accurately weigh 10 mg of the cell-penetrating peptide pardaxin peptide powder (PAR, sequence: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH, SEQ ID NO.1), and 10 mg of DSPE-PEG. 2000 -NHS was dissolved in 3 mL of anhydrous dimethylformamide (DMF), and N,N-diisopropylethylamine (DIPEA) was added. The mixture was stirred at 250 rpm for 2 h under nitrogen atmosphere in an ice bath in the dark, and the pH was adjusted to 7.4. A dialysis membrane (molecular weight cutoff: 3500 Da) was cut, and the reaction solution was poured into the membrane and sealed. The membrane was then dialyzed in 2 L of deionized water for 24 h. The solution was replaced with the same volume of deionized water, and dialyzed for another 24 h. The resulting solid, DSPE-PEG, was obtained as a white powder after freeze-drying. 2000 -PAR.

[0092] For DSPE-PEG 2000 The structure of -PAR was examined, and the results are as follows: Figure 4 As shown, DSPE-PEG 2000 The signal with a chemical shift of PAR in the δ 6.81 to 8.23 ​​ppm range clearly belongs to the characteristic peak of the amide bond; while the signal with a chemical shift in the δ 4.46 to 4.65 ppm range corresponds to the methylene signal peak adjacent to the amide bond in the PAR peptide. This result preliminarily indicates that the active ester on DSPE-PEG2000-NHS undergoes an amide reaction with the amino group on PAR, and DSPE-PEG 2000 -PAR was successfully synthesized.

[0093] Further Fourier transform infrared spectroscopy (FT-IR) analysis was performed on the raw materials and products, and the results are as follows: Figure 5 As shown, in the wavenumber range of 1680-1630 cm⁻¹ -1 The absorption band observed in the inner band corresponds to the carbonyl (C=O) stretching vibration of the amide I band; while at 1570-1510 cm⁻¹... -1 The absorption band formed within this range is the result of the coupling between the NH stretching vibration and the CN stretching vibration, i.e., the amide II band; in addition, the absorption band located at 1335-1200 cm⁻¹... -1 The absorption band is formed by the coupling of NH bending vibration and CN stretching vibration, i.e., the amide III band. This result further confirms the DSPE-PEG... 2000 Successful synthesis of PAR.

[0094] 2. Construction of a cascaded targeted drug delivery system

[0095] 2.1 Preparation of drug-loaded lipid nanoparticles

[0096] Lipid nanoparticles were prepared using a thin-film dispersion-extrusion method. 15 mg Dlin-MC3-DMA, 6 mg DSPC, 7 mg cholesterol (CHOL), and 4 mg DSPE-PEG were accurately weighed. 2000 -PAR and 25 mg DOTAP, along with 4 mg of phosphodiesterase 4 (PDE4) inhibitor A5, were added to a round-bottom flask containing 15 mL of anhydrous ethanol and chloroform (1:2). The mixture was sonicated at 37°C and 500W in a water bath until fully dissolved, then the organic solvent was removed by rotary evaporation under reduced pressure at 45°C. The flask was then placed in a vacuum drying oven and dried overnight at 50°C to remove as much residual organic solvent as possible. A suitable amount of deionized water was added for 40 min of hydration. The mixture was then sonicated for 15 min in an ultrasonic cell disruptor (60% power, 2 s sonication, 3 s pause) to disperse the system. The nanoparticles were then extruded sequentially through 400 nm, 200 nm, and 100 nm microporous membranes using a liposome manual extruder to obtain a drug-loaded lipid nanoparticle (A5@LNP) suspension. Impurities were removed by dialysis. Sucrose (final concentration 2% w / v) was added, and the mixture was freeze-dried to prepare a lyophilized formulation for later use.

[0097] Using the same preparation process, unmodified A5@NPs and unmodified A5@ENPs nanoparticles were prepared for subsequent comparative experiments.

[0098] 2.2 Synthesis of Cascaded Targeted Drug Delivery System

[0099] A fusion of exosomes and drug-loaded lipid nanoparticles was prepared using a membrane extrusion method. 0.2 mg of protein equivalent exosomes and 1 mg of A5@LNP were mixed to a final volume of 1 mL by vortexing and sonication (30% amplitude, 30 s sonication, 30 s pause, 4 sonication cycles). The exosome-A5@LNP mixture was incubated at 37 °C for 12 h, and then repeatedly extruded 10 times sequentially through 400 nm, 200 nm, and 100 nm polycarbonate membranes using a manual liposome extruder to obtain a cascaded targeted drug delivery system (A5@ELNP).

[0100] 3. Fusion validation of cascaded targeted drug delivery systems

[0101] 3.1 Fluorescence Resonance Energy Transfer (FRET)

[0102] Following the instructions of the Red Fluorescent Iodine (DiI) and Green Fluorescent Iodine (DiO) staining kits for cell membranes, respectively, lipid nanoparticles (A5@LNP) were labeled with DiO, and exosomes (Exo) were labeled with DiI, with both serving as controls. Fluorescently labeled fusion polymers A5@ELNP were prepared using the aforementioned methods. 500 μL of each of A5@ELNP and the control were placed in colorimetric tubes, and fluorescence emission spectra in the 470–600 nm wavelength range were scanned at 450 nm excitation to investigate the FRET effect of DiO and DiI fluorescence in the fusion polymers prepared by the film extrusion method.

