Pulmonary alveolar targeted non-invasive drug delivery carrier capable of achieving drug delivery through respiratory tract inhalation as well as preparation method and application of pulmonary alveolar targeted non-invasive drug delivery carrier

Abstract

The invention discloses a non-invasive pulmonary alveolar targeting carrier capable of being inhaled through a respiratory tract and a preparation method and application of the non-invasive pulmonary alveolar targeting carrier. The non-invasive pulmonary alveolar targeting carrier is mainly characterized in that a PEG grafted enzyme response substrate (the enzyme response substrate is pulmonary alveolar surface active protein SP-B / SP-C / SP-D) is synthesized firstly, and then the PEG grafted enzyme response substrate is used for modifying liposome vesicles formed by a liposome main body and cholesterol; animal-derived or synthetic alveolar surfactants are loaded in the vesicles, and the average particle size of the vesicles is 50-1000 nm. The liposome vesicle overcomes the defect that in the prior art, alveolar surfactants or liposome vesicles are difficult to reach alveolar delivery drugs in a targeted mode, alveolar active substances can be efficiently delivered, and the liposome vesicles have good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an alveolar-targeted drug carrier capable of non-invasive drug administration through the respiratory tract, and a preparation method and application thereof. Background Art

[0002] The main problem affecting the survival of premature infants after birth is pulmonary hypoplasia. Hypoplastic alveolar epithelial cells cannot secrete enough surfactant to maintain alveolar volume stability, resulting in pulmonary edema and atelectasis in the surfactant-deficient alveoli, and the inability to maintain normal ventilation and gas exchange functions. Currently, there are two treatment methods recommended by the guidelines:

[0003] Before birth, pregnant women receive a small intramuscular injection of glucocorticoids to promote fetal lung development. Glucocorticoids have numerous side effects on both the mother and the fetus, and are only used in limited quantities after birth when premature infants cannot be weaned off a ventilator.

[0004] 2. After birth, premature infants undergo endotracheal intubation and instillation of animal (porcine, bovine) or synthetic pulmonary surfactant (PS). The trachea of ​​premature infants is fragile and small, requiring specialized personnel to establish an endotracheal channel. Instilling fluid into the trachea of ​​premature infants is technically difficult, carries a high risk of complications, and has a severe complication rate exceeding 33%, requiring highly qualified personnel. This treatment approach is difficult to popularize, making it difficult for premature infants born in primary care hospitals to receive appropriate treatment. Even in high-level hospitals, the risks of this procedure are significant. Droplets instilled into the trachea can cause choking, atelectasis, and even asphyxiation in premature infants. Droplets instilled into the main bronchi cannot guarantee distribution within the terminal alveoli. Coughing up the drug out of the airway can lead to treatment failure. Alveoli that are unable to reach the droplets present a state of "segmental atelectasis." Current guidelines, considering patient tolerance and the risk of complications, recommend only a maximum of two applications. This approach cannot be implemented consistently and repeatedly throughout the developmental period, as needed, nor can it be widely adopted to meet the treatment needs of primary care hospitals.

[0005] Nebulized inhalable drug particles may offer a potential solution to this dilemma and enable postnatal PS treatment in premature infants. The specific requirements for total droplet size for nebulized inhalation therapy are: a total droplet size of 1μm to 5μm. When the particle size is <1μm, even if the droplets are inhaled into the alveoli, they are exhaled with breathing and cannot be retained in the alveoli. When the particle size is >5μm, the droplets are primarily retained in the main bronchi and have difficulty entering the alveoli at the terminal airways. If the liposome vesicles fail to reach the alveoli and immediately disintegrate, the following outcomes may occur: 1. Accumulation in the trachea or bronchi, resulting in excretion with sputum; 2. Accumulation on the alveolar epithelial surface, blocking the "air-liquid" barrier and preventing normal alveolar oxygen / carbon dioxide exchange; 3. "Transfection" into respiratory epithelial cells with the liposomes, affecting cell metabolism and producing cytotoxicity.

[0006] Therefore, the market needs a non-invasive drug delivery carrier that can target the alveoli through the respiratory tract. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above technical defects and provide an alveolar targeted drug delivery carrier that uses liposome particles to carry and achieve non-invasive inhalation drug delivery through the respiratory tract, as well as its preparation method and application.

[0008] The present invention is achieved through the following technical solutions:

[0009] A non-invasive drug delivery vehicle for alveolar targeting via the respiratory tract comprises vesicles and alveolar surfactant loaded within the vesicles. The vesicles are composed, by molar percentage, of 40-50 mol% liposome bulk, 40-50 mol% cholesterol, and 6-15 mol% PEG-grafted enzyme-responsive substrate. Based on the weight percentage of the vehicle for alveolar targeting via the respiratory tract, the vesicles are loaded with 10-30 wt% alveolar surfactant, and have an average particle size of 50 to 1000 nm.

[0010] The alveolar surfactant is selected from at least one of animal-derived alveolar surfactant and synthetic alveolar surfactant. The enzyme response substrate is selected from at least one of pulmonary surfactant protein (SP)-B (surfactant protein B), SP-C (surfactant protein C), and SP-D (surfactant protein D); and the liposome body 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).

[0011] The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of being administered via respiratory inhalation of the present invention comprises the following steps:

[0012] Step A: dissolving the enzyme response substrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 4-(dimethylamino)pyridine (DMAP) in a molar ratio of 1:(30-70):(10-50) in solvent A, under inert gas protection, and stirring at 0-5°C to activate the carboxyl group;

[0013] Step B: Based on the molar amount of the enzyme-responsive substrate, 15-25 times of mPEG-NHS was added, and the reaction was carried out at 0-5°C under inert gas protection to obtain the enzyme-responsive substrate grafted PEG;

[0014] Step C: dissolving the enzyme-responsive substrate grafted PEG, cholesterol, and liposome body in solvent B at a molar ratio of 1:(3-7):(3-7), adding pulmonary surfactant (PS), and evaporating the solvent under reduced pressure to obtain a liposome film composited with pulmonary surfactant (liposome film composited with PS);

[0015] Step D: Add PBS solution to the liposome film of the above-mentioned composite alveolar surfactant, disperse it by ultrasonic wave to form vesicles, adjust the vesicle particle size by controlling the ultrasonic treatment time, and then add physiological saline to disperse it to obtain an alveolar-targeted non-invasive drug delivery carrier that can be inhaled through the respiratory tract.

[0016] In step B, mPEG-NHS is a polyethylene glycol end, which undergoes a coupling reaction with an enzyme-responsive substrate to obtain a PEG-enzyme-responsive substrate compound (such as PEG-(SP-B)) having enzyme-related functions.

