A drug delivery system targeting lung alveolar microenvironment redox and preparation method and application thereof

Abstract

The application discloses a drug delivery system targeting lung alveolus microenvironment oxidation and reduction as well as a preparation method and application thereof, and comprises nanoparticles self-assembled by amphiphilic GSH-(oxi)PEI-PCL polymers, wherein the nanoparticles load fat-soluble drugs or fluorescent dyes, GSH in the amphiphilic GSH-(oxi)PEI-PCL polymers is connected with (oxi)PEI-PCL through a disulfide bond, and the (oxi)PEI-PCL is PEI-PCL subjected to partial oxidation. The pharmacodynamic test result shows that the drug delivery system can realize passive targeting aggregation of drugs in a lesion and microenvironment oxidation and reduction sensitive distribution, meanwhile, the drug delivery system shows an antioxidation effect in a lesion microenvironment and intracellular, realizes more accurate drug distribution effect and in-vivo treatment effect, and provides a new drug route for treatment of lung diseases such as premature lung dysplasia, bacterial / viral pneumonia or burn inhalation lung injury.

Description

Technical Field

[0001] This invention belongs to the fields of chemistry and biomedical engineering, and specifically relates to a drug delivery system that targets the redox reaction of the alveolar microenvironment, its preparation method, and its application. Background Technology

[0002] Local intraalveolar administration of glucocorticoids has long been an important and widely used treatment for various lung diseases in newborns and adults. For example:

[0003] (1) The most fatal developmental malformation in premature infants is fetal lung malformation, with the most critical aspect being the malformation of alveolar cells, especially type II alveolar epithelial cells. Glucocorticoids can promote fetal lung development. However, due to the inability to effectively administer hormones locally to the fetal lungs, the main treatment currently involves prenatal injection of glucocorticoids into the mother, which then distributes to the fetus and subsequently to the epithelial cells in the fetal lungs. A secondary treatment is to attempt to administer glucocorticoids to the infant after birth.

[0004] However, most of these systemic injection methods cannot reach the epithelial cells of the fetal lungs, resulting in low treatment efficiency; moreover, most drugs are distributed in other organs such as the liver and kidneys of the pregnant woman and fetus, causing significant side effects and even inhibiting the development and immunity of the pregnant woman / fetus / infant.

[0005] (2) In cases of widespread bacterial / viral pneumonia, burns or chemical inhalation pneumonia, respiratory allergies, cough, excessive sputum, or even bed rest after major surgery, patients are given "Budesonide inhalation suspension" and similar glucocorticoids multiple times a day under ultrasonic nebulization to improve the pathological state of alveolar epithelium and protect and restore the patient's lung function.

[0006] However, in these common nebulized medication methods, liquid corticosteroids form uniform small droplets that can enter the airway and reach the alveoli with the respiratory airflow. However, under conventional nebulization conditions, liquid drugs generally form excessively small droplets, such as those <1μm in diameter. These droplets flow smoothly throughout the airway from the bronchi to the alveoli and are eventually exhaled, resulting in extremely low alveolar retention efficiency. Even if a small number of droplets can remain in the alveoli, they are unlikely to penetrate the mucus secreted within the alveoli. Even if a small amount of drug dissolves in the mucus and comes into contact with the respiratory epithelium, it is difficult to naturally penetrate the cell membrane and be delivered or internalized into the alveolar epithelial cells. Therefore, the cell-targeted delivery efficiency of this type of inhaled corticosteroid therapy is extremely low. Most drugs enter the environment with the exhaled airflow and fail to exert their effect; most drugs retained in the airway are expelled through the respiratory tract with sputum, which can lead to refractory complications such as fungal / bacterial infections of the mouth and throat and mucosal ulcers. When passing through the airway-gastrointestinal junction, they are swallowed into the digestive tract with respiratory secretions or sputum, producing the widespread systemic side effects of oral corticosteroids.

[0007] Insufficient targeted distribution of these glucocorticoids leads to poor treatment outcomes for lung diseases; their systemic distribution also results in widespread extrapulmonary side effects. These problems plague the treatment of a wide range of patients, including, but not limited to, respiratory diseases, and urgently need to be addressed clinically.

[0008] PEI-PCL is a block copolymer composed of poly(∈-caprolactone) and polyethyleneimine. PEI-PCL has a unique structure with a hydrophilic outer PEI layer and a hydrophobic inner PCL layer, maintaining stability in water or commonly used clinical solvents such as physiological saline. Furthermore, its hydrophobic inner PCL structure is particularly suitable for loading hydrophobic glucocorticoid drugs. However, while efficiently loading glucocorticoids, the high positive charge of the outer hydrophilic PEI layer, if used directly as a drug surface, would lead to significant toxicity to any living cells that may come into contact with it.

[0009] Therefore, providing a low-toxicity drug delivery system that targets diseased alveolar cells and promotes active endocytosis of drugs by the alveolar epithelium without exhalation through the airway is of great clinical significance for alveolar oxidative stress-related lesions such as preterm pulmonary hypoplasia, bacterial / viral pneumonia, or burn aspiration lung damage. Summary of the Invention

[0010] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a drug delivery system that targets the redox reaction of the alveolar microenvironment. This drug delivery system can effectively reduce in vivo toxicity, achieve passive targeted aggregation in lesions and redox-sensitive distribution in the microenvironment, and at the same time exhibit antioxidant effects in the lesion microenvironment and cells.