[0103] The results are as follows Figure 6 As shown, when Exo is labeled with only DiI, a unique emission peak of DiI appears near 570 nm; when A5@LNP is labeled with only DiO, a separate emission peak of DiO is only observed around 510 nm. However, when Exo is fused with A5@LNP, fluorescence emission peaks of both DiO and DiI appear simultaneously. Specifically, the fluorescence signal peak of DiO-labeled A5@LNP around 510 nm is reduced, while the fluorescence signal peak of DiI-labeled Exo around 570 nm is increased. This clearly demonstrates the existence of the FRET effect, further confirming the successful fusion of Exo and A5@LNP.

[0104] 3.2 Observation of fusion degree using laser confocal microscopy

[0105] Take an appropriate amount of A5@ELNP solution labeled with DiO and DiI, and use a laser confocal microscope to further observe the degree of fusion of the two fluorophores in the fusion body.

[0106] The results are as follows Figure 7 As shown in the overlay image, orange-yellow fluorescence is observed, indicating that the green fluorescent A5@LNP has successfully fused with the red fluorescently labeled exosome.

[0107] 4. Characterization of cascaded targeted drug delivery systems

[0108] 4.1 Particle size and dispersion index

[0109] Take appropriate amounts of A5@LNP and A5@ELNP suspensions, dilute them to a certain concentration, and then sonicate them. Use a laser scattering particle size analyzer (Zetasizer Nano S90, Malvern) to determine the particle size and dispersion index (PDI) of each sample by dynamic light scattering (DLS).

[0110] 4.2 Determination of drug loading rate and encapsulation efficiency

[0111] Dissolve 1 mg of A5 in DMSO to prepare a 100 µg / mL stock solution, and pass it through a 200-800 cm⁻¹ filter.-1 A full-band ultraviolet spectroscopy scan was used to determine the wavelength corresponding to the maximum absorption peak, and a standard curve was plotted based on the absorbance values ​​of different concentrations of A5 at this wavelength. 1 mg of A5@LNP or A5@ELNP was dissolved in DMSO to form a suspension and then sonicated to break the emulsion. The absorbance value at the maximum absorption wavelength of A5 was measured using an ultraviolet spectrophotometer, and the drug content was calculated based on the standard curve. The drug loading rate (%DL) and encapsulation efficiency (%EE) were calculated using the following formulas:

[0112]

[0113] In the formula, W d1 W represents the amount of drug encapsulated in the drug-loaded nanoparticles. dt W represents the total amount of drug added to the formulation. T This refers to the mass of the total solids added to the formulation.

[0114] The results are shown in Table 2. The particle size of A5@ELNP was measured to be 110.00±7.00 nm, slightly larger than that of A5@LNP (107.33±1.53 nm). These results demonstrate the successful fusion between exosomes and drug-loaded lipid nanoparticles. During the re-extrusion molding process, the drug loading rate was significantly improved, jumping from 4.58%±0.01% to 8.00%±0.05%.

[0115] Table 2. Results of particle size, dispersion index, drug loading rate, and encapsulation efficiency.

[0116]

[0117] 4.3 Zeta potential

[0118] Exo suspension and appropriate amounts of A5@LNP and A5@ELNP suspensions that had been appropriately diluted and sonicated were taken, and their surface Zeta potentials were measured using a Zetasizer Nano ZS-90 laser scattering particle size analyzer. The system stabilization time of the samples was 60 s, the running time was 120 s, and multiple measurements were taken, with the average value being recorded.

[0119] The results are as follows Figure 8 As shown, the potential of the originally negatively charged Exo fused with the originally positively charged A5@LNP composed of cationic lipids changed significantly. The potential of the fusion product A5@ELNP was -6.25±10.09 mV, lower than that of A5@LNP (+54.37±9.13 mV), but higher than that of Exo (-22.67±6.01 mV), proving the fusion was successful.

[0120] 4.4 Transmission electron microscopy morphology analysis

[0121] After appropriate dilution of A5@LNP and A5@ELNP suspensions, the samples were sonicated for 3 min, and the morphological characteristics of each sample were observed by TEM.

[0122] The results are as follows Figure 9 and Figure 10 As shown, A5@ELNP exhibits a clear spherical morphology and a typical core-shell nanostructure. Compared to A5@LNP, A5@ELNP shows a significant film corona formation on its outer shell, a characteristic consistent with the typical behavior of fusion composites.

[0123] 4.5 Ultraviolet Spectroscopy Analysis

[0124] The UV spectrophotometer was used to measure the full-band UV spectra of A5, A5@LNP and A5@ELNP samples in the range of 200-800 nm to verify whether the small molecule drug A5 was successfully loaded into the nanoparticles.

[0125] The results are as follows Figure 11 As shown, the characteristic absorption peak of A5 is located at approximately 320 nm. In the UV-Vis absorption spectra of both A5@LNP and A5@ELNP, characteristic absorption peaks corresponding to A5 were observed. However, due to the presence of lipid components, the characteristic absorption peaks of A5 in A5@LNP and A5@ELNP shifted, a phenomenon known as the "redshift," with these peaks moving to around 380 nm.

[0126] In summary, the experimental results show that the small molecule compound A5 has been successfully loaded into nanoparticles.

[0127] 5. In vitro release

[0128] To simulate the drug release effect in vivo, appropriate amounts of A5 raw material and A5@ELNP containing an equivalent amount of drug were weighed, dissolved into 20 mL suspensions, and placed into dialysis bags with a molecular weight cutoff of 3500 Da. At 37°C, using 10 mL of PBS buffer at pH 7.4 as the medium, samples were taken at different time points (sampling times of 2, 4, 6, 8, 12, 24, 30, and 48 h) to determine the concentration of released drug and plot the drug release curve.