[0017] Specifically, the enzyme-responsive substrate is selected from at least one of SP-B, SP-C, and SP-D, and has a molecular weight range of 34,000-70,000 Da. The liposome host is selected from at least one of dipalmitoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. The molecular weight of the mPEG-NHS is in the range of 1,000-3,000 Da.

[0018] In step A, the amount of solvent A is 1-20 times the total weight of other substances in step A;

[0019] Optionally, the solvent A is selected from at least one of acetonitrile / water solution (the volume ratio may be 1:3-5) and DMSO / water solution (the volume ratio may be 1:3-5).

[0020] Specifically, the reaction time of step B is 48-96 hours.

[0021] In step B, after the PEG grafted enzyme response substrate is generated by reaction, it is dialyzed through a dialysis bag and then freeze-dried to obtain a PEG grafted enzyme response substrate raw material.

[0022] Specifically, the solvent B is selected from at least one of dichloromethane, chloroform, and a chloroform / methanol composite solvent (the volume ratio may be 1:1-3). The amount of solvent B added is 5-30 times the weight of the liposome body.

[0023] Specifically, in step C, the conditions for removing the solvent under reduced pressure are a temperature of 30-45° C. and a pressure of 10-150 MPa.

[0024] In step C, the concentration of alveolar surfactant is 30-40 mg / mL.

[0025] In step C, the enzyme-responsive end of the enzyme-responsive substrate grafted onto PEG serves as a sensitive response end to form a liposome film with cholesterol and the liposome body, effectively compounding the alveolar surfactant during the formation of the liposome film.

[0026] In step D, the amount of PBS solution added is 0.1-10 times the mass of the liposome film; the PBS solution is not added all at once, but 5-50% of the volume of the PBS solution is added every minute. The purpose is to form fully dispersed liposome vesicles with relatively uniform particle size through self-assembly. The grafted PEG chain segment significantly increases the stability of the liposome vesicles.

[0027] In step D, the ultrasonic treatment time ranges from 3 to 15 minutes. The longer the ultrasonic treatment time, the smaller the particle size of the vesicles.

[0028] PBS solution is a phosphate buffered saline solution, and the pH range can be 7.2-7.6.

[0029] The alveolar-targeted non-invasive drug delivery carrier capable of being administered through respiratory tract inhalation of the present invention is used to prepare medicines for treating lung diseases associated with alveolar surfactant deficiency.

[0030] The present invention has the following beneficial effects:

[0031] The present invention utilizes liposomes and cholesterol to form vesicles with a particle size of 50 to 1000 nm. The vesicles are embedded with PEG-enzyme response substrates, and the vesicles are loaded with alveolar surfactant (PS). The PS suspension, which can only be instilled through endotracheal intubation and may temporarily or long-term block the airway, is carried by liposome vesicles to achieve non-invasive inhalation administration. Invasive operations and complications are avoided, and continuous and repeated non-invasive administration is achieved. By precisely controlling the particle size of the vesicles in the droplets to 50 to 1000 nm, the droplet size after atomization is 1 μm to 5 μm, which is suitable for reaching the alveoli. Avoid droplets that are too large or too small, which reduces the efficiency of alveolar distribution. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Schematic diagram of the alveolar-targeted non-invasive drug delivery vehicle through the respiratory tract of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0034] The raw materials used in the present invention come from the following sources:

[0035] mPEG-NHS: Mn = 2000 Da, purchased from Sigma-Aldrich;

[0036] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide, purchased from Sigma-Aldrich;

[0037] DMAP: 4-(dimethylamino)pyridine, purchased from Sigma-Aldrich;

[0038] SP-B: molecular weight 68.5 kDa, Recombinant Human SP-B / Surfactant Protein B GST (N-Term) Protein, Bio-Techne;

[0039] SP-C: molecular weight 47.3 kDa, Recombinant Human Prosurfactant Protein C GST (N-Term) Protein (Catalog # H00006440-P01), Bio-Techne;

[0040] SP-D: molecular weight 35.4 kDa, Recombinant Human SP-D Protein, CF (1920-SP-050), Bio-Techne;

[0041] Anti-SP-B antibody: Anti-Surfactant Protein B (Mature) antibody [RM370] (ab271345), abcam.

[0042] ACE2 enzyme: molecular weight is approximately 84kDa, (ab287935), abcam.

[0043] The RBD segment of the human coronavirus spike protein: molecular weight is approximately 34kDa, SARS-CoV-2 Spike Glycoprotein RBD (Active) (ab273065), abcam.

[0044] DPPC: molecular weight 734, dipalmitoylphosphatidylcholine, purchased from Sigma-Aldrich;

[0045] PC: molecular weight 385, phosphatidylcholine, purchased from Sigma-Aldrich;

[0046] PE: molecular weight 744, phosphatidylethanolamine, purchased from Sigma-Aldrich;

[0047] Phosphatidylserine: molecular weight 792, purchased from Sigma-Aldrich;

[0048] Cholesterol: molecular weight 386, purchased from Sigma-Aldrich;

[0049] Kelisu: bovine alveolar surfactant PS, 35 mg / ml, purchased from Shuanghe Modern Pharmaceutical;

[0050] Solvents such as acetonitrile and dichloromethane were from Sigma-Aldrich;

[0051] Preparation technology of embodiment and comparative example:

[0052] Example 1: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), and the enzyme response substrate is SP-B;

[0053] Step A: 0.01 mmol of surfactant protein B, 5 mmol of EDC and 5 mmol of DMAP were dissolved in 10 mL of acetonitrile aqueous solution (acetonitrile: water = 1:1), and stirred at 500 rpm for 2 h in an ice-water bath under N2 protection to activate the carboxyl group.

[0054] Step B: Add 0.2 mmol of mPEG-NHS and react for 72 h; place the reaction solution in a dialysis bag (MWCO = 3.5 kDa), dialyze for 72 h, and freeze-dry to obtain the product PEG-(SP-B).

[0055] Step C: Dissolve 5 μmol of PEG-(SP-B), 25 μmol of cholesterol, and 25 μmol of dipalmitoylphosphatidylcholine (DPPC) in 5 mL of dichloromethane, then add 0.2 mL (concentration of 35 mg / ml) of injectable bovine alveolar surfactant (PS) dropwise, and evaporate the solvent to dryness by vacuum rotary evaporation to form a composite PS film.

[0056] Step D: Subsequently, 2 mL of PBS solution was added dropwise to the composite PS film at a rate of 20% per minute and ultrasonically dispersed at 45 W power (see the table for specific time). Finally, 2 mL of saline (0.9% NaCl) solution was added to dissolve and disperse the DPPC-PS-cholesterol-(SP-B)-vesicles. The volume was adjusted to 5 mL and stored at 4°C to obtain drug delivery vehicle 1.