[0011] This invention is achieved through the following technical solution:

[0012] In a first aspect, the present invention provides a drug delivery system targeting the redox reaction of the alveolar microenvironment. The drug delivery system includes nanoparticles formed by the self-assembly of an amphiphilic GSH-(oxi)PEI-PCL polymer. The nanoparticles are loaded with a lipophilic drug or a fluorescent dye. In the amphiphilic GSH-(oxi)PEI-PCL polymer, GSH and (oxi)PEI-PCL are linked by disulfide bonds. The (oxi)PEI-PCL is a partially oxidized PEI-PCL.

[0013] In this invention, PEI-PCL is an amphiphilic block copolymer formed by crosslinking polyethyleneimine (PEI) and polycaprolactone (PCL), and its chemical structure is as follows, where x = 100-200 and y = 40-50:

[0014]

[0015] The invention described herein can be obtained either by self-production using conventional methods or by commercial purchase.

[0016] The present invention (oxi) PEI-PCL represents partially oxidized PEI-PCL, which is the chemical structure described above. The unit is oxidized to unit.

[0017] Preferably, the oxidation degree of the (oxi)PEI-PCL is 30% to 70% (for example, it can be 30%, 32%, 35%, 38%, 40%, 43%, 45%, 48%, 50%, 53%, 55%, 58%, 60%, 62%, 65%, 69% or any range between the above proportions).

[0018] The degree of oxidation of (oxi)PEI-PCL described in this invention represents the percentage of oxidized units out of the total number of units.

[0019] Based on the microenvironmental characteristics of increased oxidative stress in alveoli during alveolar inflammation or developmental abnormalities, this invention further selects glutathione (GSH), a negatively charged and most important physiological antioxidant in organisms, to modify PEI-PCL. GSH can neutralize the high positive charge of PEI, making it sensitive to the oxidative microenvironment. It can react with reactive oxygen species (ROS), simultaneously exerting an antioxidant effect and reducing ROS in the microenvironment. Furthermore, it detaches after the reaction, gradually exposing the positive charge of PEI.

[0020] The modified GSH-(oxi)PEI-PCL of this invention has a low surface positive potential, which ensures low cytotoxicity during distribution. However, GSH reduces the overall surface potential of the drug delivery system, resulting in extremely low transfection and drug delivery efficiency to oral, pharyngeal, and airway epithelial cells during distribution. Once GSH-(oxi)PEI-PCL enters the oxidative stress microenvironment of diseased alveoli, the outermost GSH reacts with ROS, gradually detaching while antagonizing local oxidative stress inflammation. This exposes PEI and its positive potential, gradually enhancing its electrical stimulation, transfection-promoting, and endocytosis-promoting effects on the surrounding alveolar cell membranes until successful endocytosis is stimulated in alveolar epithelial cells. After endocytosis, the GSH on the cell surface further exerts its antioxidant effect, PEI-PCL disintegrates, releasing the loaded glucocorticoid drug, thereby achieving targeted drug delivery of glucocorticoids to diseased alveolar cells.

[0021] Preferably, the surface potential of the drug delivery system is 12–24 mV (for example, it can be 12 mV, 13 mV, 14 mV, 15 mV, 17 mV, 19 mV, 20 mV, 22 mV, 24 mV or any range between the above values).

[0022] Preferably, the average particle size of the drug delivery system is 60-400 nm, more preferably 80-300 nm (e.g., 85 nm, 100 nm, 120 nm, 150 nm, 170 nm, 190 nm, 200 nm, 230 nm, 250 nm, 275 nm, 290 nm, 300 nm, or any range between the above values). Drug delivery carriers within this particle size range, after being processed by clinical and conventional ultrasonic nebulization equipment and conditions, can form droplets of 1-5 μm in size, facilitating their delivery to the airways and terminal alveoli, resulting in good distribution and contributing to further enhancing the redox-sensitive release effect within the microenvironment.

[0023] Preferably, the lipid-soluble drug is a glucocorticoid, such as dexamethasone or prednisone.

[0024] Secondly, the present invention also provides a method for preparing the above-mentioned drug delivery system targeting alveolar microenvironment redox reaction, comprising the following steps:

[0025] (1) PEI-PCL was partially oxidized to (oxi)PEI-PCL by H2O2 oxidation method;

[0026] (2) Thioglycolic acid was used to thiolate (oxi)PEI-PCL to obtain SH-(oxi)PEI-PCL;

[0027] (3) SH-(oxi)PEI-PCL was oxidized with GSH to obtain an amphiphilic GSH-(oxi)PEI-PCL polymer;

[0028] (4) Assemble amphiphilic GSH-(oxi)PEI-PCL polymer and lipid-soluble anti-inflammatory drugs into a drug delivery system that targets the redox reaction of the alveolar microenvironment.