[0129] The results are as follows Figure 12 As shown, at the same time points, the drug release rate in A5@ELNP was significantly lower than that in free A5. This demonstrates that A5@ELNP has a significant sustained-release effect, effectively prolonging the drug's residence time in the body, thereby reducing the frequency of administration and improving patient compliance.

[0130] 6. Free radical scavenging ability test

[0131] 6.1 DPPH free radical scavenging ability

[0132] Weigh an appropriate amount of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) powder and prepare a 0.1 mM DPPH working solution using anhydrous ethanol as the solvent. Mix samples of different concentrations with the DPPH working solution at a 1:1 volume ratio as the sample assay group. Incubate at 37℃ in the dark for 30 min. Add 200 μL to a 96-well microplate and measure the absorbance at 517 nm using a microplate reader. Use 200 μL of each sample mixed with an equal volume of anhydrous ethanol as the sample control group. Use VC (20 μM) as the positive control and hydrocortisone (HC) as the drug control. Calculate the DPPH free radical scavenging rate using the following formula:

[0133] ;

[0134] In the formula, A0 is the absorbance of the DPPH working solution; A1 is the absorbance of the sample control group; and A2 is the absorbance of the sample test group.

[0135] 6.2 ABTS free radical scavenging

[0136] The ABTS free radical scavenging ability was determined according to the instructions of the ABTS free radical scavenging ability test kit (Solepro, catalog number: BC4775), with VC (20 μM) as a positive control and HC as a drug control.

[0137] The results are as follows Figure 13 As shown, compared with HC, a conventional drug for treating acute lung injury, high-dose A5@ELNP exhibited statistically significant differences in DPPH and ABTS free radical scavenging capabilities. Specifically, the DPPH scavenging ability of A5@ELNP showed a clear dose-dependent characteristic, with an antioxidant capacity of 83.06% at a concentration of 20 μM, comparable to the results of the positive control VC at the same dose. Furthermore, the free radical scavenging capacity of A5@ELNP was generally higher than that of A5@LNP at the same dose, indicating that A5@ELNP fused with exosomes significantly enhances both DPPH and ABTS antioxidant capabilities.

[0138] Example 3: Functional Validation of a Cascaded Targeted Drug Delivery System

[0139] The main reagent materials involved in this embodiment are shown in Table 3.

[0140] Table 3 Main Reagent Materials

[0141]

[0142] I. In vitro functional verification

[0143] 1. Biocompatibility testing

[0144] 1.1 Cytotoxicity Detection

[0145] With 5×10 3 Raw 264.7 cells were seeded into 96-well plates at a density of cells / well and cultured at 37°C with 5% CO2 until the density reached approximately 80%. The original culture medium was replaced with 100 μL of fresh medium containing different concentrations of the drug, and incubation was continued for 24 h under the same conditions. The experimental groups were as follows: blank control group, model group (lipopolysaccharide LPS, 1 μg / mL), positive control group (HC, 0.05 mg / mL, i.e., 140 μM), A5 group (4 μM, 10 μM, and 20 μM), A5@LNP group (4 μM, 10 μM, and 20 μM), and A5@ELNP group (4 μM, 10 μM, and 20 μM). Then, 10 μL of CCK-8 solution was added to each well, mixed well, and incubated at 37°C for 40 min. Cell viability was measured by the CCK-8 assay to evaluate the cytotoxicity of the drug.

[0146] The results are as follows Figure 14 As shown, LPS, as an inducer of inflammation in a cell model, can promote the proliferation of Raw264.7 cells to some extent. Meanwhile, the toxicity of A5 or A5@LNP increases with increasing doses, but the toxicity of A5@ELNP at the same concentration is lower. This indicates that the strategy of introducing exosomes to form fusion bodies effectively reduces the surface charge of the original particles, thereby reducing their potential toxic side effects.

[0147] 1.2 Live / Dead Cell Staining

[0148] With 5×10 3Raw 264.7 cells were seeded into 96-well plates at a density of cells / well and cultured at 37°C with 5% CO2 until the density reached approximately 80%. The original culture medium was replaced with 100 μL of fresh medium containing different concentrations of the drug, and incubation was continued for 24 h under the same conditions. The experimental groups were as follows: blank control group, model group (lipopolysaccharide LPS, 1 μg / mL), positive control drug hydrocortisone group (HC, 140 μM), A5 group (2 μM, 4 μM, 10 μM, and 20 μM), A5@LNP group (2 μM, 4 μM, 10 μM, and 20 μM), and A5@ELNP group (2 μM, 4 μM, 10 μM, and 20 μM). Live / dead cell staining was performed using the Calcein / PI cell viability and cytotoxicity assay kit to evaluate the biocompatibility of the samples. The number of live and dead cells was observed under a fluorescence microscope and quantitatively analyzed using ImageJ.

[0149] The results are as follows Figure 15 and Figure 16 As shown, the staining results of live / dead cells are consistent with the results of the CCK-8 experiment.

