Drug delivery system for targeting in-alveolar stem cells as well as preparation method and application of drug delivery system

Abstract

The invention discloses a drug delivery system for targeting stem cells in pulmonary alveoli as well as a preparation method and application of the drug delivery system. The drug delivery system comprises nano-liposome vesicles, PEG-angiotensin converting enzyme II substrate polypeptide with a targeting function and pulmonary alveolar surface active substances, the PEG-angiotensin converting enzyme II substrate polypeptide with the targeting function is embedded into a membrane material of the nano-liposome vesicle, and the alveolar surface active substance is wrapped in the nano-liposome vesicle. According to the delivery system, efficient targeted disintegration in the pulmonary alveoli can be achieved, the pulmonary alveolar surface active substances contained in the vesicles can be generally and uniformly distributed in the pulmonary alveoli, and the treatment effect on the pulmonary dysplasia of the premature infant is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the fields of chemistry and biomedical engineering, and particularly relates to a drug delivery system targeting alveolar stem cells, and a preparation method and application thereof. Background Art

[0002] Pulmonary hypoplasia in premature infants is a common complication of premature birth. Its main pathological manifestation is insufficient alveolar surfactant, which leads to insufficient ventilation and even asphyxiation in premature infants. Pulmonary surfactant is a bioactive substance whose function is to maintain the surface tension of the alveoli and prevent alveolar collapse and fluid accumulation. In addition, the lungs and bronchi of premature infants are not fully developed. Their bronchi and blood vessels are smaller and more fragile, and there is less lung tissue, which leads to limited gas exchange and respiratory function. Moreover, insufficient alveolar surfactant in premature infants leads to insufficient alveolar tension and stability, resulting in breathing difficulties and impaired lung function. This makes premature infants more susceptible to respiratory diseases such as pneumonia and respiratory distress syndrome.

[0003] Currently, the most common treatment methods in clinical practice are: (1) Before birth, pregnant women receive a small dose of intramuscular injection of "glucocorticoids" to target the fetal lungs and promote lung development, but this is not recommended after birth. This type of drug has many side effects on pregnant women and fetuses; (2) After birth, animal (pig, cow) or synthetic pulmonary surfactant (PS) is dripped through an invasively inserted infant tracheal tube. This requires specially qualified personnel to establish an intratracheal channel before liquid dripping can be performed. This medical operation is difficult, technically demanding, and dangerous. The rate of severe complications such as choking, asphyxiation, and atelectasis is greater than 33%. This method drips into the main bronchus, and the distribution in the terminal alveoli cannot be guaranteed. Moreover, the drug in the droplet is coughed out of the airway, resulting in poor efficacy. Some alveoli that the droplet cannot reach cannot be treated and still present a state of "segmental atelectasis". The current guidelines, considering the poor tolerance of the fragile airway of premature infants and the extremely high probability of complications, recommend a maximum of two applications. It is not possible to continue and repeatedly treat during the developmental period according to the patient's condition. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a drug delivery system targeting alveolar stem cells. This drug delivery system can achieve efficient targeted disintegration drug release in the alveoli under a non-invasive inhalation administration method. The alveolar surfactant contained in the vesicles can be uniformly distributed in the alveoli, significantly improving the treatment effect of premature infants' pulmonary dysplasia while reducing iatrogenic trauma to the respiratory tract of premature infants.

[0005] The present invention is achieved through the following technical solutions:

[0006] A drug delivery system targeting alveolar stem cells comprises nanoliposome vesicles, a PEG-angiotensin converting enzyme II substrate polypeptide with a targeting function, and an alveolar surfactant. The PEG-angiotensin converting enzyme II substrate polypeptide with a targeting function is embedded in the membrane material of the nanoliposome vesicles, the alveolar surfactant is wrapped inside the nanoliposome vesicles, and the sequence of the angiotensin converting enzyme II substrate polypeptide is selected from Mca-YVADAP-Lys(Dnp)-OH, Mca-Ala-Pro-Lys(Dnp)-OH, or other similar functional sequences.

[0007] The alveolar stem cells described in the present invention are type II alveolar epithelial cells (AEC2), which are resident stem cells of the alveoli. They have angiotensin-converting enzyme 2 (ACE2) on their cell membrane surface and can secrete alveolar surfactant PS (composed of phospholipids, pulmonary surfactant protein (SP)-A / B / C / D, etc.).

[0008] The membrane material of the nanoliposome vesicles of the present invention is composed of cholesterol and phospholipids. The phospholipid is selected from at least one of dipalmitoylphosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), phosphatidylcholine (1,2-diacyl-sn-glycero-3-phosphocholine, PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), with dipalmitoylphosphatidylcholine (DPPC) being more preferred. DPPC is a natural, major component of pulmonary surfactant (PS). Using DPPC as the membrane material can further supplement PS, which is urgently needed for pulmonary hypoplasia.