[0029] In the process of partially oxidizing PEI-PCL to (oxi)PEI-PCL using the H2O2 oxidation method, this invention obtains (oxi)PEI-PCL with different degrees of oxidation by adjusting the amount of H2O2 added or controlling the reaction time.

[0030] Preferably, in step (1), the molar ratio of PEI-PCL to H2O2 is 1:8-15.

[0031] Thirdly, the present invention also provides the application of the above-mentioned drug delivery system targeting alveolar microenvironment redox in the preparation of drugs for the prevention or treatment of lung diseases.

[0032] The lung diseases described in this invention include, but are not limited to, premature pulmonary dysplasia, bacterial pneumonia, viral pneumonia, or burn aspiration lung damage.

[0033] The drug described in this invention is administered via nebulized inhalation. The particle size of the drug delivery system after loading the glucocorticoid drug is precisely controlled to ensure that the median aerodynamic particle size of the droplets formed after processing by conventional nebulized inhalation equipment remains stable at 1–5 μm. If the droplet size is <1 μm, the droplets flow smoothly throughout the airway from the bronchi to the alveoli and are eventually exhaled, resulting in extremely low alveolar retention efficiency. If the droplet size is >5 μm, the droplets are mainly retained in the main bronchus and rarely enter the airway terminals to reach the alveoli.

[0034] This invention first controls the average particle size of the drug delivery system to 60-400 nm, and then screens the ultrasonic atomization conditions of the drug solution to ensure that the atomized droplet particle size can be stably controlled between 1 and 5 μm. This is suitable for reaching the alveoli through the main bronchus, while avoiding the droplets being exhaled from the alveoli by the airflow due to being too small, thus effectively improving alveolar retention and alveolar distribution efficiency.

[0035] This invention provides a localized drug droplet administration via nebulization, which remains in the alveoli, avoiding the systemic side effects of intravenous glucocorticoids. After entering the diseased alveoli, the drug droplet achieves GSH-responsive anti-inflammatory effects in the oxidative stress microenvironment, and targeted internalization of the drug by alveolar epithelial cells after PEI positive charge exposure. This prevents the unphagocytized glucocorticoid drug from being expelled through the airway with respiratory secretions, thus avoiding refractory complications such as fungal / bacterial infections and mucosal ulcers in the oral cavity and pharynx. It also avoids the widespread systemic side effects of oral glucocorticoids caused by swallowing the drug with respiratory secretions or sputum at the airway-gastrointestinal junction.

[0036] Compared with the prior art, the present invention has the following technical effects:

[0037] This invention first obtains (oxi)PEI-PCL by moderately oxidizing PEI-PCL, which reduces its in vivo toxicity and excessively high surface positive potential, facilitating low-toxicity drug delivery and treatment. Furthermore, a reducing, negatively charged small molecule, GSH, is introduced onto the surface of the oxidized (oxi)PEI-PCL via disulfide bonds, further reducing the surface potential of the drug delivery carrier and thus effectively reducing in vivo toxicity. As the most important antioxidant group in vivo, GSH can detach in the inflammatory microenvironment under significant oxidative stress, revealing a positively charged core while simultaneously achieving microenvironment-targeted drug release and anti-oxidative therapy. After the drug is internalized by alveolar cells, the GSH remaining on the carrier and carried into the cells can exert an antioxidant effect within the alveolar cells.

[0038] Pharmacodynamic testing results show that the drug delivery system of this invention enables passive targeted accumulation of drugs within lesions and redox-sensitive distribution in the microenvironment, while exhibiting antioxidant effects in both the lesion microenvironment and cells, achieving a more precise drug distribution effect and in vivo therapeutic effect. This overcomes the problems of insufficient targeted delivery to diseased cells and side effects on organs and tissues outside the lesion when intravenous or inhaled glucocorticoids are needed in clinical situations involving alveolar oxidative stress-related lesions such as premature pulmonary dysplasia, bacterial pneumonia, viral pneumonia, or burn aspiration lung damage. It provides a new drug delivery route for the treatment of lung diseases such as premature pulmonary dysplasia, bacterial / viral pneumonia, or burn aspiration lung damage. Detailed Implementation

[0039] The present invention will be further illustrated below through specific embodiments. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments.

[0040] The main raw materials for this invention are sourced from the following sources:

[0041] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, Cat. 22572-40-3) Sigma-Aldrich N-hydroxysuccinimide (NHS, Cat. 6066-82-6) Sigma-Aldrich Polyethyleneimine-polycaprolactone (PEI-PCL, mW = 10 kDa) Qiyue Biotechnology Polyethyleneimine-polycaprolactone (PEI-PCL, mW = 15 kDa) Qiyue Biotechnology <![CDATA[Hydrogen peroxide (H2O2, Cat. 7722-84-1)]]> Sigma-Aldrich Thioglycolic acid (Cat. 68-11-1) Sigma-Aldrich Long-chain carbocyanine dye (DiR, Cat. 100068-60-8) Sigma-Aldrich Dexamethasone (Cat.50-02-2) Sigma-Aldrich Prednisone (Cat. 53-03-2) Sigma-Aldrich Glutathione (GSH, reduced form) (Cat. 70-18-8) Sigma-Aldrich <![CDATA[Chloroform (CHCl3) (Cat. No. 67-66-3)]]> Sigma-Aldrich <![CDATA[Ethyl alcohol (CH3CH2OH) (Cat. No. 64-17-5)]]> Sigma-Aldrich Tween 80 (Cat.9005-65-6) Sigma-Aldrich