[0057] Example 2: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), the enzyme response substrate is SP-D, and the process except the ultrasonic time is the same as that of Example 1. The amount of raw materials used is the same as that of Example 1, and finally the drug delivery carrier 2 is obtained.

[0058] Example 3: The liposome main body is phosphatidylethanolamine (PE), the enzyme response substrate is SP-B, the process except for the ultrasonic time is the same as that of Example 1, and the amount of raw materials used is the same as that of Example 1, and finally the drug delivery carrier 3 is obtained.

[0059] Example 4: The liposome main body is phosphatidylcholine (PC), the enzyme response substrate is SP-B, the process except for the ultrasonic time is the same as that of Example 1, and the amount of raw materials used is the same as that of Example 1, and finally the drug delivery carrier 4 is obtained.

[0060] Example 5: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), the enzyme response substrate is SP-B, and the process parameters except for the ultrasonic time are the same as those in Example 1, except that PEG-(SP-B) is 3.6 μmol, and finally the drug delivery carrier 5 is obtained.

[0061] Example 6: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), the enzyme response substrate is SP-C, the process except the ultrasonic time is the same as Example 1, the amount of raw materials used is the same as Example 1, and finally the drug delivery carrier 6 is obtained.

[0062] Example 7: The liposome main body is phosphatidylserine, the enzyme response substrate is SP-B, the process except for the ultrasonic time is the same as Example 1, and the amount of raw materials used is the same as Example 1, and finally the drug delivery carrier 7 is obtained.

[0063] Example 8: The main body of the liposome is phosphatidylserine, and the enzyme response substrate is SP-D;

[0064] Step A: 0.01 mmol of surfactant protein D, 7 mmol of EDC and 1 mmol of DMAP were dissolved in 5 mL of DMSO aqueous solution (acetonitrile:water=1:4), and stirred at 300 rpm for 1 h in an ice-water bath under N2 protection to activate the carboxyl group.

[0065] Step B: Add 0.15 mmol of mPEG-NHS and react for 72 h; place the reaction solution in a dialysis bag (MWCO = 3.5 kDa), dialyze for 72 h, and freeze-dry to obtain the product PEG-(SP-D).

[0066] Step C: 5 μmol of PEG-(SP-D), 15 μmol of cholesterol, and 35 μmol of phosphatidylserine were dissolved in 8 mL of dichloromethane. 0.2 mL (concentration of 35 mg / ml) of injectable bovine alveolar surfactant (PS) was slowly added dropwise. The solvent was evaporated to dryness by vacuum rotary evaporation to form a composite PS film.

[0067] Step D: Then, 3 mL of PBS solution was added dropwise to the phosphatidylserine-PS-cholesterol-(SP-D)-vesicles at a rate of 40% / min, and ultrasonically dispersed (time see the table). Finally, 2 mL of normal saline (0.9% NaCl) solution was added to dissolve and disperse the phosphatidylserine-PS-cholesterol-(SP-D)-vesicles, and the volume was adjusted to 5 mL. The solution was stored at 4°C to obtain drug delivery vehicle 8.

[0068] Comparative Example 1: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), the PEG-modified part is anti-SP-B, the amount of PEG-(anti-SP-B) is 5 μmol, and the process except for the ultrasonic time is the same as that of Example 1. The amounts of other raw materials are the same as those of Example 1, and finally the drug delivery carrier 9 is obtained.

[0069] Comparative Example 2: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), the enzyme response substrate is SP-B, the amount of PEG-(SP-B) added in step B is 0.5 μmol, the process except the ultrasonic time is the same as Example 1, and the other raw materials are the same as Example 1, and finally the drug delivery carrier 10 is obtained.

[0070] Comparative Example 3: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), and the PEG-modified part is a recombinant protein of ACE2 enzyme. The process is the same as that of Example 1 except for the ultrasonic time, and the amount of raw materials is the same as that of Example 1. Finally, the drug delivery carrier 11 is obtained.

[0071] Comparative Example 4: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), and the PEG-modified part is the RBD segment of the human coronavirus spike protein. The process is the same as that in Example 1 except for the ultrasound time, and the amount of raw materials is the same as that in Example 1. Finally, the drug delivery carrier 12 is obtained.

[0072] Comparative Example 5: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), but does not contain PEG grafted enzyme-responsive substrate modification: Specifically, 25 μmol of cholesterol and 25 μmol of dipalmitoylphosphatidylcholine were dissolved in 5 mL of dichloromethane, and then 0.2 mL (concentration of 35 mg / ml) of injectable bovine alveolar surfactant PS was slowly added dropwise. The solvent was evaporated to dryness under vacuum rotary evaporation to form a DPPC-PS-cholesterol-complex liposome membrane. Subsequently, 2 mL of PBS solution was added dropwise to the DPPC-PS-cholesterol-complex liposome membrane at a rate of 0.5 mL / min, and ultrasonic dispersion was performed (time is shown in the table). Finally, 2 mL of normal saline (0.9% NaCl) solution was added to dissolve and disperse the DPPC-PS-cholesterol-complex liposome membrane, and the volume was fixed to 5 mL. The membrane was stored at 4°C to obtain drug delivery vehicle 13.

[0073] Comparative Example 6: The liposome main body is dipalmitoylphosphatidylcholine (DPPC), the enzyme response substrate is SP-B, and it does not contain PEG. The specific process is as follows: 5 μmol of SP-B, 10 mg (25 μmol) of cholesterol, and 25 μmol of dipalmitoylphosphatidylcholine (DPPC) are dissolved in 5 mL of dichloromethane. Then, 0.2 mL (concentration of 35 mg / mL) of injectable bovine alveolar surfactant PS is slowly added dropwise. The solvent is evaporated to dryness by vacuum rotary evaporation to form PS-loaded vesicles. Subsequently, 2 mL of PBS solution is added dropwise to the PS-loaded vesicles at a rate of 0.5 mL / min, and ultrasonic dispersion is performed (time is shown in the table). Finally, 2 mL of physiological saline (0.9% NaCl) solution is added to dissolve and disperse the PS-loaded vesicles. The volume is fixed to 5 mL and stored at 4°C to obtain drug delivery vehicle 14.

[0074] Table 1: Test results of Examples and Comparative Examples

[0075]

[0076]

[0077] Table 1 continued:

[0078]

[0079] Among them, the treatment effect evaluation of the healthy control group was PaO2=98.6mmHg, lung compliance=3.5mL / cmH2O, renal function BUN=21.35mg / dL, and liver function ALT=37.2U / L.