[0150] 2. Cell uptake experiment

[0151] Dissolve FITC-WGA powder in 5 μL of anhydrous DMSO to obtain a 5 mg / mL stock solution. Dilute the FITC-WGA stock solution with HBSS (phenol red-free) to a final concentration of 20 μg / mL in the FITC-WGA cell membrane probe working solution. Perform fluorescent labeling using DiI. Generate Raw 264.7 cells at a concentration of 5 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / dish in confocal dishes. When the cells adhered and grew to approximately 80% confluence, some cells were stimulated with lipopolysaccharide (LPS, 1 μg / mL) for 24 h, while the remaining cells continued normal culture. Simultaneously, different fluorescently labeled nanoparticles were added to each dish for 2 h. After removing the culture medium containing the fluorescent nanoparticles, the cells were washed with PBS, carefully scraped off in the same direction using a cell scraper, resuspended in 1 mL of PBS, filtered through a 40 μm cell filter, and analyzed using flow cytometry.

[0152] The results are as follows Figure 17As shown, the uptake levels, from lowest to highest, are A5@NPs < A5@ENPs < A5@LNP < A5@ELNP, indicating a trend of increasing uptake with increasing modification level. Compared to normal Raw 264.7 cells, LPS-induced inflammatory cells showed a significant increase in the uptake of the same drug. This result suggests that cell-penetrating peptides facilitate drug entry into cells, and exosome fusion further enhances cellular drug uptake. The A5@ELNP exhibits superior targeting performance against inflammatory cells.

[0153] 3. Anti-inflammatory activity assay

[0154] Raw 264.7 cells were seeded into cell culture plates, cell slides, or confocal cell culture dishes and incubated at 37°C with 5% CO2 until the cell density reached 80%. The experimental groups were as follows: blank control group, model group (LPS, 1 μg / mL), positive control group (HC, 0.05 mg / mL, i.e., 140 μM), A5 group (10 μM and 20 μM), A5@LNP group (10 μM and 20 μM), and A5@ELNP group (10 μM and 20 μM). After pretreatment with the respective drug for 1 h, cells in each group were stimulated with LPS (1 μg / mL) for 24 h. The blank control group received no treatment, while the model group cells were induced with 1 μg / mL LPS for 24 h. Cell supernatants were collected, centrifuged at 12000×g for 5 min at 4°C, and the levels of NO and the inflammatory factors IL-6 and TNF-α in the supernatant were measured.

[0155] The results are as follows Figure 18 As shown, compared with the normal control group, the levels of inflammation-related substances in the model group cells were significantly increased, indicating that the inflammation model was successfully established. After treatment with different drugs, the levels of NO, TNF-α, and IL-6 in each group showed varying degrees of reduction compared with the model group. The A5@ELNP group showed the most significant improvement, and under the same drug concentration conditions, the therapeutic effect of A5@ELNP was more obvious than that of A5@LNP.

[0156] 4. DNA damage detection

[0157] Cells were treated according to the method described in section "3. Anti-inflammatory activity detection", and the ability of different drugs to repair DNA damage was detected using a DNA damage detection kit via γ-H2AX immunofluorescence assay.

[0158] The results are as follows Figure 19As shown, almost no γ-H2AX fluorescent focal points were observed in the control group cells, indicating that the DNA was in an undamaged state. In contrast, the number of γ-H2AX focal points, a DNA damage marker, was significantly increased in the model group, indicating a significant increase in the degree of DNA damage. After treatment with A5@ELNP, the number of intracellular γ-H2AX fluorescent focal points was significantly reduced, with only a small amount remaining, indicating that A5@ELNP has a significant effect in alleviating DNA damage. A5 is a poorly water-soluble drug, and the organic cosolvents used in preparing its solution may cause some degree of damage to cells. At the same concentration, the fluorescence intensity of both A5@LNP and A5@ELNP was lower than that of free A5. This indicates that loading A5 into a nanomedicine delivery system with an internally hydrophobic and externally hydrophilic structure can not only significantly improve the solubility and bioavailability of A5, but also effectively reduce the amount of organic solvent used, thereby reducing potential cytotoxic side effects.

[0159] 5. Apoptosis

[0160] Cells were treated according to the method described in section "3. Anti-inflammatory activity detection". Apoptosis was detected using the Annexin V-FITC / PI double staining apoptosis detection kit and then analyzed by flow cytometry.

[0161] The results are as follows Figure 20 As shown, the apoptosis level in the model group was significantly increased, indicating that LPS can induce apoptosis in Raw 264.7 cells. After drug treatment, the number of apoptotic cells decreased in all groups, with the A5@ELNP group showing the most significant improvement, highlighting the potential of drug nanotechnology in enhancing efficacy.

[0162] 6. Antioxidant stress level

[0163] 6.1 Total Antioxidant Capacity (T-AOC) Detection

[0164] Cells were treated according to the method in section "3. Anti-inflammatory activity detection". The original culture medium in the pretreated cells was discarded. The cells were washed twice with PBS for 1 min each time. The T-AOC level was detected according to the instructions of the Total Antioxidant Capacity (T-AOC) Detection Kit.

[0165] 6.2 Detection of catalase (CAT) activity

[0166] Cells were treated according to the method described in section 3, "Anti-inflammatory Activity Detection," and CAT activity was detected according to the instructions of the Catalase (CAT) Activity Assay Kit.

[0167] 6.3 Detection of Superoxide Dismutase (SOD) Activity

[0168] Cells were treated according to the method described in section "3. Anti-inflammatory activity detection", and SOD activity was detected according to the instructions of the superoxide dismutase (SOD) activity detection kit.

[0169] The results are as follows Figure 21 As shown, all treatment groups enhanced the activity of two enzymes closely related to oxidative stress, CAT and SOD, and improved the total antioxidant capacity. The effect of A5@ELNP (concentration of 20 μM) was the most prominent.