[0042] Example 1:

[0043] Synthesis of S1 and (oxi)PEI-PCL

[0044] The PEI-PCL solution (10 mM) was prepared by dissolving 0.1 g of PEI-PCL in 1.0 mL of ultrapure water. 1 mL of PEI-PCL (10 kDa, 10 mM, pH = 9) and 1 mL of freshly prepared H₂O₂ (0.1 M) aqueous solution were mixed under vigorous stirring. The final solution (2 mL) was stirred at 35 °C and 1000 rpm for 1 h to form (oxi)PEI-PCL. Finally, the solution was filtered using a 3 kDa cutoff ultrafiltration tube to remove excess H₂O₂, and lyophilized to obtain a pale yellow solid (oxi)PEI-PCL.

[0045] Synthesis of S2 and SH-(oxi)PEI-PCL

[0046] Weigh 9.2 mg of mercaptoacetic acid (10 mM) and dissolve it in 10 mL of ultrapure water. Add excess EDC (44.5 mg, 15 mM) and NHS (17 mg, 15 mM) carboxyl activator and stir for 2 h to fully activate the carboxyl groups of mercaptoacetic acid. Then add 1 g of (oxi)PEI-PCL (10 mM) and stir at 35 °C for 24 h. The reaction solution is then dialyzed for two days in a dialysis bag with a molecular cutoff of 3.4 kDa in ultrapure water. After lyophilization, SH-(oxi)PEI-PCL is obtained and stored at 4 °C for later use.

[0047] Synthesis of S3 and GSH-(oxi)PEI-PCL

[0048] GSH-(oxi)PEI-PCL polymer is obtained by oxidizing the active thiol groups of SH-(oxi)PEI-PCL with the thiol groups on glutathione GSH. The specific procedure is as follows: Weigh SH-(oxi)PEI-PCL (5g, 0.5mmol) and mix it with glutathione (1.54g, 5mmol) in a 100mL round-bottom flask containing 20mL of aqueous solution. Stir and react at 23℃ for 18h. After the reaction, dialyze the product in pure water for 1 day using a dialysis bag (molecular cutoff: 0.5kDa) to remove unreacted GSH. The product is then lyophilized to obtain GSH-(oxi)PEI-PCL.

[0049] Synthesis of GSH-(oxi)PEI-PCL nanoparticles loaded with dexamethasone and fluorescent dye DiR (S4)

[0050] Nanoparticles loaded with a lipid-soluble fluorescent dye and dexamethasone were prepared by solvent evaporation as follows: Amphiphilic polymer GSH-(oxi)PEI-PCL (0.15 g, 1 mmol), DiR (50 mg, 50 μmol), and dexamethasone (8 mg, 20 μmol) were dissolved in 6 mL of a 1:2 mixture of chloroform and ethanol. The organic phase solution was then added dropwise to a 0.1% w / v Tween 80 aqueous solution while simultaneously ultrasonically dispersing and stirring at 60 W for 5 min. After stirring, the emulsion was placed in a rotary evaporator to evaporate most of the organic solvent under reduced pressure. Subsequently, the solution was placed in a dialysis bag (molecular cutoff: 3.4 kDa) and dialyzed in ultrapure water for 24 h to remove the remaining organic solvent and raw materials. The resulting nanoparticle suspension was centrifuged at 10,000 rpm for 15 min to collect the nanoparticles, finally yielding drug-loaded nanoparticles.

[0051] Example 2:

[0052] The difference from Example 1 is that the concentration of H2O2 used for PEI-PCL oxidation in S1 is 0.08M; and in S4, ultrasonic dispersion and stirring are performed at a power of 75W for 5 minutes.

[0053] Example 3:

[0054] The difference from Example 1 is that the molecular weight of PEI-PCL used in S1 is 15kDa; the concentration of H2O2 used for PEI-PCL oxidation in S1 is 0.15M; and in S4, ultrasonic dispersion and stirring are performed at a power of 45W for 5 minutes.

[0055] Example 4:

[0056] The difference from Example 1 is that the PEI-PCL used in S1 has a molecular weight of 15 kDa; and the loaded drug is prednisone.

[0057] Comparative Example 1:

[0058] Synthesis of S1 and (oxi)PEI-PCL

[0059] The PEI-PCL solution (10 mM) was prepared by dissolving 0.1 g of PEI-PCL in 1.0 mL of ultrapure water. 1 mL of PEI-PCL (10 kDa, 10 mM, pH = 9) and 1 mL of freshly prepared H₂O₂ (0.1 M) aqueous solution were mixed under vigorous stirring. The final solution (2 mL) was stirred at 35 °C and 1000 rpm for 1 h to form (oxi)PEI-PCL. Finally, the solution was filtered using a 3 kDa cutoff ultrafiltration tube to remove excess H₂O₂, and lyophilized to obtain a pale yellow solid (oxi)PEI-PCL.