[0080] In the premature non-treatment group, PaO2=69.3 mmHg, lung compliance=1.3 mL / cmH2O, renal function BUN=21.33 mg / dL, and liver function ALT=37.5 U / L.

[0081] Comments on the distribution experiment:

[0082] The experimental data from Examples 1-8 demonstrate that the liposome body is a natural, primary component of alveolar surfactant, which self-assembles with cholesterol and a PEG-enzyme-responsive substrate compound to form vesicles. These vesicles are loaded with bovine alveolar surfactant and ultrasonically dispersed into particles with an average size of 50-1000 nm. This vesicle structure and particle size ensure that the liposomes are distributed to the airway terminals during aerosol inhalation. Upon contact with natural enzymes within the alveoli that physiologically degrade alveolar surfactant proteins, the PEG-linked polypeptide / protein degrades, the liposomes efficiently disintegrate, and the alveolar surfactant is evenly released into the alveoli. This is demonstrated by high relative fluorescence intensity in frozen pathological sections of lung tissue, indicating effective drug retention in the lungs.

[0083] The enzyme-sensitive response substrate of Comparative Example 1 is an antibody to Surfactant Protein B, an important protein in the alveolar surfactant. The vesicles contain antibody proteins to Surfactant Protein B, which cannot be specifically degraded by the natural enzymes of the alveolar surface protein SP-B in the alveoli. During the process of aerosol inhalation and distribution to the airway terminals, after contacting the natural enzymes of the alveolar surface protein in the alveoli, they cannot be specifically degraded, nor can they induce obvious liposome disintegration, resulting in extremely low efficiency of alveolar surfactant release. This is manifested in that the relative fluorescence intensity of frozen pathological sections of lung tissue is significantly lower than that of Example 1, and the drug retention effect in the lungs is poor. Compared with Example 1, this group of drugs does not have a specific natural enzyme substrate for alveolar surface protein in the alveoli, and the degradation efficiency and speed in the lungs are lower. The liposomes containing the antibody protein to Surfactant Protein B that cannot disintegrate are discharged through the trachea with airway secretions, resulting in a lower relative fluorescence intensity in frozen pathological sections of lung tissue. The liposomes retained in the alveoli are almost unable to disintegrate and release the drug, and the drug distribution in the alveoli is significantly lower than that in Example 1. However, the antibody protein of Surfactant Protein B is a polypeptide / protein that can be non-specifically degraded by proteolytic enzymes in the surface secretions of the alveoli and bronchioles, disintegrating at an extremely low rate and very slowly, releasing the drug. Therefore, the relative fluorescence intensity of its frozen lung tissue pathological sections is slightly higher than that of Comparative Example 5. Moreover, although the degradation efficiency of the antibody of Surfactant Protein B is extremely low, its content is relatively high. After a long period of retention in the alveoli, the drug that is not excreted with the respiratory secretions may be degraded by non-specific enzymes and eventually disintegrated, thereby not significantly blocking gas exchange at the "gas-liquid barrier"; nor will it continue to contact the alveolar epithelial cell membrane and induce large-scale endocytosis of the alveolar epithelial cells, leading to cytotoxicity.

[0084] The liposomes in Comparative Example 2 consist primarily of dipalmitoylphosphatidylcholine (DPPC), a natural component of pulmonary surfactant, and the polypeptide substrate is Surfactant Protein B. The ratio of PEG-(SP-B) to phospholipid is reduced to 1 / 10 of that in Example 1. This structured liposome contains minimal substrate for degradation by natural enzymes of alveolar surface proteins within the alveoli. During aerosol inhalation and distribution to the terminal airways, the PEG-linked polypeptide degrades upon contact with natural enzymes of alveolar surface proteins within the alveoli. However, due to the minimal amount of PEG-linked polypeptide, its degradation has minimal impact on liposome stability, resulting in extremely low liposome disintegration efficiency and surfactant release efficiency. This is manifested by significantly lower relative fluorescence intensity in frozen lung tissue pathological sections than in Example 1, indicating poor drug distribution within the lungs. Compared to Example 1, this group of drugs has fewer enzyme substrates, resulting in lower efficiency and rate of degradation within the lungs, slower liposome disintegration, and undisintegrated liposomes are expelled through the trachea with airway secretions, resulting in lower relative fluorescence intensity in frozen lung tissue pathological sections. However, due to its certain disintegration efficiency, the overall drug retention effect is better than that of Comparative Example 5. The liposomes retained in the alveoli can achieve a low proportion of drug release by relying on less polypeptide degradation, and the drug distribution in the alveoli is significantly lower than that in Example 1, but significantly higher than that in Comparative Example 5. Moreover, although the ratio of PEG-(SP-B) to phospholipid is reduced, the drug that is not excreted with respiratory secretions can still be finally degraded after a long period of retention in the alveoli, so as not to significantly block the gas exchange of the "gas-liquid barrier"; nor will it cause large-scale transfection and endocytosis of alveolar epithelial cells after continuous contact with the alveolar epithelial cell membrane, leading to cytotoxicity.

[0085] The liposome body of Comparative Example 3 is the natural main component of alveolar surfactant, dipalmitoylphosphatidylcholine (DPPC), and the enzyme-sensitive response substrate is the angiotensin-converting enzyme 2 (ACE2) enzyme on the alveolar epithelial cell membrane. This structured liposome contains the ACE2 enzyme itself and cannot be specifically degraded by the natural enzymes of alveolar surface protein in the alveoli or the ACE2 enzyme on the alveolar epithelial cell membrane. During the process of aerosol inhalation distribution to the airway terminal, after contacting the natural enzymes of alveolar surface protein in the alveoli, it cannot be degraded, nor can it induce obvious liposome disintegration, and the efficiency of alveolar surfactant release is extremely low. The relative fluorescence intensity of frozen pathological sections of lung tissue is significantly lower than that of Example 1, and the drug retention effect in the lung is poor; compared with Example 1, this group of drugs does not have the natural enzyme substrate of alveolar surface protein in the alveoli, and the degradation efficiency and speed in the lung are low. The liposomes that cannot disintegrate are discharged through the trachea with airway secretions, resulting in a low relative fluorescence intensity of frozen pathological sections of lung tissue. The liposomes retained in the alveoli are almost unable to disintegrate and release the drug, and the drug distribution in the alveoli is significantly lower than that in Example 1. However, ACE2 itself is a polypeptide / protein and can be non-specifically degraded by proteolytic enzymes in the surface secretions of the alveoli and bronchioles, releasing the drug at a very low rate. Therefore, the relative fluorescence intensity of the frozen pathological sections of the lung tissue is slightly higher than that of Comparative Example 5. Moreover, although the degradation efficiency of PEG-ACE2 is extremely low, its content is relatively large. After a long period of retention in the alveoli, some of the drugs that are not discharged with the respiratory secretions may still be finally degraded by non-specific enzymes, so as not to significantly block the gas exchange of the "gas-liquid barrier"; nor will it cause large-scale transfection and endocytosis of alveolar epithelial cells after continuous contact with the alveolar epithelial cell membrane, leading to cytotoxicity.