[0170] II. In vivo functional verification

[0171] 1. Pharmacokinetics and Tissue Distribution

[0172] 1.1 Pharmacokinetic Analysis

[0173] A pharmacokinetic analysis methodology and drug standard curves were established. SPF-grade Balb / c mice (6-8 weeks old, weighing 18-22 g) were randomly divided into two groups of three mice each. After acclimatization for 7 days, A5 (1 mg / kg) or A5@ELNP (1 mg / kg, calculated as A5) was injected via the tail vein. Mice were fasted for 12 h before administration but allowed free access to water. Feeding resumed 4 h after administration. Blood samples of 150 μL were collected from the fundus venous plexus of the mice at 0 h before administration and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration. The collected samples were placed in anticoagulant tubes containing EDTA-K2 and centrifuged at 6800×g for 10 min at 4°C. The drug concentration in the plasma after centrifugation was determined by liquid chromatography-mass spectrometry (LC-MS) for pharmacokinetic analysis.

[0174] The results are shown in Table 4. The half-lives in the blood of A5 and A5@ELNP after intravenous injection were 6.45±3.11 h and 6.48±4.11 h, respectively. Although the half-life of the A5@ELNP group was slightly longer, the difference was not statistically significant. At the same dosage, there was a significant difference in the maximum detectable drug concentration in the serum of the two groups, with the concentration in the A5@ELNP group being much lower than that in the A5 group. This may be due to the targeting properties of A5@ELNP, which allows the drug to be specifically delivered to the lungs and "retained" by the pulmonary vascular network, resulting in a significant reduction in the drug concentration in the blood.

[0175] Table 4. Changes in pharmacokinetic parameters in Balb / c mouse serum over time (n=3)

[0176]

[0177] 1.2 Organizational Distribution

[0178] SPF-grade Balb / c mice were randomly divided into two groups of nine each. After a 7-day acclimatization period, the mice were treated according to the "1.1 Pharmacokinetic Analysis" procedure. At designated sampling points (0.25 h, 2 h, and 6 h), the mice were sacrificed, and five organs (heart, lung, liver, spleen, and kidney) were harvested, fixed, homogenized at low temperature, and centrifuged at 4°C and 13,000 rpm for 10 min. The supernatant was then collected, and the drug concentration in the homogenate supernatant was determined according to the established drug LC-MS methodology and standard curve.

[0179] The results are as follows Figure 22 As shown, the highest drug concentrations in lung tissue were observed in the A5@ELNP group at 0.25, 2, and 6 h, demonstrating its excellent lung targeting ability. At the same time points, the drug concentrations in lung tissue of the A5@ELNP group were also significantly higher than those of the A5 group. This result confirms the good targeting ability of the drug delivery system and strongly supports the idea that nanomedicine delivery systems can enable A5 to accumulate and remain highly in the lungs.

[0180] 1.3 In vivo imaging

[0181] Male Balb / c mice were randomly divided into four groups (DiD group, A5@LNP group, A5@ELNP group, and LPS+A5@ELNP group), with three mice in each group. After a 7-day acclimatization period, the mice were dehaired using a hair removal cream. The LPS+A5@ELNP group received intratracheal infusion of LPS to establish an acute lung injury model, while the other groups received no treatment. Mice in each group were injected with the corresponding fluorescently labeled drug via the tail vein. At different time points (0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours), the mice were anesthetized, and photographs were taken using a small animal in vivo imaging system. The fluorescence signal intensity was detected using a small animal in vivo optical imaging system.

[0182] The results are as follows Figure 23 As shown, compared with the free fluorescein DiD group, the fluorescently labeled A5@LNP group, A5@ELNP group, and LPS + A5@ELNP group all exhibited significant lung targeting, indicating that the drug can target the lungs regardless of the presence of lung inflammation. Two hours after administration, fluorescence began to gradually shift to and accumulate in the liver. Even after 24 hours, fluorescent signals could still be detected in the lungs, but the lung fluorescence intensity of the LPS + A5@ELNP group was weaker than that of the A5@LNP group and the A5@ELNP group.

[0183] 1.4 Fluorescence distribution of isolated organs

[0184] Based on the in vivo imaging results, mice were sacrificed at the time point with the highest targeting activity, and their brains, hearts, livers, spleens, lungs, and kidneys were harvested. A small animal in vivo optical imaging system was used to detect the distribution of each drug in different tissues. Next, the harvested mouse lungs were embedded in Tissue-Tek OCTs, and cryosections were performed at 8 μm using a cryostat. Cell nuclei were stained with DAPI. Drug distribution within the lung tissue sections was observed using a fluorescence microscope.

[0185] The results are as follows Figure 24 As shown, the lung fluorescence in the A5@ELNP group was more concentrated and less dispersed in other organs.

[0186] Finally, the lung tissue from each group was sectioned, counterstained with nuclear dye, and observed under a fluorescence microscope. The results are as follows: Figure 25 As shown, the drug can be distributed in various areas of the lungs, with the most concentrated distribution near the bronchi.