[0009] In the drug delivery system of the present invention, based on the total molar amount of the membrane material of the nanoliposome vesicle, the chimeric amount of the PEG-angiotensin converting enzyme II substrate polypeptide is 5-20 mol%, preferably 10-15 mol%.

[0010] ACE2 enzymes are evenly distributed on the epithelial cell membranes in the alveoli. The present invention uses mPEG-NHS as the polyethylene glycol end and angiotensin converting enzyme II (ACE2) polypeptide substrate (ACE2pep1) as the sensitive response end to synthesize a PEG-angiotensin converting enzyme II substrate polypeptide with targeting function, which is embedded into the membrane material of the nanoliposome vesicle. When the nanoliposome vesicles reach the alveoli, under the action of the ACE2 enzyme evenly distributed on the epithelial cell membranes in the alveoli, the PEG-angiotensin converting enzyme II substrate polypeptide is degraded, resulting in efficient disintegration of the liposomes and uniform release of alveolar surfactant in the alveoli, thereby achieving the purpose of efficient treatment. PEG enhances the stability of the liposomes, allowing the nanoliposome vesicles to disintegrate in larger quantities in the alveoli at the enzyme-responsive site, while remaining stable in other lung environments.

[0011] The alveolar surfactants described in the present invention include bovine lung surfactant (such as Colisol) or porcine lung phospholipid injection (Coulso) and other drugs with alveolar surfactant functions.

[0012] The invention loads the natural medicine bovine lung or porcine lung surfactant in the delivery system, and after targeted release in the alveoli, it has a significant therapeutic effect on premature infants with pulmonary hypoplasia.

[0013] The particle size of the delivery system of the present invention is 50-1000 nm, preferably 300-500 nm.

[0014] The present invention also provides a method for preparing the above-mentioned drug delivery system targeting alveolar stem cells, comprising the following steps:

[0015] (1) Angiotensin-converting enzyme II polypeptide substrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 4-(dimethylamino)pyridine were added to an acetonitrile aqueous solution and magnetically stirred in an ice-water bath under N2 protection to activate the polypeptide carboxyl group;

[0016] (2) reacting the activated angiotensin-converting enzyme II polypeptide substrate with mPEG-NHS to obtain a PEG-angiotensin-converting enzyme II substrate polypeptide (PEG-ACE2pep) with targeting function;

[0017] (3) PEG-angiotensin converting enzyme II substrate polypeptide, cholesterol, and phospholipid are dissolved in dichloromethane, and then alveolar surfactant is added. The solvent is removed by rotary evaporation to form a composite liposome membrane;

[0018] (4) An aqueous medium is added to the composite liposome membrane and ultrasonically dispersed to obtain a drug delivery system targeting alveolar stem cells.

[0019] Preferably, in step (3), the amount of the PEG-ACE II substrate polypeptide added is 5-20 mol%, preferably 10-15 mol%, of the total molar amount of cholesterol and phospholipid.

[0020] The present invention also provides the use of the above-mentioned drug delivery system targeting intra-alveolar stem cells in the preparation of a drug for treating premature infants with pulmonary hypoplasia, wherein the drug is administered by atomization inhalation, and the droplet size after atomization is 1 μm to 5 μm. When the droplet size is less than 1 μm, even if the droplets are inhaled into the alveoli, they are exhaled with the breath and cannot be retained in the alveoli. When the droplet size is greater than 5 μm, the droplets are mainly retained in the main bronchi and it is difficult to enter the alveoli at the end of the airway. The present invention precisely controls the particle size of the vesicles in the droplets to 50 to 1000 nm, so that the droplet size after atomization is 1 μm to 5 μm, which is suitable for reaching the alveoli, and avoids the droplets being too large or too small to reduce the efficiency of alveolar inhalation distribution.

[0021] Compared with the prior art, the present invention has the following technical effects:

[0022] The present invention uses liposome vesicles to load alveolar surfactant, which can achieve non-invasive inhalation drug delivery without relying on endotracheal intubation, thus avoiding invasive operations and complications.

[0023] After the nanoliposome vesicles in the delivery system of the present invention reach the alveoli, they come into contact with the ACE2 enzyme evenly distributed on the epithelial cell membrane in the alveoli. Under the action of the enzyme, the PEG-angiotensin-converting enzyme II substrate polypeptide is degraded, thereby achieving efficient disintegration of the liposomes, preventing the indisintegrable liposomes from accumulating on the surface of the respiratory epithelium, blocking the "gas-liquid" barrier, and thus blocking the normal oxygen / carbon dioxide exchange of the alveoli. It also prevents the liposome vesicles that cannot disintegrate immediately from carrying drugs and transfecting into the respiratory epithelial cells, causing potential toxicity.