[0086] The liposome body of comparative example 4 is the natural main component of alveolar surfactant, dipalmitoylphosphatidylcholine (DPPC), and the enzyme-sensitive response substrate is the specific structure of human coronavirus SARS-CoV-2 for targeting and anchoring ACE-2 enzyme protein on the type 2 epithelial cell membrane on the alveolar surface, and the cell receptor binding domain (Receptor binding domain, RBD) of the viral spike protein. This structural liposome includes the SARS-CoV-2 spike protein RBD segment. The RBD segment can bind to the ACE-2 enzyme protein on the surface of the alveolar epithelial cell membrane itself, thereby promoting ACE-2, RBD and its connected drugs to be endocytosed together into the cytoplasm, but cannot be specifically degraded outside the cell membrane by the natural enzymes of the alveolar surface protein in the alveoli. In the process of aerosol inhalation distribution to the airway terminal, after contacting the natural enzymes of the alveolar surface protein in the alveoli, it is impossible to degrade, nor can it induce obvious liposome disintegration, and the efficiency of alveolar surfactant release is extremely low. However, the RBD segment can specifically bind to ACE-2 on the alveolar epithelial cell membrane of type 2, prompting the drug-containing liposomes to be internalized together with ACE-2, which is manifested as the relative fluorescence intensity of the frozen pathological sections of lung tissue is lower than that of Example 1, but significantly higher than that of Comparative Example 5. Compared with Example 1, this group of drugs does not have a natural enzyme substrate for alveolar surface material proteins in the specific alveoli, but by promoting epithelial cell endocytosis, it achieves targeted retention in the lungs, resulting in a fluorescence intensity higher than that of Comparative Example 5. And its retention in the lungs is epithelial cell endocytosis, and its alveolar retention is positively correlated with the cell membrane expression of ACE-2. Therefore, the relative fluorescence intensity of the frozen pathological sections of lung tissue is higher than that of Comparative Example 5 and other comparative examples. However, this RBD-ACE-2 binding endocytosis method is limited by the amount of ACE-2 protein on the surface of alveolar epithelial cells. After saturated endocytosis of a large number of RBDs, the ACE-2 on the cell membrane is significantly reduced. Before the supplemented ACE-2 is expressed on the cell membrane, the binding efficiency with the RBD decreases, and the amount of drug endocytosis is limited. Liposomes that fail to be internalized are excreted through the trachea with airway secretions, resulting in a lower relative fluorescence intensity in frozen lung tissue pathological sections than in Example 1. Liposomes containing the spike protein RBD segment, after prolonged retention within the alveoli, may eventually come into contact with the alveolar epithelial cell membrane and be internalized into the alveolar epithelial cells. This large-scale internalization of drug components that should otherwise be secreted by alveolar epithelial cells may lead to cytotoxicity.

[0087] In the distribution of Comparative Example 4, it is worth noting that the relative fluorescence intensity of the frozen pathological sections of the oral mucosa in this group is much higher than that of all the examples and all other comparative examples. This is because the oral mucosal epithelium, like the lung epithelial cell membrane, highly expresses ACE2. When the ACE2 on the oral mucosal cell membrane comes into contact with the aerosolized drug, it is bound by the RBD segment of the aerosolized drug, and then guides the liposome drug into the oral mucosal epithelial cells in an endocytic manner through RBD-ACE-2 binding, resulting in an increase in fluorescence intensity and inducing cytotoxicity.

[0088] The liposomes in Comparative Example 5 consist primarily of dipalmitoylphosphatidylcholine (DPPC), a natural component of alveolar surfactant. They are not linked to an enzyme-sensitive polypeptide substrate and lack any groups available for enzymatic degradation within the lungs. During aerosol inhalation, even if liposomes with this structure reach the airway terminals and come into contact with natural enzymes in the alveolar surface material proteins within the alveoli, they are unable to specifically degrade the enzyme-sensitive groups. The liposomes are difficult to disintegrate, and the drug cannot be released into the alveoli. This is manifested by extremely low relative fluorescence intensity in frozen lung tissue pathology sections. Compared to Example 1, this group of drugs lacks an enzyme substrate, resulting in extremely low efficiency and speed of degradation within the lungs. Liposome disintegration is almost negligible, and undegradable liposomes may be expelled through the trachea with airway secretions, resulting in low relative fluorescence intensity in frozen lung tissue pathology sections. Some liposomes retained in the alveoli cannot be degraded in airway secretions, and prolonged retention may block gas exchange at the "gas-liquid barrier." They may even trigger extensive endocytosis by alveolar epithelial cells after continued contact with the alveolar epithelial cell membrane, leading to cytotoxicity.

[0089] The enzyme-sensitive response substrate of Comparative Example 6 does not contain a PEG segment, and its connection stability with the liposome body is extremely poor. After being combined with the liposome, the stability of the liposome itself is poor. Drugs with this structure rupture in large quantities during the ultrasonic atomization process, making it difficult to maintain morphological stability and ensure that the atomized droplets are of a size suitable for alveolar distribution. After the liposomes rupture, the PS contained in them is exhaled into the airway during exhalation, and alveolar targeted distribution cannot be achieved. Therefore, the alveolar distribution efficiency detected by drug fluorescence is much lower than that of Example 1.