[0060] Synthesis of (oxi)PEI-PCL nanoparticles loaded with S2, dexamethasone, and fluorescent dye DiR

[0061] Nanoparticles loaded with a lipid-soluble fluorescent dye and dexamethasone were prepared by solvent evaporation as follows: The amphiphilic polymer (oxi) PEI-PCL (0.1 g, 1 mmol), DiR (50 mg, 50 μmol), and dexamethasone (8 mg, 20 μmol) were dissolved in 6 mL of a 1:2 mixture of chloroform and ethanol. The organic phase solution was then added dropwise to a 0.1% w / v Tween 80 aqueous solution while simultaneously ultrasonically dispersing and stirring at 60 W for 5 min. After stirring, the emulsion was placed in a rotary evaporator to evaporate most of the organic solvent under reduced pressure. Subsequently, the solution was placed in a dialysis bag (molecular cutoff: 3.4 kDa) and dialyzed in ultrapure water for 24 h to remove the remaining organic solvent and raw materials. The resulting nanoparticle suspension was centrifuged at 10,000 rpm for 15 min to collect the nanoparticles, finally yielding drug-loaded nanoparticles.

[0062] Comparative Example 2:

[0063] Synthesis of S1 and SH-PEI-PCL

[0064] Weigh 9.2 mg of mercaptoacetic acid (10 mM) and dissolve it in 10 mL of ultrapure water. Add excess EDC (44.5 mg, 15 mM) and NHS (17 mg, 15 mM) carboxyl activator and stir for 2 h to fully activate the carboxyl groups of mercaptoacetic acid. Then add 1 mL of PEI-PCL (10 mM) and stir at 35 °C for 24 h. Subsequently, dialyze the reaction solution in ultrapure water for two days using a dialysis bag with a molecular cutoff of 3.4 kDa. Freeze-dry to obtain SH-PEI-PCL, store at 4 °C for later use.

[0065] Synthesis of S2 and GSH-PEI-PCL

[0066] GSH-PEI-PCL polymer is obtained by oxidizing the active thiol groups of SH-PEI-PCL with the thiol groups on glutathione GSH. The specific procedure is as follows: SH-(oxi)PEI-PCL (5g, 0.5mmol) and glutathione (1.54g, 5mmol) are stirred in a 100mL round-bottom flask containing 20mL of aqueous solution and reacted at 23°C for 18h. After the reaction, unreacted GSH is removed by dialyzing in pure water for 1 day using a dialysis bag (molecular cutoff: 0.5kDa). The product is then lyophilized to obtain GSH-PEI-PCL.

[0067] Synthesis of S3 GSH-PEI-PCL nanoparticles loaded with prednisolone and fluorescent groups

[0068] Nanoparticles loaded with a lipid-soluble fluorescent dye and dexamethasone were prepared by solvent evaporation as follows: Amphiphilic polymer GSH-PEI-PCL (0.15 g, 1 mmol), DiR (50 mg, 50 μmol), and dexamethasone (8 mg, 20 μmol) were dissolved in 6 mL of a 1:2 mixture of chloroform and ethanol. The organic phase solution was then added dropwise to a 0.1% w / v Tween 80 aqueous solution while simultaneously ultrasonically dispersing and stirring at 60 W for 5 min. After stirring, the emulsion was placed in a rotary evaporator to evaporate most of the organic solvent under reduced pressure. Subsequently, the solution was placed in a dialysis bag (molecular cutoff: 3.4 kDa) and dialyzed in ultrapure water for 24 h to remove the remaining organic solvent and raw materials. The resulting nanoparticle suspension was centrifuged at 10,000 rpm for 15 min to collect the nanoparticles, finally yielding drug-loaded nanoparticles.

[0069] Comparative Example 3:

[0070] The difference from Example 1 is that in S4, ultrasonic dispersion and stirring are performed at a power of 75W for 10 minutes.

[0071] Comparative Example 4:

[0072] The difference from Example 1 is that in S4, ultrasonic dispersion and stirring are performed at a power of 45W for 3 minutes.

[0073] Comparative Example 5:

[0074] The difference from Example 1 is that the concentration of H2O2 used for PEI-PCL oxidation in S1 is 0.05M.

[0075] Comparative Example 6:

[0076] The difference from Example 1 is that the concentration of H2O2 used for PEI-PCL oxidation in S1 is 0.3M.

[0077] I. Characterization of Drug Delivery Systems

[0078] Potential testing method: A Zeta potential analyzer (Brookhaven ZetaPALS) was used, with the temperature set at 25℃, and the electric field strength was tested in automatic mode (5-20V / cm) with at least 3 repetitions and the average value was taken.

[0079] Particle size testing method: The particle size of the sample was tested using a Zeta-Plus potentiometric particle size analyzer (Brooken Haven). The incident laser wavelength was λ = 532 nm, the incident angle was θ = 90°, and the temperature was 25℃. The average value of three measurements was taken.