[0024] The nanoliposome vesicles of the present invention can efficiently disintegrate on the surface of the alveoli expressing angiotensin-converting enzyme II in the lungs and be evenly distributed in the alveoli of the entire lung. The alveolar surfactant contained in the vesicles can achieve universal and uniform distribution of the alveoli, avoid segmental atelectasis, and significantly improve the therapeutic effect on premature infants with pulmonary dysplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the structure of the drug delivery system targeting alveolar stem cells prepared in Example 1. DETAILED DESCRIPTION

[0026] The present invention will be further described below by way of specific embodiments. The following examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples.

[0027] The main raw material sources of the present invention are as follows:

[0028]

[0029]

[0030] Example 1:

[0031] 0.05 mmol of angiotensin-converting enzyme II (ACE2) peptide substrate 1 (ACE2pep1), with the sequence Mca-YVADAP-Lys(Dnp)-OH (synthetic peptide substrate 1 for ACE-2 enzyme) and a molecular weight of 1145, was dissolved with 5 mmol of EDC and 5 mmol of DMAP in 10 mL of acetonitrile-water solution (acetonitrile:water = 1:1). Under nitrogen protection, the mixture was stirred magnetically at 500 rpm in an ice-water bath for 2 hours to activate the peptide carboxyl groups. After 2 hours, 0.5 mmol of mPEG-NHS was added, and the reaction was continued for 72 hours. After completion of the reaction, the reaction solution was placed in a dialysis bag (MWCO = 3.5 kDa), dialyzed for 72 hours, and freeze-dried to obtain the product PEG-(ACE2pep1).

[0032] PEG-(ACE2pep1) 45 mg (7.5 μmol), cholesterol 10 mg (25 μmol) and dipalmitoylphosphatidylcholine (DPPC) 20 mg (25 μmol) were dissolved in 5 mL (25 μmol) of dichloromethane. 0.2 mL of 35 mg / ml bovine lung surfactant (PS) for injection (Colisu) was slowly added dropwise in a rotary evaporation vessel and dissolved in dichloromethane. Vacuum rotary evaporation was continued to evaporate the solvent to dryness to form a composite liposome membrane.

[0033] Subsequently, PBS solution (2 mL) was added dropwise at a rate of 0.5 mL / min to the liposome membrane formed by the above-mentioned PEG-(ACE2pep1), DPPC, cholesterol, and injectable PS. After the addition was completed, ultrasonic treatment was carried out for 10 minutes at a frequency of 45 W ultrasonic power and 2 seconds on and off. Then, physiological saline (0.9% NaCl solution) was added to dissolve and disperse the mixture, and the volume was adjusted to 2 mL. Subsequently, a liposome extruder was assembled, and the above-mentioned suspension was added. It was first extruded through a polycarbonate membrane with a pore size of 1000 nm, followed by a polycarbonate membrane with a pore size of 500 nm, to prepare the delivery system vesicles.

[0034] Figure 1 The schematic diagram of the structure of the prepared delivery system is shown. Figure 1It can be seen that the delivery system of the present invention includes nanoliposome vesicles, PEG-angiotensin converting enzyme II substrate polypeptide with targeting function and alveolar surfactant. The PEG-angiotensin converting enzyme II substrate polypeptide with targeting function is embedded in the membrane material of the nanoliposome vesicles, and the alveolar surfactant is wrapped inside the nanoliposome vesicles.

[0035] Example 2:

[0036] Dipalmitoylphosphatidylcholine (DPPC) was replaced by phosphatidylethanolamine (PE), and the pore sizes of the polycarbonate membrane used for liposome extrusion in the final step were 1000 nm, 500 nm, and 50 nm, respectively. The rest was the same as in Example 1.

[0037] Example 3:

[0038] The artificial synthetic polypeptide substrate 1 of ACE-2 enzyme was replaced with the artificial synthetic polypeptide substrate 2 of ACE2 enzyme (ACE2pep2), whose sequence was Mca-Ala-Pro-Lys(Dnp)-OH and molecular weight was 696.7, and PEG-(ACE2pep2) was synthesized. The rest was the same as in Example 1.

[0039] Example 4:

[0040] Dipalmitoylphosphatidylcholine (DPPC) was replaced by phosphatidylcholine (PC), the pore size of the polycarbonate membrane used for liposome extrusion in the last step was 1000 nm, and the rest was the same as in Example 1.

[0041] Example 5:

[0042] The molar amount of PEG-ACE II substrate polypeptide in the liposome membrane was adjusted to 10 mol %. The pore sizes of the polycarbonate membrane extruded from the liposomes in the final step were 1000 nm, 500 nm and 120 nm. The rest was the same as in Example 1.

[0043] Example 6:

[0044] Dipalmitoylphosphatidylcholine (DPPC) was replaced by phosphatidylserine (PS), and the rest was the same as in Example 1.

[0045] Comparative Example 1:

[0046] The artificial synthetic polypeptide substrate 1 of ACE-2 enzyme was replaced with the recombinant protein of ACE2 enzyme to synthesize PEG-ACE2, and the rest was the same as in Example 1.