[0090] Comments on efficacy trials:

[0091] The main body of the liposomes of Examples 1-8 is dipalmitoylphosphatidylcholine (DPPC), the natural main component of alveolar surfactant, or its analogs phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS); the liposome targeting polypeptide substrates are Surfactant Protein B protein, Surfactant Protein C protein, and Surfactant Protein D protein. These proteins can be naturally degraded by naturally occurring specific enzymes in the lungs. This structure can ensure that the liposomes are distributed to the airway terminals during aerosol inhalation, and the PEG-linked polypeptide or its analogs are degraded after contacting the natural enzymes of the alveolar surface material proteins in the alveoli in the lungs, resulting in efficient disintegration of the liposomes and uniform release of alveolar surface material into the alveoli. After the carrier and the alveolar surfactant contained therein are effectively retained and evenly dispersed in the lungs, they play the expected role of reducing alveolar surface tension, increasing lung compliance, maintaining relatively stable alveolar volumes, preventing atelectasis, and preventing pulmonary edema. Then, it plays a role in promoting alveolar development and improving ventilation and gas exchange function. And the sensitive groups of the drug liposome shell can eventually be degraded by the natural enzymes of the alveolar surface material proteins in the alveoli. The drugs that are not discharged with the respiratory secretions can achieve final disintegration after being retained in the alveoli, so as not to significantly block the gas exchange of the "gas-liquid barrier" and produce obvious toxic and side effects. After the drug disintegrates in time, it will not trigger endocytosis into the alveolar epithelial cells, thus avoiding causing cytotoxicity. Compared with the non-treatment group, after the full course of treatment with the embodiment drugs, the PaO2 of the ventilation and gas exchange function after long-term alveolar development is significantly improved, and is even very close to the healthy control group (Example 1, Example 6). After the drug treatment in this group, compared with the non-treatment group, the lung compliance reflecting the degree of improvement of premature alveolar collapse, fibrosis and other lesions is also significantly improved, and is even very close to the healthy control group.

[0092] The liposome body of Comparative Example 1 is the natural main component of alveolar surfactant, dipalmitoylphosphatidylcholine (DPPC), and the enzyme-sensitive response substrate is an antibody to Surfactant Protein B, an important protein in alveolar surfactant. This structural liposome contains the antibody protein of Surfactant Protein B, which cannot be specifically degraded by the natural enzymes of alveolar surface material proteins in the alveoli. During the process of aerosol inhalation distribution to the airway terminals, after contacting the natural enzymes of alveolar surface material proteins in the alveoli in the lungs, it cannot be degraded, nor can it induce obvious liposome disintegration, and the efficiency of alveolar surfactant release is extremely low. Compared with the non-treated group, after a full course of treatment with this group of drugs, the PaO2 reflecting the ventilation and gas exchange function after long-term development of the alveoli was only slightly improved, but compared with the healthy control group and the embodiment, the gap was larger. After treatment with this group of drugs, the lung compliance reflecting the degree of improvement of lesions such as premature alveolar collapse and fibrosis was not significantly improved, and the gap was larger than that of the healthy control group and the embodiment. This indicates that this group of drugs failed to effectively release alveolar surfactant in the alveoli after treatment, and failed to achieve the effect of evenly distributing alveolar surfactant in the alveoli, reducing alveolar surface tension, increasing lung compliance, maintaining relatively stable alveolar volumes, preventing atelectasis, and preventing pulmonary edema. Furthermore, it failed to promote alveolar development and improve ventilation function. Although the degradation efficiency of Surfactant Protein B antibodies is extremely low, its content is relatively high. After a long period of retention in the alveoli, some of the drugs that are not excreted with respiratory secretions may still be finally degraded by non-specific enzymes, thus not significantly blocking gas exchange at the "gas-liquid barrier" and producing significant toxic side effects.

[0093] The liposome body of Comparative Example 2 is the natural main component of alveolar surfactant, dipalmitoylphosphatidylcholine (DPPC), the polypeptide substrate is Surfactant Protein B protein, and the dosage ratio of PEG-(SP-B) to phospholipid is reduced to 2 / 3 of that of Example 1. The liposomes of this structure contain very little substrate for the natural enzyme degradation of alveolar surface material proteins in the alveoli. During the process of aerosol inhalation distribution to the airway terminal, after contacting the natural enzymes of alveolar surface material proteins in the alveoli, the PEG-linked polypeptide is degraded. However, due to the very low content of the polypeptide linked to PEG, its degradation has little effect on the stability of the liposome, the liposome disintegration efficiency is very low, and the alveolar surfactant release efficiency is very low. Compared with the non-treated group, after a full course of treatment with this group of drugs, the PaO2 reflecting the ventilation and gas exchange function after long-term alveolar development was slightly improved, but compared with the healthy control group and the embodiment, the gap is large. After treatment with this group of drugs, the lung compliance reflecting the degree of improvement of premature alveolar collapse, fibrosis and other lesions was also only slightly improved, but compared with the healthy control group and the embodiment, the gap is large. This suggests that this group of drugs failed to effectively release alveolar surfactant within the alveoli after treatment, failing to achieve the effects of evenly distributing alveolar surfactant within the alveoli, reducing alveolar surface tension, increasing lung compliance, maintaining relatively stable alveolar volumes, and preventing atelectasis and pulmonary edema. Furthermore, they failed to promote alveolar development and improve ventilation and gas exchange.

[0094] The liposome main body of comparative example 3 is the natural main component of alveolar surfactant, dipalmitoylphosphatidylcholine (DPPC), and the enzyme sensitive response substrate is ACE-2 enzyme protein itself.This structure liposome includes ACE-2 enzyme protein itself, can not be for the natural enzyme specific degradation of alveolar surface material protein in alveoli.During atomization inhalation distribution to airway terminal process, after contacting the natural enzyme of alveolar surface material protein in alveoli in lung, it is impossible to degrade, and it is also impossible to induce obvious liposome disintegration, and alveolar surfactant release efficiency is extremely low.Compared to non-treatment group, after the full course of treatment of this group of medicines, the PaO2 of ventilation and gas exchange function after the long-term development of alveoli is reflected.It is larger than that of healthy control group and embodiment.After this group of drug treatment, the lung compliance reflecting the improvement degree of lesions such as premature alveolar collapse, fibrosis is only slightly improved, but it is larger than that of healthy control group and embodiment. This indicates that this group of drugs failed to effectively release alveolar surfactant within the alveoli after treatment, and failed to achieve the effects of evenly distributing alveolar surfactant within the alveoli, reducing alveolar surface tension, increasing lung compliance, maintaining relatively stable alveolar volumes, preventing atelectasis, and preventing pulmonary edema. Furthermore, it failed to promote alveolar development and improve ventilation function. Although the "PEG-ACE-2 enzyme protein" response fragment used in this group of drugs has an extremely low degradation efficiency, its content is relatively high. After a long period of retention in the alveoli, some of the drugs that are not excreted with respiratory secretions may still be finally degraded by non-specific enzymes, thus preventing significant toxic side effects from significantly blocking gas exchange at the "gas-liquid barrier."