[0080] The method for testing the oxidation degree of (oxi)PEI-PCL: PEI-PCL before and after oxidation was characterized using hydrogen atomic NMR spectroscopy. The number of PEI units and the number of unoxidized PEI units in (oxi)PEI-PCL were calculated by integrating the characteristic peaks. The characteristic peak of PCL near δ = 4.05 ppm was integrated and normalized to calculate the number of PCL units and further verified by estimating the number of PEI units through the integration ratio. For PEI-PCL before and after oxidation, the characteristic peak of PEI was taken from the hydrogen NMR spectrum near δ = 2.6-2.8 ppm, and the integration was calculated to obtain the total number of PEI units in PEI-PCL before oxidation and the number of unoxidized PEI units in (oxi)PEI-PCL after oxidation. The oxidation degree was calculated according to the following formula.

[0081]

[0082] Table 1

[0083] II. Effect Evaluation

[0084] A premature mouse model was established, and the mice were given spray treatment in a constant temperature incubator.

[0085] 1. Near-infrared in vivo fluorescence (NIRF) molecular imaging experiments to evaluate the respiratory tract-specific delivery function of drugs.

[0086] Model establishment: Healthy adult SPF-grade Sprague-Dawley (SD) rats (250-300g, purchased from Guangdong Provincial Medical Laboratory Animal Center) were mated in pairs (male to female ratio 2:1) during estrus. Females with vaginal plugs observed were marked as day 0 of gestation (D0). Premature rats were anesthetized and delivered via cesarean section at 20 days of gestation. Drugs for each group were resuspended in PBS and placed in a nebulizer. The treatment dose was controlled at 0.1 mg PCL equivalent per dose of nanomedicine. Premature rats were randomly assigned to groups for treatment and placed in a self-made oxygen chamber connected to the nebulizer, fed with suckling mice. The humidity in the oxygen chamber was maintained at 50-60%, and the temperature at 24-25℃, for 12 hours 24 hours a day and 12 hours a night. Free access to food and water was provided, and the cages were opened for 15 minutes daily for cleaning.

[0087] 2. To evaluate the efficiency of inhaled drug distribution in the alveoli and trachea, 1 hour after inhalation of the fluorescently labeled drug, the airway (including the trachea and left and right bronchi) was separated, and lung tissue from both sides of the left and right bronchi was collected and examined with a live fluorescence instrument to evaluate the fluorescence intensity in the alveoli and assess the targeted distribution efficacy in the alveoli.

[0088] 3. To evaluate the effect of inhaled drugs on promoting lung development, the body weight of premature rats and the wet weight of the lungs after removing non-lung tissue were measured 21 days after treatment. The lung coefficient was calculated as lung wet weight / body weight. The lung coefficient reflects lung development in rodents.

[0089] 4. To evaluate the ability of inhaled drugs to promote lung cell development and secretion of alveolar surfactant after maturation, the secretion levels of representative proteins of alveolar surfactant in the alveoli were measured. Premature rats in each group were anesthetized and fixed 21 days after treatment. The trachea was isolated from the neck, and after intubation, preheated saline was injected, which was then slowly injected into the lungs. After the rat lungs gradually swelled and turned pale, the perfusion fluid was slowly aspirated, repeated 5 times to obtain bronchoalveolar lavage fluid (BALF). The BALF was collected in centrifuge tubes, and the content of surfactant protein D (SP-D) was measured by ELISA strictly according to the kit instructions.

[0090] The test results of each embodiment and comparative example are shown in Table 2-3:

[0091] Table 2

[0092]

[0093]

[0094] Table 3

[0095]

[0096] As shown in Tables 2-3 above, the drug delivery system of Comparative Example 1 lacks GSH, resulting in a lack of corresponding oxidative stress-induced inflammatory alveolar microenvironment. This leads to insufficient targeted distribution efficiency in lung lesions, primarily in the alveoli. Drugs are expelled into the main airways via pulmonary secretions, resulting in insufficient drug distribution within the alveolar lesions. Furthermore, the lack of GSH results in a high surface potential and significant cytotoxicity. After drug phagocytosis into alveolar cells, the lack of GSH may reduce the intracellular antioxidant effect, thus decreasing the therapeutic effect or even causing toxicity. This is manifested in the following ways compared to the previous example: a significant decrease in total fluorescence intensity in lung tissue and a significant increase in total fluorescence intensity in the airways; poor treatment efficacy within the alveoli; significantly lower post-treatment body weight; significantly higher lung wet weight; significantly higher lung coefficient; and insufficient production and low content of SP-D, an important component of alveolar surfactant.

[0097] In Comparative Example 2, the positively charged PEI-PCL group in the drug delivery system was unoxidized, exhibiting a high surface potential and significant cytotoxicity, damaging alveolar cells. Its extremely high surface potential led to a tighter binding to the negatively charged alveolar cell membrane and stimulated endocytosis, resulting in strong alveolar cell phagocytosis of the drug. However, after entering the cells, it did not produce a good therapeutic effect. This is because unoxidized PEI-PCL produces high intracellular toxicity, antagonizing the therapeutic effect of the therapeutic drug, thus reducing the therapeutic effect and even causing further damage due to toxicity. Compared to the previous example, the enhanced alveolar phagocytosis resulted in a stronger distribution in lung tissue, with no significant difference in distribution within the airways, potentially limiting the therapeutic effect; post-treatment body weight was significantly lower, lung wet weight was significantly higher, lung coefficient was significantly higher, and the content of SP-D, an important component of alveolar surfactant, was lower.