[0047] Comparative Example 2:

[0048] The amount of PEG-angiotensin converting enzyme II substrate polypeptide added was reduced, and the molar amount of PEG-angiotensin converting enzyme II substrate polypeptide in the liposome membrane was adjusted to 1 mol %. The rest was the same as in Example 1.

[0049] Comparative Example 3:

[0050] The artificial synthetic substrate 1 of ACE-2 enzyme was replaced with the viral spike protein RBD segment (SARS-CoV-2 Spike RBD) to synthesize PEG-RBD, and the rest was the same as in Example 1.

[0051] Comparative Example 4:

[0052] The PEG-angiotensin converting enzyme II substrate polypeptide was not added, and the rest was the same as in Example 1.

[0053] Comparative Example 5:

[0054] The artificial synthetic polypeptide substrate 1 of ACE-2 enzyme was replaced with surface anti-SP-A antibody to synthesize PEG-anti (SP-A), and the rest was the same as in Example 1.

[0055] 1. Determination of particle size

[0056] The average particle size of the sample was measured using a Zeta-Plus potentiometric particle size analyzer (Brooken Haven) at an incident laser wavelength of λ = 532 nm, an incident angle of θ = 90°, and a temperature of 25° C. The measurement results are shown in Table 1.

[0057] Table 1: Examples and Comparative Examples and Particle Size Detection

[0058] Grouping membrane materials The part embedded in the membrane Final particle size of product (nm) Example 1 Cholesterol-DPPC PEG-(ACE2pep1) 426.1 Example 2 Cholesterol-PE PEG-(ACE2pep1) 56.5 Example 3 Cholesterol-DPPC PEG-(ACE2pep2) 427.2 Example 4 Cholesterol-PC PEG-(ACE2pep1) 898.7 Example 5 Cholesterol-DPPC PEG-(ACE2pep1) 126.0 Example 6 Cholesterol-PS PEG-(ACE2pep1) 935.3 Comparative Example 1 Cholesterol-DPPC PEG-ACE2 423.2 Comparative Example 2 Cholesterol-DPPC PEG-(ACE2pep1) 427.4 Comparative Example 3 Cholesterol-DPPC PEG-RBD 389.9 Comparative Example 4 Cholesterol-DPPC -- 416.8 Comparative Example 5 Cholesterol-DPPC PEG-anti(SP-A) 420.9

[0059] 2. Distribution Experiment

[0060] 1. Establishment of premature pulmonary hypoplasia model and grouping treatment:

[0061] Healthy adult SPF Sprague-Dawley (SD) rats weighing 250-300 g (purchased from the Guangdong Medical Laboratory Animal Center) were co-bred at a ratio of 2:1 male to female during estrus. Females were diagnosed as pregnant on the second day of gestation if a vaginal plug was observed and sperm was positive in vaginal specimens. This was designated as gestational day 0 (D0). Pregnant rats underwent premature cesarean section on D18, and the pups were harvested to establish a premature birth model. Pups born naturally on D22-23 served as normal controls.

[0062] After immobilizing and inducing anesthesia in pregnant mice, cesarean sections were performed to remove premature mice. Mother mice that had given birth naturally within 2 to 3 days before the cesarean section served as nurse mice. The premature mice were then placed in the nurse cages, and the nurses' feeding habits were closely monitored. The premature mice and nurse mice were simultaneously placed in an oxygen chamber connected to a nebulizer and randomly assigned to treatment groups.

[0063] The process of aerosol treatment:

[0064] Ultrasonic nebulization: Add sufficient liposome solution into the nebulizer cup of the small animal ultrasonic nebulizer (ZK-WHQ) and mix well. Connect the power switch. After the indicator light comes on, turn on the mist switch and adjust the flow knob. After the mist particles are uniform, connect the solution to the feeding box. Apply for 30 minutes each time, once every hour, for 21 consecutive days.

[0065] Research Groups:

[0066] Treatment group: premature rats, nebulized and inhaled the PBS suspension of the delivery system of Example and Comparative Example all day long.

[0067] Premature control: premature mice, PBS solution, nebulized inhalation throughout the day.

[0068] Normal control: naturally delivered pups, PBS solution, nebulized inhalation throughout the day.