[0095] The liposome body of comparative example 4 is the natural main component of alveolar surfactant, dipalmitoylphosphatidylcholine (DPPC), and the enzyme-sensitive response substrate is the specific structure of the human coronavirus SARS-CoV-2 for targeting and anchoring the ACE-2 enzyme protein on the alveolar surface epithelial cell membrane, the RBD segment of the spike protein. This structural liposome contains the SARS-CoV-2 spike protein RBD segment. The RBD segment can bind to the cell membrane surface ACE-2 enzyme protein, thereby promoting ACE-2, RBD and its connected drugs to be endocytosed into the cytoplasm together, but cannot be used for the natural enzymes of the alveolar surface material protein in the alveoli to specifically degrade outside the cell membrane. This group of drugs does not have a natural enzyme substrate for the alveolar surface material protein in the specific alveoli, but by targeting and anchoring the ACE-2 enzyme itself, it promotes epithelial cell endocytosis and achieves targeted retention in the lungs. The degradation efficiency and speed in the alveoli are extremely low, and the distribution amount in the alveolar epithelial cells is extremely high. After liposomes containing the spike protein RBD segment bind to ACE-2 and are internalized into alveolar epithelial cells, the cells are forced to internalize large quantities of the drug components that should otherwise be secreted and excreted, resulting in significant cytotoxicity. Compared to the untreated group, after a full course of treatment with this drug, the PaO2 of alveolar ventilation and gas exchange, which is the function of long-term alveolar development, not only did not improve, but instead significantly deteriorated and decreased. Lung compliance, which reflects the degree of improvement in alveolar collapse and fibrosis, also deteriorated significantly after treatment with this drug. This suggests that this drug treatment failed to effectively release alveolar surfactant within the alveoli, failing to achieve the desired effect of evenly distributing the surfactant within the alveoli, reducing alveolar surface tension, increasing lung compliance, maintaining relatively stable alveolar volumes, and preventing atelectasis and edema. Furthermore, it failed to promote alveolar development and improve gas exchange and gas exchange. This suggests that the drug induced large-scale internalization of the drug by alveolar epithelial cells, resulting in cytotoxicity to these cells and, in turn, significant toxic side effects in the lung tissue and systemic tissue.

[0096] The liposomes in Comparative Example 5 consist primarily of dipalmitoylphosphatidylcholine (DPPC), a natural component of alveolar surfactant. They are not linked to enzyme-sensitive polypeptide substrates and lack any groups available for enzymatic degradation. During aerosol inhalation, even if liposomes with this structure reach the airway terminals and come into contact with natural enzymes on alveolar surface proteins within the alveoli, they are unable to specifically degrade the enzyme-sensitive groups, resulting in poor liposome disintegration and drug release into the alveoli. This group of drugs lacks enzyme substrates, resulting in extremely low degradation efficiency and speed within the lungs. Liposome disintegration is almost negligible, and undegradable liposomes are expelled through the trachea with airway secretions. Some liposomes retained in the alveoli cannot be degraded in airway secretions, and prolonged retention can block gas exchange at the "gas-liquid barrier." They may even trigger massive endocytosis by alveolar epithelial cells after continued contact with the alveolar epithelial cell membrane, leading to cytotoxicity. Compared to the untreated group, after a full course of treatment with this drug, the PaO2, reflecting the ventilation and gas exchange function of the alveoli after long-term development, did not improve but instead worsened. After treatment with this group of drugs, lung compliance, which reflects the degree of improvement in premature alveolar collapse and fibrosis, also worsened. This suggests that these drugs failed to effectively release alveolar surfactant within the alveoli, failing to evenly distribute alveolar surfactant within the alveoli, thereby reducing alveolar surface tension, increasing lung compliance, maintaining relatively stable alveolar volumes, and preventing atelectasis and pulmonary edema. Furthermore, they failed to promote alveolar development and improve ventilation. Instead, they restricted animal development by blocking gas exchange across the "air-liquid barrier" and were toxic to alveolar epithelial cells by inducing endocytosis, resulting in significant side effects.

[0097] The enzyme-sensitive response substrate of Comparative Example 6 does not contain a PEG segment, and its connection stability with the liposome body is extremely poor. After being incorporated into the liposomes, the liposomes themselves are less stable. Drugs with this structure rupture in large quantities during ultrasonic atomization, making it difficult to maintain a stable morphology and ensure that the atomized droplets are of a size suitable for alveolar distribution. After the liposomes rupture, the contained PS is exhaled out of the airways during exhalation, preventing targeted alveolar distribution. Compared to the untreated group, after a full course of treatment with this drug, the PaO2, reflecting the ventilation and gas exchange function of the alveoli after long-term development, did not significantly improve, and the difference was significant compared to the healthy control group and the examples. After treatment with this drug, lung compliance, which reflects the degree of improvement in premature alveolar collapse, fibrosis, and other lesions, was significantly different from the healthy control group and the examples. This suggests that after treatment with this drug, the alveolar surfactant was not effectively delivered to the alveoli, and failed to achieve the desired effect of evenly distributing the surfactant in the alveoli to reduce alveolar surface tension, increase lung compliance, maintain relatively stable alveolar volumes, prevent atelectasis, and prevent pulmonary edema. Furthermore, it cannot promote alveolar development and improve ventilation function. Although the response fragments used in this group of drugs are not PEG-modified and have extremely low binding stability, their content is relatively high. After a long period of retention in the alveoli, some of the drugs that are not excreted with respiratory secretions may still be degraded by nonspecific enzymes, triggering drug release, thereby preventing the significant obstruction of gas exchange at the "gas-liquid barrier" and causing significant toxic side effects.

[0098] Toxicity experiment review:

[0099] The alveolar targeted delivery system of alveolar surfactant prepared by the present invention (Examples 1-7, and Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 6) did not show liver, kidney, and oral mucosal toxicity after long-term aerosol inhalation. However, Comparative Examples 4 and 5, due to the gas exchange that blocks the "gas-liquid barrier", limit the development of animals, and due to the induction of alveolar epithelial cell endocytosis, cause obvious lung toxicity and side effects on alveolar epithelial cells and other factors. In addition to the lungs, oral mucosa highly expresses ACE2. In the oral ulcer severity score, Comparative Example 4 inevitably contacts the oral mucosa of animals due to the liposomes after aerosolization. The SARS-CoV-2 spike protein RBD segment on the surface of the liposomes will bind to ACE2 on the oral mucosal epithelial cell membrane, resulting in RBD-ACE2 targeted binding. After the drug is transfected into the epithelial cells in large quantities, the oral mucosal epithelial cells engulf a large amount of drugs containing alveolar surfactant, produce obvious cytotoxicity, and lead to an increase in oral ulcers.

[0100] Test methods for embodiments and comparative examples:

[0101] (1) Vesicle particle size: The average particle size of the samples 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. Each sample was repeated three times.

[0102] (2) Establishment of premature pulmonary hypoplasia model and group treatment:

[0103] Healthy adult SPF Sprague-Dawley (SD) rats weighing 300-300 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 the vaginal specimen. 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 infant model. Pups born naturally on D22-23 served as normal controls.