[0187] 2. Pharmacological efficacy experiment in an acute lung injury (ALI) model

[0188] SPF-grade Balb / c mice (6-8 weeks old, weighing 18-22 g) were randomly divided into 6 groups: a blank control group, a model group, a positive control group (hydrocortisone injection HC, 1.5 mg / kg), and a drug-treated group (A5, A5@LNP, A5@ELNP, all calculated as A5, 1.5 mg / kg), with 8 mice in each group. The mice were acclimatized for 7 days in an environment of approximately 20℃ and 60% humidity, and were fasted and deprived of water for 12 hours before modeling. Mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital at a dose of 0.2 mL / 10 g, and then 50 μL of LPS solution (4 mg / mL, dissolved in physiological saline, 10 mg / kg) was instilled intratracheally to establish an acute lung injury mouse model. Mice in the blank control group received an equal volume of physiological saline instilled intratracheally. Two hours after modeling, each drug was injected via the tail vein, while the blank control group and the model group were injected with the same volume of physiological saline. Twenty-four hours after modeling, the in vivo efficacy was evaluated, and the changes in mouse body weight before and after modeling were recorded.

[0189] 2.1 Determination of total cell count in bronchoalveolar lavage fluid

[0190] The bronchoalveolar lavage (left lung) was 1 mL of 0.9% saline via endotracheal intubation, and the bronchoalveolar lavage fluid was collected. 0.5 mL of the lavage fluid was centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was collected for protein content determination. The precipitate was resuspended in 3 mL of saline, centrifuged at 1500 rpm for 10 min at 4°C, and resuspended again. 45 μL of the cell suspension was pipetted onto a smear and fixed, and total cell count was performed under a 200x inverted microscope.

[0191] The results are as follows Figure 26 As shown, compared with the control group, the total number of cells in the bronchoalveolar lavage fluid of the model group increased significantly, indicating that the ALI model was successfully constructed. The cell count in each treatment group decreased, approaching that of the control group, and there was a statistically significant difference from the model group. The total number of cells in the A5@ELNP treatment group was the lowest compared with other treatment groups, indicating that it had the best efficacy.

[0192] 2.2 Cytokine Level Detection

[0193] The level of the pro-inflammatory factor TNF-α in the supernatant of bronchoalveolar lavage fluid was quantitatively detected using an ELISA kit.

[0194] The results are as follows Figure 27 As shown, with the successful construction of the ALI model, TNF-α levels were significantly upregulated. However, the A5@ELNP group effectively reduced TNF-α levels.

[0195] 2.3 Evaluation of Oxidative Stress Indicators

[0196] (1) T-AOC detection

[0197] Weigh 0.1 g of right lung lobe tissue, add 1 mL of pre-cooled extraction solution, add 2 grinding steel balls, and place in a low-temperature homogenizer (4℃, 60 Hz, homogenization for 40 s, 15 s intervals, 6 runs) to prepare lung tissue homogenate. Centrifuge at 4℃, 10000 rpm for 10 min, collect the supernatant, and perform T-AOC detection.

[0198] (2) CAT and SOD activity detection

[0199] Weigh 0.1 g of right lung lobe tissue, add 1 mL of pre-cooled 1×Lysis Buffer working solution, and prepare tissue homogenate using a low-temperature homogenizer. Centrifuge at 10000×g for 15 min at 4℃, carefully aspirate the supernatant, and place it on ice for analysis.

[0200] (3) ROS detection

[0201] Weigh 0.1 g of right lung lobe tissue, add 1 mL of Extraction Buffer and 10 µL of Reagent II, and prepare a tissue homogenate using a low-temperature homogenizer. Centrifuge at 4℃, 600×g for 5 min, and collect the supernatant. Centrifuge at 4℃, 11000×g for 10 min; the precipitate is the lung tissue mitochondria. Resuspend in 200 µL of Reagent I and determine the protein concentration. Detect the mitochondrial ROS production rate in each group of mouse samples according to the instructions of the mitochondrial reactive oxygen species (ROS) production rate assay kit.

[0202] The results are as followsFigure 28 As shown, after treatment with various drugs, there were increases in SOD and CAT activity, enhanced T-AOC levels, and decreased ROS content. The A5@ELNP treatment group showed the best results, demonstrating that the drug delivery system prepared in this invention can significantly improve drug bioavailability.

[0203] 2.4 H&E staining

[0204] Left lung tissue was taken, fixed with 4% paraformaldehyde tissue fixative, stained with hematoxylin and eosin (H&E), and the pathological sections were observed to evaluate the efficacy of drug treatment.

[0205] The results are as follows Figure 29 As shown, in the A5@ELNP treatment group, the lung tissue capsule was smooth; there were no obvious abnormalities in the structure of the bronchi at all levels; a small number of granulocytes were infiltrated in the alveolar walls (blue arrows), and there was mild thickening of the alveolar walls to a moderate extent; no obvious abnormalities were observed in the blood vessels. This demonstrates that A5@ELNP has a protective effect against lung injury.

[0206] 2.5 Immunohistochemistry

[0207] Immunohistochemistry was used to detect the expression of macrophage M1 marker iNOS, M2 marker Arg-1, and neutrophil infiltration marker MPO in lung tissue. Lung tissue sections were dewaxed and hydrated, and antigen retrieval was performed using a microwave thermal retrieval method. Endogenous peroxidase was blocked by incubation in 3% H2O2 solution for 30 min in the dark, followed by PBS shaking and washing. Serum blocking was performed at room temperature for 30 min, then removed. Diluted primary antibody was added, and the sections were incubated overnight at 4°C. The next day, primary antibody residue was removed by PBS shaking and washing, and the corresponding secondary antibody was added. The sections were co-incubated in the dark for 1 h, followed by washing. After DAB staining, cell nuclei were counterstained with hematoxylin, dehydrated with graded ethanol, mounted with neutral resin, and the distribution of positive signals was observed under an optical microscope. Semi-quantitative analysis was performed using ImageJ software.