[0069] 2. Evaluation of drug alveolar targeted delivery and distribution after treatment

[0070] One hour after inhalation of the CY7 fluorescently labeled drug, lung tissues were collected from premature mice in each group, and the main airways, including the trachea, left and right main bronchi, and segmental bronchi, were completely removed; the remaining lung tissue from the main airways was separated and removed. The fluorescence intensity of the lung tissue without the airways was compared using an in vivo fluorescence device to show the specific distribution efficiency of the drug; the fluorescence intensity of the main airways was compared to show the nonspecific distribution efficiency of the drug in the lungs. Corneal tissues of premature mice were cut and examined using an in vivo fluorescence device to show the nonspecific distribution efficiency of the drug in the cornea that may be contacted by the atomized gas. The evaluation of fluorescence intensity was based on the relative value of Example 1. The test results are shown in Table 2:

[0071] Table 2 Detection of drug distribution effect in the lungs

[0072]

[0073] The above results indicate that the polymer embedded in the membrane material in Comparative Example 1 is PEG-ACE2. The ACE-2 enzyme it contains cannot be specifically degraded by the ACE-2 enzyme in the alveolar epithelial cell membrane. Consequently, the degradation efficiency and rate in the lungs are low. Undisintegrated liposomes are expelled through the trachea with airway secretions, resulting in lower relative fluorescence intensity in deprived lung tissue and higher relative fluorescence intensity in the main airway tissue. Some liposomes retained in the alveoli are almost unable to disintegrate and release the drug, and are subsequently excreted with sputum. The drug distribution within the alveoli is significantly lower than that in Example 1.

[0074] Comparative Example 2 contains less PEG-ACE2pep1 available for ACE-2 enzyme degradation, and the degradation efficiency and speed in the lungs are low. The liposomes disintegrate slowly, and the liposomes that cannot disintegrate are discharged through the trachea with the airway secretions, resulting in a lower relative fluorescence intensity of the lung tissue to the airway and a higher relative fluorescence intensity of the main airway tissue. However, since it has a certain disintegration efficiency, the overall drug retention effect is better than that of Comparative Example 4. The part of the liposomes retained in the alveoli can rely on less polypeptide degradation to achieve a low proportion of drug release, and the drug distribution in the alveoli is significantly lower than that in Example 1, but significantly higher than that in Comparative Example 4.

[0075] Comparative Example 3 The polymer embedded in the membrane material is PEG-RBD, which contains the human coronavirus SARS-CoV-2 spike protein cell receptor binding domain (Receptor binding domain, RBD) that can target the specific structure of the ACE-2 enzyme protein on the alveolar epithelial cell membrane. The RBD segment binds to the ACE-2 enzyme protein on the surface of the alveolar epithelial cell membrane, thereby promoting ACE-2, RBD and its connected drugs to be endocytosed into the cytoplasm together, but cannot be used for the ACE-2 enzyme to be specifically degraded outside the cell membrane. During the process of aerosol inhalation distribution to the airway terminal, after contacting the ACE-2 enzyme on the alveolar epithelial cell membrane, it cannot be degraded, nor can it induce obvious liposome disintegration, and the drug release efficiency in the alveoli is extremely low. However, the RBD segment can specifically bind to the ACE-2 on the alveolar type 2 epithelial cell membrane and the main airway endothelial cell membrane, prompting the liposome containing the drug to be endocytosed together with ACE-2, which is manifested as the relative fluorescence intensity of the lung tissue to the airway is lower than that of Example 1, but significantly higher than that of Comparative Example 4. The way it is retained in the lungs is by epithelial cell endocytosis, and its retention in the alveoli is positively correlated with the cell membrane expression of ACE-2, so the relative fluorescence intensity of the main airway tissue is higher than that of Comparative Example 4. However, this endocytic mode of RBD-ACE-2 binding is limited by the amount of ACE-2 enzyme protein on the surface of alveolar epithelial cells. After a large amount of RBD saturatedly induces endocytosis, the ACE-2 on the cell membrane is significantly reduced. Before the supplemented ACE-2 is re-expressed on the epithelial cell membrane, the cell membrane binding efficiency of RBD and the amount of drug endocytosis are limited. Liposomes that fail to be endocytosed are discharged through the trachea with airway secretions. In addition, the RBD segment can specifically bind to the ACE-2 enzyme protein on the main airway endothelial cell membrane, prompting the liposomes containing the drug to be endocytosed together with ACE-2 in the main airway epithelium, which together leads to the relative fluorescence intensity of the main airway tissue being higher than that of Example 1. After the liposomes containing the spike protein RBD segment are retained in the alveoli for a long time, they may eventually continue to contact the alveolar epithelial cell membrane and then non-specifically endocytose into the alveolar epithelial cells. Alveolar epithelial cells internalize large amounts of drug components that should be secreted out of the cells, which may lead to cytotoxicity. In the distribution study of Comparative Example 3, it is particularly noteworthy that the relative fluorescence intensity of the corneal tissue in this group is much higher than that of all other examples and all other comparative examples. This is because the corneal epithelium, like the lung epithelial cell membrane, highly expresses ACE-2. The RBD segment can specifically bind to ACE-2 on the corneal epithelial cell membrane, prompting the liposomes containing the drug to be internalized along with ACE-2, causing the corneal cells to swallow a large amount of liposomes and alveolar surfactant that are not needed for metabolism, inducing corneal inflammation.