[0098] Comparative Example 3's drug delivery system has a smaller particle size, which results in a significantly lower drug loading efficiency compared to the examples, leading to poorer therapeutic effects. This is manifested in the fact that, compared to the examples, there is no significant difference in drug distribution within lung tissue and airways, but the drug content is lower, potentially limiting the therapeutic effect; compared to the examples, post-treatment body weight is lower, lung wet weight is higher, lung coefficient is higher, and the content of SP-D, an important component of alveolar surfactant, is lower, indicating insufficient production of it by alveolar epithelial cells.

[0099] The drug delivery system in Comparative Example 4 had a larger particle size, which resulted in lower in vivo drug loading stability and leakage of the drug from the carrier after lyophilization and suspension, leading to poor therapeutic efficacy. The larger particle size also caused highly unstable droplets after nebulization, failing to effectively distribute the drug to the alveoli at the end of the airway; the uneven droplet size and even breakage resulted in a large amount of drug being exhaled into the airway, hindering effective distribution to the airways and alveoli. This manifested as a significant decrease in total fluorescence intensity in lung tissue and airway compared to the previous example; significantly lower post-treatment body weight, significantly higher lung wet weight, significantly higher lung coefficient, and a lower overall content of SP-D, a key component of alveolar surfactant, in the lavage fluid.

[0100] In Comparative Example 5, the positively charged PEI-PCL group in the drug delivery system underwent only mild oxidation, resulting in a higher surface potential than in the Example but lower than in the unoxidized Comparative Example 2. After GSH dissociates in the alveoli due to microenvironmental response, the carrier itself exhibits higher cytotoxicity than in the Example but lower than in Comparative Example 2, showing significant toxicity to alveolar cells. Its high surface potential allows for tight binding to the negatively charged alveolar cell membrane, thereby stimulating endocytosis. Drug phagocytosis into alveolar cells is highly efficient, but after entering the cells, incompletely oxidized PEI-PCL produces high intracellular toxicity, antagonizing the therapeutic effect of the therapeutic drug and reducing the overall therapeutic effect. This manifests as no significant difference in distribution within lung tissue and airways compared to the Example, but potentially limited therapeutic efficacy; lower post-treatment body weight, higher lung wet weight, higher lung coefficient, and lower content of SP-D, a key component of alveolar surfactant.

[0101] The excessively high oxidation level of the core in the drug delivery system of Comparative Example 6 resulted in an extremely low core potential. This property led to lower cytotoxicity of the core after GSH dissociated in response to the lung microenvironment. Conversely, this low-potential core structure caused it to stimulate the negatively charged alveolar endothelial cell membrane, resulting in lower drug endocytosis capacity. Once the drug entered the alveoli, its retention within alveolar cells was ultimately reduced. Moreover, the actual measured drug loading efficiency of this structure was significantly lower than that of the Example, failing to carry sufficient drug into the alveolar endothelial cells and thus failing to achieve the expected therapeutic effect. This manifested as lower distribution in lung tissue compared to the Example, with no significant difference in airway distribution, but a significantly limited therapeutic effect; lower post-treatment body weight, higher lung wet weight, higher lung coefficient, and lower levels of SP-D, an important component of surfactant secreted by alveolar epithelial cells.

[0102] In Examples 1-4, oxidized (oxi)PEI-PCL is used. This reduces its in vivo toxicity and lowers its excessively high surface positive potential, facilitating low-toxicity drug delivery and treatment. Furthermore, the surface of (oxi)PEI-PCL is modified by linking the most important antioxidant group, GSH, to the surface via disulfide bonds, further reducing the drug's surface potential. GSH can detach in the inflammatory microenvironment under significant oxidative stress, revealing a positively charged core while simultaneously providing anti-inflammatory effects. This achieves microenvironment-targeted drug release and microenvironmental antioxidant therapy. After the drug is endocytosed by cells, the residual GSH can exert an antioxidant effect within the cells. The preferred particle size range for this drug delivery system is 80–300 nm. Within this particle size range, the inhalable droplets formed are uniform and stable, with droplet size controllable between 1–5 μm, facilitating delivery to the airways and terminal alveoli, resulting in good distribution and further enhancing the redox-sensitive release effect within the microenvironment. The overall structure of the delivery system enables passive targeted accumulation of drugs within lesions and redox-sensitive distribution within the microenvironment, while also exhibiting antioxidant effects in both the lesion microenvironment and cells. These effects, combined with the loaded therapeutic drugs, achieve more precise drug distribution and in vivo therapeutic effects. Compared to the untreated premature rat group, treated rats showed a significant increase in body weight, a significant decrease in lung wet weight, and a significant decrease in lung coefficient; their overall growth trend was close to that of normal rats.