[0208] The results are as follows Figure 30 and Figure 31 As shown, in mice treated with A5@ELNP, the expression levels of iNOS and MPO were significantly decreased, while the expression level of Arg-1 was significantly increased. This indicates that A5@ELNP may effectively reverse the inflammatory immune microenvironment in the lungs and significantly reduce the infiltration of neutrophils into inflamed lung tissue by inducing the transformation of M1 macrophages into M2 macrophages with anti-inflammatory properties.

[0209] 3. In vivo safety evaluation

[0210] 3.1 Complete blood count (CBC)

[0211] Blood was collected from mice using the fundus venous plexus sampling method, and anticoagulated whole blood was collected using EDTA anticoagulant tubes. After sampling, the blood was gently inverted several times to ensure thorough mixing with the anticoagulant before performing routine blood tests.

[0212] The results are as follows Figure 32 As shown, in mice treated with PBS, LPS, A5@LNP, or A5@ELNP, except for the A5@LNP treatment group where the platelet count was slightly below the normal reference range, all other routine blood analysis indicators remained stably within the normal range. This indicates that the drug dosage and modeling agent dosage used in this invention are at reasonable and safe levels.

[0213] 3.2 Analysis of H&E staining pathological sections of major organs (heart, liver, spleen, and kidneys)

[0214] The results are as follows Figure 33 As shown, no significant lesions or pathological changes were observed in the heart, liver, spleen, and kidneys of any group of mice. This indicates that the drug maintained good safety within the dosage range and predetermined treatment period of this invention.

[0215] III. Exploring the Mechanism of Cascaded Targeted Drug Delivery Systems in the Treatment of Acute Lung Injury

[0216] 1. Mitochondria and endoplasmic reticulum Ca 2+ Measurement

[0217] First, prepare a 1 mM 2-APB stock solution. Accurately weigh 200 mg of Pluronic® F-127 and dissolve it in anhydrous DMSO. Heat and stir until completely dissolved to prepare a 20% (w / v) stock solution. Dissolve 50 μg of Fluo-4 AM calcium ion fluorescent probe powder, Rhod-2 AM calcium ion fluorescent probe powder, and Mag-Fluo-4 AM endoplasmic reticulum calcium ion fluorescent probe powder in 22.79 μL, 22.24 μL, and 30.58 μL of anhydrous DMSO, respectively, to prepare stock solutions with a concentration of 2 mM for each. Then, take an appropriate amount of each calcium ion fluorescent probe stock solution and mix thoroughly with the 20% Pluronic® F-127 stock solution at a volume ratio of 1:1 to obtain a 1 mM stock solution.

[0218] Raw 264.7 cells were cultured at 5 × 10⁶ cells per dish. 5Cells were seeded at a density of [number] cells / dish in 35 mm glass-bottom confocal cell culture dishes. When cells adhered and reached approximately 60% confluence, they were pretreated for 1 h with 10 μM of the IP3R inhibitor 2-APB and 20 μM of A5@ELNP (as A5). Subsequently, cells were stimulated with 1 μg / mL LPS for 24 h. The blank control group received no intervention, while the model group cells were induced with 1 μg / mL LPS for 24 h. Finally, flow cytometry was used to measure the calcium content of each group. 2+ Changes in content.

[0219] 1.1 Mitochondrial Ca 2+ Measurement

[0220] Discard the original culture medium after pretreatment, wash twice with PBS, add 500 μL of serum-free cell culture medium diluted to 4 µmol / L Rhod-2 AM working solution, and incubate at room temperature in the dark for 30 min. Collect cells, wash twice with PBS, resuspend cells in 500 μL PBS, filter through a 40 μm cell sieve, and measure mitochondrial calcium by flow cytometry. 2+ level.

[0221] The results are as follows Figure 34 As shown, after treatment with A5@ELNP co-incubation with inflammatory cells, the intracellular mitochondrial Ca2+... 2+ The signal was also significantly attenuated. The results suggest that the nanoparticles may play a positive role in alleviating inflammatory responses by restoring the homeostasis of the mitochondrial-associated endoplasmic reticulum membrane (MAM).

[0222] 1.2 Endoplasmic reticulum Ca 2+ Measurement

[0223] Endoplasmic reticulum Ca2+ determination using Mag-Fluo-AM 2+ Levels. Pretreated cells were incubated with 4 µmol / L Mag-Fluo-AM working solution at 37°C for 30 min, then the dye working solution was removed, the cells were washed twice with PBS, and the cells were resuspended in 500 μL PBS for evaluation by flow cytometry.

[0224] The results are as follows Figure 35 As shown, after treatment with 2-APB or A5@ELNP, the Ca in the endoplasmic reticulum... 2+ The fluorescence signal intensity rebounded and was higher than that of the control group in terms of Ca. 2+ The fluorescence signal intensity indicates that 2-APB or A5@ELNP can inhibit the outflow of calcium ions from the endoplasmic reticulum. During inflammation, the intracellular calcium ion concentration increases, resulting in a higher calcium ion concentration in the endoplasmic reticulum of cells treated with 2-APB or A5@ELNP compared to the control group.

[0225] 2. Detection of intracellular mitochondrial ROS

[0226] The results are as follows Figure 36 As shown, abnormal intracellular calcium ion flow rapidly increased ROS levels. Treatment with 2-APB effectively prevented calcium transfer from the endoplasmic reticulum to the mitochondria, leading to a significant decrease in ROS levels. Similarly, A5@ELNP treatment showed a similar decreasing trend in ROS levels. Based on these findings, it is hypothesized that A5@ELNP can effectively scavenge ROS by restoring the dynamic balance of intracellular calcium ions, thereby achieving its anti-inflammatory effect.