[0076] Comparative Example 4 is not linked to a PEG-angiotensin converting enzyme II substrate polypeptide, and does not have any groups that can be specifically degraded by enzymes in the lungs. The liposomes are difficult to disintegrate after being inhaled into the alveoli, and the drug cannot be released into the alveoli. The non-degradable liposomes may be discharged through the trachea with the airway secretions, resulting in a low relative fluorescence intensity of the lung tissue that has been degraded to the airways; and efficient alveolar distribution cannot be achieved. The drug discharged through the trachea with the airway secretions causes the relative fluorescence intensity of the main airway tissue to be extremely low.

[0077] In Comparative Example 5, the polymer embedded in the membrane material is PEG-anti (SP-A). The Surfactant Protein A antibody protein contained therein cannot be specifically degraded by the ACE-2 enzyme, and the degradation efficiency and speed in the lung are low. The liposomes of the Surfactant Protein A antibody that cannot disintegrate are discharged through the trachea with the airway secretions, resulting in a low relative fluorescence intensity of the lung tissue that has gone to the airway, and a high relative fluorescence intensity of the main airway tissue. The liposomes retained in the alveoli can hardly disintegrate to release the drug, and the drug distribution in the alveoli is significantly lower than that in Example 1. However, the Surfactant Protein A antibody itself is a protein and can be non-specifically degraded by the proteolytic enzymes in the surface secretions of the alveoli and small bronchi, disintegrating at a very low ratio and very slow speed to release the drug, so its relative fluorescence intensity of the main airway tissue is lower than that of Comparative Example 4.

[0078] 3. Evaluation of treatment effect

[0079] To show the efficacy of treatment on pulmonary ventilation and gas exchange function, as well as the oxygen content in the blood of premature mice: arterial blood samples were collected from the right common carotid artery of the pups using a heparin-treated polyethylene catheter. The PaO2 in the sample was analyzed using an automatic blood gas analyzer. The experimental mice were anesthetized with an intraperitoneal injection of 0.5 mL / kg chloral hydrate. After anesthesia, they were fixed, the trachea was cut, and endotracheal intubation was performed. The detection instrument - BuxCo small animal pulmonary function experimental platform system was connected to the open part of the airway of the experimental mouse to detect the tidal volume of the premature mouse (referring to the volume of gas inhaled or exhaled each time when the animal breathes in a calm state). These indicators can show the therapeutic effect of treatment on pulmonary ventilation and gas exchange function, as well as the oxygen content in the blood of the animal. The test results are shown in Table 3:

[0080] Table 3: Tidal volume and blood oxygen concentration function test

[0081] Grouping Tidal volume (mL) <![CDATA[PaO2(mmHg) <!-- 7 -->]]> Healthy control group 0.72 98.3 Premature non-treatment group 0.35 65.7 Example 1 0.68 95.2 Example 2 0.65 93.9 Example 3 0.66 94.8 Example 4 0.64 94.3 Example 5 0.55 85.4 Example 6 0.64 91.7 Comparative Example 1 0.36 67.0 Comparative Example 2 0.39 69.8 Comparative Example 3 0.3 61.3 Comparative Example 4 0.32 61.2 Comparative Example 5 0.35 65.8

[0082] The polymer embedded in the membrane material of Comparative Example 1 is PEG-ACE2, and the ACE-2 enzyme protein it contains itself cannot be specifically degraded by the ACE-2 enzyme protein of the epithelial cell membrane. During the process of aerosol inhalation distribution to the airway terminal, after contact with the ACE-2 enzyme, it cannot be degraded, nor can it induce obvious liposome disintegration, and the drug release efficiency is extremely low. Compared with the non-treated group, after a full course of treatment with this group of drugs, the tidal volume reflecting the long-term development of the alveoli did not improve significantly, and the gap was larger than that of the healthy control group and the embodiment. The PaO2 reflecting the alveolar ventilation function was only slightly improved, but the gap was larger than that of the healthy control group and the embodiment. This shows that after treatment with this group of drugs, the alveolar surfactant was not effectively released in the alveoli, and failed to play the role of promoting alveolar development and improving ventilation function after the alveolar surfactant was evenly distributed in the alveoli, reducing alveolar surface tension, increasing lung compliance, maintaining the relative stability of alveolar volume, preventing atelectasis, and preventing pulmonary edema.

[0083] Comparative Example 2 contains very little polypeptide substrate for ACE-2 enzyme degradation, and its degradation has little effect on the stability of the liposomes, the liposome disintegration efficiency is extremely low, and the drug release efficiency is extremely low. Compared with the non-treated group, after a full course of treatment with this group of drugs, the tidal volume reflecting the long-term development of the alveoli was slightly improved, but there was a large gap compared to the healthy control group and the example. After treatment with this group of drugs, PaO2, which reflects the alveolar ventilation function, was only slightly improved, but there was a large gap compared to the healthy control group and the example.