[0104] 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. Both the premature mice and the nurses were placed simultaneously in an oxygen chamber connected to a nebulizer and randomly assigned to treatment groups.

[0105] The process of aerosol treatment:

[0106] 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.

[0107] Research Groups:

[0108] Treatment group: Premature rats, drug suspension in PBS, nebulized inhalation throughout the day.

[0109] Premature control: premature mice, PBS solution, nebulized inhalation throughout the day.

[0110] Normal control: naturally delivered pups, PBS solution, nebulized inhalation throughout the day.

[0111] (3) Evaluation of drug alveolar targeted delivery function and distribution after treatment:

[0112] The drug was fluorescently labeled with CY7 according to the method published in our group's previous research [Redox Biology, 2021, 42:101908]. Three hours after aerosol inhalation of the CY7 fluorescently labeled drug, lung tissue, lung tissue, and oral mucosal tissue from each group were collected and frozen pathological sections were prepared. The fluorescence intensity of the sections from Example 1 was used as a baseline to evaluate the fluorescence intensity of the tissue sections from the other examples and comparative examples.

[0113] (4) Evaluation of treatment effect

[0114] To demonstrate the therapeutic effect on pulmonary ventilation and systemic oxygen levels in premature rats, arterial blood samples were collected from the right common carotid artery of the rats using a heparinized polyethylene catheter. PaO2 in the samples was analyzed using an automated blood gas analyzer.

[0115] Experimental mice were anesthetized with an intraperitoneal injection of 0.5 mL / kg chloral hydrate. Following anesthesia, the rats were immobilized, the trachea was cut, and endotracheal intubation was performed. A dedicated instrument, the RC Airway Resistance and Lung Compliance Measurement System, was connected to the airway opening of the experimental mice to measure the dynamic lung compliance (Cdyn, in mL / cmH2O) of the newborn rats. This indicator reflects changes in lung elasticity, such as preterm atelectasis, microvesicular atrophy, fibrosis, edema, and inflammation.

[0116] (5) Evaluation of therapeutic toxicity:

[0117] To assess the hepato-renal toxicity of inhaled drugs in animals, liver function markers were tested. After the treatment period, blood was drawn from the tail vein of the rats to measure blood urea nitrogen (BUN), a marker of renal function, and alanine transaminase (ALT), a marker of liver function. The assays were performed using a Hitachi 7600 fully automated biochemical analyzer. The oral cavity of premature mice was observed after anesthetization in the same manner as above, and the severity of ulcer was evaluated according to the indicators reported in the literature [[1] Standard for the preparation of oral ulcer animal model (draft) [J]. Pharmacology and Clinical Medicine of Traditional Chinese Medicine, 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) yellow-white pseudomembrane covering the ulcer surface; (4) depression in the center and neatly raised edges; (5) swollen lips and drooling; (6) reduced food intake and weight loss. The above-mentioned apparent indicators were quantitatively scored and divided into 3 levels: none, mild, and significant, with scores of 0, 1, and 2, respectively.

Claims

1. A non-invasive alveolar-targeted drug delivery vehicle capable of inhalation administration via the respiratory tract, characterized in that: The invention comprises vesicles and alveolar surfactant loaded in the vesicles. The vesicles are composed of 40-50 mol% liposome main body, 40-50 mol% cholesterol and 6-15 mol% enzyme response substrate grafted PEG in molar percentage; in terms of weight percentage of alveolar targeted non-invasive drug delivery carrier through the respiratory tract, 10-30wt% of alveolar surfactant is loaded, and the average particle size of the vesicles is 50~1000nm.

2. The alveolar-targeted non-invasive drug delivery carrier capable of inhalation administration through the respiratory tract according to claim 1, characterized in that: The enzyme response substrate is selected from at least one of SP-B, SP-C, and SP-D; the liposome body is selected from at least one of dipalmitoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine; and the alveolar surfactant is selected from at least one of animal-derived alveolar surfactants and synthetic alveolar surfactants.

3. The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of inhalation administration via the respiratory tract according to claim 1 or 2, characterized in that: The following steps are involved: Step A: dissolving the enzyme response substrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 4-(dimethylamino)pyridine in a molar ratio of 1:(30-70):(10-50) in solvent A, under inert gas protection, and stirring at 0-5°C to activate the carboxyl group; Step B: Based on the molar amount of the enzyme-responsive substrate, 15-25 times of mPEG-NHS was added, and the reaction was carried out at 0-5°C under inert gas protection to obtain the enzyme-responsive substrate grafted PEG; Step C: dissolving the enzyme-responsive substrate grafted with PEG, cholesterol, and liposome main body in solvent B at a molar ratio of 1:(3-7):(3-7), adding alveolar surfactant, and evaporating the solvent under reduced pressure to obtain a liposome film composited with alveolar surfactant; Step D: Add PBS solution to the liposome film of the above-mentioned composite alveolar surfactant, disperse it by ultrasonic wave to form vesicles, adjust the vesicle particle size by controlling the ultrasonic treatment time, and then add physiological saline to disperse it to obtain an alveolar-targeted non-invasive drug delivery carrier that can be inhaled through the respiratory tract.

4. The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of inhalation administration through the respiratory tract according to claim 3, characterized in that: The enzyme response substrate is selected from at least one of SP-B, SP-C, and SP-D, and has a molecular weight range of 34,000-70,000 Da; the liposome body is selected from at least one of dipalmitoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine; and the molecular weight range of the mPEG-NHS is 1,000-3,000 Da.

5. The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of inhalation through the respiratory tract according to claim 3, characterized in that: The solvent A is selected from at least one of acetonitrile / water solution and DMSO / water solution.

6. The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of inhalation administration through the respiratory tract according to claim 3, characterized in that: The reaction time of step B is 48-96 hours.

7. The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of inhalation administration through the respiratory tract according to claim 1, characterized in that: In step C, the conditions for removing the solvent under reduced pressure are a temperature of 30-45° C. and a pressure of 10-150 MPa; and the concentration of the alveolar surfactant is 30-40 mg / mL.

8. The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of inhalation administration through the respiratory tract according to claim 3, characterized in that: The solvent B is selected from at least one of dichloromethane, chloroform, and chloroform / methanol.

9. The method for preparing the alveolar-targeted non-invasive drug delivery carrier capable of inhalation administration through the respiratory tract according to claim 3, characterized in that: In step D, the ultrasonic treatment time ranges from 3 to 15 minutes.

10. Use of the drug delivery carrier according to claims 1-2, characterized in that: Used for preparing medicines for treating lung diseases associated with alveolar surfactant deficiency.

Images