[0227] 3. Detection of changes in mitochondrial membrane potential (MMP)

[0228] The levels of mitochondrial membrane potential (MMP) in cells were detected using two probes, JC-1 and Disc 3 (5). Following the instructions of the enhanced mitochondrial membrane potential assay kit, JC-1 (200×) stock solution was mixed with a dedicated staining buffer at a volume ratio of 1:20 to prepare the JC-1 staining working solution. The original culture medium in the pretreated cells was aspirated, the cells were washed twice with PBS, and 2 mL of JC-1 staining working solution was added to resuspend the cell pellet. The cells were incubated at 37°C for 20 min. Staining was terminated, the supernatant was removed after centrifugation, the cells were washed twice with JC-1 staining buffer, and the pellet was collected and resuspended in 500 μL of buffer for flow cytometry analysis. A 5 μM Disc 3 (5) working solution was prepared. Following the same method as JC-1, the collected cells were incubated with the Disc 3 (5) working solution at 37°C in the dark for 20 min, the dye was washed off, and the cells were resuspended for flow cytometry analysis.

[0229] The results are as follows Figure 37 and Figure 38As shown, under normal physiological conditions, mitochondria in the control group generally maintained an intact membrane potential. The fluorescent probe JC-1 formed J-type aggregates in the mitochondrial matrix through a polarity-dependent mechanism, exhibiting characteristic red fluorescent clusters (highly aggregated state characterizing normal membrane potential). Only a small number of cell populations showed mitochondrial membrane potential depolarization, causing the probe to be diffusely distributed in monomeric form, manifesting as a green fluorescent signal (JC-1 monomeric state indicating membrane potential depletion). Fluorescence heterogeneity analysis confirmed that the integrity of mitochondrial function dominated in the control group. However, when cells were stimulated by LPS, the mitochondrial membrane potential decreased rapidly, the red fluorescence significantly weakened, while the green fluorescence significantly increased, indicating that mitochondrial function was severely affected. In addition, this trend of decreasing mitochondrial membrane potential was effectively alleviated after treatment with 2-APB and A5@ELNP. Specifically, 2-APB could inhibit changes in mitochondrial membrane potential caused by endoplasmic reticulum stress, while A5@ELNP exhibited significant anti-inflammatory activity, helping to prevent further decrease in mitochondrial membrane potential. When mitochondrial membrane potential was detected using the Disc 3 (5) probe, the fluorescence signal intensity detected by flow cytometry increased when the mitochondrial membrane potential decreased. LPS-induced cell inflammation resulted in a significant increase in fluorescence signal intensity, reflecting the decrease in mitochondrial membrane potential. However, after cell treatment with 2-APB or A5@ELNP, the detected fluorescence signal weakened. This change not only indicates that the downward trend in mitochondrial membrane potential was effectively curbed, but also corroborates the results obtained using the JC-1 method, further confirming the pharmacological activities of 2-APB and A5@ELNP. These two substances exerted a positive influence on changes in mitochondrial membrane potential by inhibiting endoplasmic reticulum stress and exerting anti-inflammatory effects, respectively, thus providing effective experimental evidence for the treatment of inflammation-related diseases.

[0230] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a cascaded targeted drug delivery system, characterized in that, Includes the following steps: (1) Pardaxin peptide with DSPE-PEG 2000 - Co-incubation with NHS yields peptide-modified lipids; (2) The peptide-modified lipids, Dlin-MC3-DMA, DSPC, cholesterol, DOTAP and phosphodiesterase 4 inhibitor were mixed and dissolved, and drug-loaded lipid nanoparticles were prepared by thin film dispersion-extrusion method. (3) The drug-loaded lipid nanoparticles are co-incubated with exosomes by membrane extrusion to prepare the cascade targeted drug delivery system.

2. The preparation method according to claim 1, characterized in that, In step (1), the pardaxin peptide and the DSPE-PEG 2000 The mass ratio of -NHS is 1:

1.

3. The preparation method according to claim 1, characterized in that, In step (1), the co-incubation conditions are as follows: co-incubation at 250 rpm for 2 h in anhydrous dimethylformamide solvent under the protection of N,N-diisopropylethylamine and nitrogen.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the peptide-modified lipids, Dlin-MC3-DMA, DSPC, cholesterol and DOTAP is 4:15:6:7:

25.

5. The preparation method according to claim 1, characterized in that, In step (2), the conditions for mixing and dissolving are: in a mixed solvent of anhydrous ethanol: chloroform = 1:2, the mixture is sonicated in a water bath until dissolved, and then the organic solvent is removed.

6. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the drug-loaded lipid nanoparticles to exosomes is 1:0.

2.

7. The preparation method according to claim 1, characterized in that, In step (3), the co-incubation conditions are as follows: after mixing the drug-loaded lipid nanoparticles with exosomes, incubate at 37°C for 12 h.

8. The preparation method according to any one of claims 1-7, characterized in that, The phosphodiesterase 4 inhibitor is A5, and its structural formula is as follows: ； The exosomes are human adipose-derived mesenchymal stem cell exosomes.

9. A cascaded targeted drug delivery system prepared by the preparation method according to any one of claims 1-8.

10. The use of the cascade targeted drug delivery system as described in claim 9 in the preparation of a medicament for treating acute lung injury or an anti-inflammatory drug.

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