[0084] The polymer embedded in the membrane material of Comparative Example 3 is PEG-RBD, which contains the RBD segment of the SARS-CoV-2 spike protein. The RBD segment can bind to the ACE-2 enzyme protein on the cell membrane surface, thereby promoting the endocytosis of ACE-2, RBD and its connected drugs into the cytoplasm, but it cannot provide the ACE-2 enzyme with specific degradation outside the cell membrane to achieve the release of the drug in the alveoli. Compared with the non-treatment group, after a full course of treatment with this group of drugs, the tidal volume of premature mice not only did not improve, but extremely deteriorated and decreased significantly. After treatment with this group of drugs, PaO2 also deteriorated extremely. This first shows that after treatment with this group of drugs, the alveolar surfactant in this group of drugs failed to be effectively released in the alveoli, and failed to play the role of evenly distributing the alveolar surfactant in the alveoli, reducing the alveolar surface tension, increasing lung compliance, maintaining the relative stability of the large and small alveolar volumes, preventing atelectasis, and preventing pulmonary edema, and promoting alveolar development and improving ventilation function. This secondly indicates that it induces a large amount of drug endocytosis in alveolar epithelial cells, resulting in cytotoxicity to alveolar epithelial cells and main airway epithelial cells, which may cause obvious lung tissue and systemic toxic side effects.

[0085] Comparative Example 4, without the PEG-ACE II substrate peptide attached, lacked any groups available for enzymatic degradation, making the liposomes difficult to disintegrate and preventing drug release into the alveoli. Compared to the untreated group, after a full course of treatment with this drug, tidal volume, a reflection of long-term alveolar development, not only did not improve but actually deteriorated. PaO2, a measure of alveolar ventilation and gas exchange function, also worsened.

[0086] In Comparative Example 5, the polymer embedded in the membrane material was PEG-anti(SP-A), and the antibody protein Surfactant Protein A contained therein was not specifically degraded by the ACE-2 enzyme. Compared to the untreated group, after a full course of treatment with this drug, tidal volume, reflecting long-term alveolar development, showed only a slight improvement, but significantly lower than the healthy control group and the examples. PaO2, reflecting alveolar ventilation function, did not improve significantly after treatment with this drug, and significantly lower than the healthy control group and the examples.

[0087] The polymer embedded in the membrane material of Examples 1-6 is a PEG-angiotensin-converting enzyme II substrate polypeptide. During aerosol inhalation, the PEG-angiotensin-converting enzyme II substrate polypeptide is degraded upon contact with the ACE-2 enzyme in the lungs, resulting in efficient liposome disintegration and uniform release of alveolar surfactant into the alveoli. After the drug and the alveolar surfactant contained in it are effectively retained and evenly dispersed in the lungs, they achieve the desired effects of reducing alveolar surface tension, increasing lung compliance, maintaining relatively stable alveolar volumes, preventing atelectasis and pulmonary edema, and promoting alveolar development and improving ventilation. Furthermore, the sensitive groups on the outer shell of the drug liposomes can ultimately be degraded by the ACE-2 enzyme. Drugs that are not excreted with respiratory secretions can be retained in the alveoli and eventually disintegrate, thus preventing significant toxic side effects by significantly blocking gas exchange at the "gas-liquid barrier." The timely disintegration of the drug prevents endocytosis into alveolar epithelial cells, thus avoiding cytotoxicity. Compared to the non-treatment group, after a full course of treatment with the Example drugs, the tidal volume was significantly improved, even very close to the healthy control group (Example 1, Example 3). After drug treatment, the PaO2, reflecting alveolar ventilation function, was also significantly improved compared to the non-treatment group, even very close to the healthy control group.

[0088] IV. Evaluation of Therapeutic Toxicity

[0089] To assess the potential pulmonary toxicity after inhaled drug treatment, lung histological sections were obtained after anesthesia and lung inflammation scores were calculated: 0: no pathological findings; 1: scattered infiltration of single polymorphonucleuleukocytes (PMNs) with intact tissue architecture; 2: scattered infiltration of a few or small groups of PMNs with mild exudates, a small amount of intravascular congestion, or occasional hemorrhage; 3: extensive infiltration of numerous PMNs, which may cluster locally or infiltrate around the vessel wall, or even numerous PMNs in the blood within the lumen; exudates and hemorrhage in the alveolar cavity, with occasional congestion or focal atelectasis; 4: destruction of normal lung tissue architecture, necrosis of the lung parenchyma, PMN nuclear fragmentation, nuclear condensation, or nuclear dissolution, and increased cytoplasmic eosinophilia. To assess the potential corneal toxicity of aerosolized inhaled drugs, horizontal scans of the anterior segment optical coherence tomography (ASCO) were performed in all groups of premature rats after anesthesia, and central corneal thickness was measured. The weight of premature mice in each group was measured 21 days after treatment to reflect the overall growth and development level of the premature mice. The test results are shown in Table 4:

[0090] Table 4: Toxicity evaluation results

[0091] Grouping Weight (g) Pneumonia Inflammation Score Corneal thickness (μm) Healthy control group 58.3 0 129.3 Preterm non-treatment group 35.2 1.0 129.3 Example 1 57.1 0 129.3 Example 2 57.0 0 129.4 Example 3 56.8 0 129.3 Example 4 56.2 0 129.5 Example 5 52.9 0 129.2 Example 6 56.4 0 129.6 Comparative Example 1 37.1 0 129.5 Comparative Example 2 37.5 0 129.3 Comparative Example 3 31.2 3.0 162.1 Comparative Example 4 33.2 1.0 129.8 Comparative Example 5 36.5 0 129.7

[0092] From the above results, it can be seen that in Examples 1-6, Comparative Example 2, Comparative Example 1, and Comparative Example 5, no lung toxicity and corneal toxicity were observed after long-term aerosol inhalation, and the weight indicators reflecting overall development all showed different degrees of improvement.

[0093] However, in Comparative Examples 4 and 3, the lung inflammation scores were significantly higher than that in Example 1 because the blocking of the gas exchange of the "gas-liquid barrier" restricted the development of the animals and caused obvious lung toxic side effects due to factors such as inducing endocytosis of alveolar epithelial cells.

[0094] In the corneal thickness scoring, in Comparative Example 3, since the atomized liposome drug diffuses in the breeding box and inevitably contacts the animal cornea, the SARS-CoV-2 spike protein RBD segment on the surface of the liposome will bind to the ACE-2 enzyme protein on the surface of the conjunctival and corneal epithelial cells and transfect in large quantities, causing the ocular epithelial cells to phagocytize a large amount of drugs containing alveolar surfactant, resulting in obvious cytotoxicity and a significant pathological increase in corneal thickness.

Claims

1. A drug delivery system targeting alveolar stem cells, characterized in that: The drug delivery system includes nanolipids The invention relates to a plasmid vesicle, a PEG-angiotensin converting enzyme II substrate polypeptide with a targeting function, and an alveolar surfactant. The PEG-angiotensin converting enzyme II substrate polypeptide with a targeting function is embedded in the membrane material of the nanoliposome vesicle, the alveolar surfactant is wrapped inside the nanoliposome vesicle, and the sequence of the angiotensin converting enzyme II substrate polypeptide is selected from at least one of Mca-YVADAP-Lys(Dnp)-OH and Mca-Ala-Pro-Lys(Dnp)-OH.

2. The drug delivery system targeting alveolar stem cells according to claim 1, characterized in that The alveolar stem cells are alveolar type II epithelial cells.

3. The drug delivery system targeting alveolar stem cells according to claim 1, characterized in that The membrane material of the nanoliposome vesicle is composed of cholesterol and phospholipids, and the phospholipids are selected from at least one of dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine, and are more preferably dipalmitoylphosphatidylcholine.

4. The drug delivery system targeting alveolar stem cells according to claim 1, characterized in that Based on the total molar amount of the membrane material of the nanoliposome vesicle, the chimeric amount of the PEG-angiotensin converting enzyme II substrate polypeptide is 5-20 mol%, preferably 10-15 mol%.

5. The drug delivery system targeting alveolar stem cells according to claim 1, characterized in that The alveolar surfactant includes bovine lung surfactant for injection, porcine lung phospholipid injection or artificially synthesized components containing alveolar surfactant.

6. The drug delivery system targeting alveolar stem cells according to claim 1, characterized in that The particle size of the delivery system is 50-1000 nm, preferably 300-500 nm.

7. The method for preparing the drug delivery system targeting alveolar stem cells according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Add the peptide substrate of angiotensin-converting enzyme II, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-(dimethylamino)pyridine to an acetonitrile aqueous solution and stir magnetically in an ice-water bath under N2 protection to activate the peptide carboxyl group; (2) reacting the activated angiotensin-converting enzyme II polypeptide substrate with mPEG-NHS to obtain a PEG-angiotensin-converting enzyme II substrate polypeptide with targeting function; (3) PEG-angiotensin converting enzyme II substrate peptide, cholesterol, and phospholipid are dissolved in dichloromethane, and then alveolar surfactant is added. The solvent is removed by rotary evaporation to form a composite liposome membrane; (4) Adding aqueous medium to the composite liposome membrane and ultrasonically dispersing it will yield a drug delivery system targeting alveolar stem cells.

8. The preparation method according to claim 7, characterized in that In step (3), the amount of the PEG-ACE II substrate polypeptide added is 5-20 mol%, preferably 10-15 mol%, of the total molar amount of cholesterol and phospholipid.

9. Use of the drug delivery system targeting alveolar stem cells according to any one of claims 1 to 6 in the preparation of a drug for treating premature infants with pulmonary hypoplasia.

10. The use according to claim 9, characterized in that The drug is administered by atomization inhalation, and the droplet size after atomization is 1 μm to 5 μm.

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