[0103] III. Toxicity Evaluation

[0104] 1. To assess the hepatotoxicity of inhaled drugs in animals, liver function markers were detected: After the treatment course, blood was collected from the tail vein of young rats to detect alanine aminotransferase (ALT). The instrument used was a Hitachi 7600 fully automated biochemical analyzer.

[0105] 2. To assess the toxicity of inhaled drugs to the oral mucosa of animals, the oral mucosa toxicity of premature rats was assessed 21 days after treatment: the severity of ulcers was evaluated according to the indicators reported in the literature [Specifications for the Preparation of Animal Models of Oral Ulcers (Draft) [J]. Chinese Materia Medica Pharmacology and Clinical Practice, 2017, 33(06):162-163.DOI:10.13412 / j.cnki.zyyl.2017.06.044.]: (1) redness and swelling of the ulcer mucosa; (2) ulceration of the ulcer mucosa; (3) yellowish-white pseudomembrane covering the ulcer surface; (4) central depression and neat raised edges; (5) swelling of the lips and drooling; (6) reduced food intake and weight loss. The above apparent indicators were quantified and scored into 3 levels: none, mild, and significant, with scores of 0, 1, and 2 respectively.

[0106] 3. To assess the toxicity of inhaled drugs to the lung tissue of animals, after 21 days of treatment, lung tissue from premature rats was fixed with paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and the structural and morphological changes of the lung tissue were observed under a light microscope. After HE staining, the pneumonia score of premature rats was assessed as follows: 0: no pathological manifestations; 1: scattered infiltration of single polymorphonuclea leukocytes (PMNs), with intact tissue structure; 2: scattered infiltration of a small number or small groups of PMNs, slight exudation, slight intravascular congestion or occasional hemorrhage; 3: extensive infiltration of large numbers of PMNs, which may locally aggregate into clusters, or infiltrate around the blood vessel walls, and even large numbers of PMNs are seen in the blood within the lumen; exudation and hemorrhage are seen in the alveolar cavities, with occasional congestion or focal atelectasis; 4: destruction of normal lung tissue structure, necrosis of lung parenchyma, visible PMN nuclear fragmentation, nuclear condensation or nuclear dissolution, and enhanced eosinophilic staining of the cytoplasm.

[0107] The results of each embodiment are shown in Table 4:

[0108] Table 4

[0109]

[0110]

[0111] As can be seen from the results of Examples 1-4 in Table 4, the drug delivery system prepared by the present invention did not cause any obvious toxic side effects on solid organs such as the liver and lungs, or the oral mucosa that inevitably comes into contact with the drug during inhalation.

Claims

1. A drug delivery system targeting the redox activity of the alveolar microenvironment, characterized in that, The drug delivery system comprises nanoparticles formed by the self-assembly of an amphiphilic GSH-(oxi)PEI-PCL polymer. The nanoparticles are loaded with a lipophilic drug or a fluorescent dye. In the amphiphilic GSH-(oxi)PEI-PCL polymer, GSH and (oxi)PEI-PCL are linked by disulfide bonds. The (oxi)PEI-PCL is partially oxidized PEI-PCL. The oxidation degree of the (oxi)PEI-PCL is 30% to 70%. The potential of the drug delivery system is 12 to 24 mV.

2. The drug delivery system targeting alveolar microenvironment redox according to claim 1, characterized in that, The average particle size of the drug delivery system is 60-400 nm.

3. The drug delivery system for targeting alveolar microenvironment redox according to claim 2, characterized in that, The average particle size of the drug delivery system is 80-300 nm.

4. The drug delivery system targeting alveolar microenvironment redox according to claim 1, characterized in that, The lipid-soluble drug is a glucocorticoid, selected from at least one of dexamethasone or prednisone.

5. A method for preparing a drug delivery system targeting alveolar microenvironment redox according to any one of claims 1-4, characterized in that, Includes the following steps: (1) PEI-PCL was partially oxidized to (oxi)PEI-PCL using the H2O2 oxidation method; (2) Thioglycolic acid was used to thiolate (oxi)PEI-PCL to obtain SH-(oxi)PEI-PCL; (3) The amphiphilic GSH-(oxi)PEI-PCL polymer was obtained by oxidizing SH-(oxi)PEI-PCL with GSH; (4) Assemble amphiphilic GSH-(oxi)PEI-PCL polymer and lipid-soluble drugs into a drug delivery system that targets the redox of the alveolar microenvironment.

6. The preparation method according to claim 5, characterized in that, In step (1), the molar ratio of PEI-PCL to H2O2 is 1:8-15.

7. The use of the drug delivery system targeting alveolar microenvironment redox according to any one of claims 1-4 in the preparation of medicaments for the prevention or treatment of lung diseases.

8. The application according to claim 7, characterized in that, The lung diseases mentioned include premature pulmonary dysplasia, bacterial pneumonia, viral pneumonia, or burn aspiration lung damage.

9. The application according to claim 7, characterized in that, The drug is administered via nebulized inhalation, and the median aerodynamic particle size of the droplets during nebulized inhalation is 1-5 μm.

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