Environmental response type nano-carrier for inhalation administration and preparation method of environmental response type nano-carrier

Abstract

The invention relates to an environmental response type nano-carrier for inhalation administration and a preparation method, the average particle size of the nano-carrier is 30-700 nm, and the nano-carrier comprises alveolar surface active substances and environmental response lipid; the molar ratio of the alveolar surface active substance to the environment-responsive lipid is (40-99): (1-60). The invention also provides a nano-drug combined preparation using the environmental response type nano-carrier, the nano-drug combined preparation comprises a drug, and the drug is encapsulated in the environmental response type nano-carrier; or the drug is loaded on the environmental response type nano-carrier. The environment-responsive nano-carrier and the nano-drug combined preparation thereof can be used for preparing drugs for treating lung diseases, the mucus penetrating effect is remarkable, and drugs can be provided for the lung through inhalation administration, so that the drugs can reach far-end lung tissue and are responsively released at the focus.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an environment-responsive nanocarrier for inhalation drug administration and a preparation method thereof. Background Art

[0002] The lungs are directly connected to the external environment and have a large specific surface area and rich blood supply, so they are easily invaded by pathogenic microorganisms. Many tumors often metastasize to the lungs through various pathways. However, the drug utilization rate of traditional oral drugs in the lungs is poor, which poses a challenge to clinical treatment.

[0003] At present, several inhaled drugs have been reported in this field, mainly including the following categories: Steroid drugs (Steroids): such as fluticasone, beclomethasone, etc., used to control inflammation and asthma and other diseases. Long-acting bronchodilators (Long-acting Beta-agonists, LABA): such as salmeterol, formoterol, etc., used to treat asthma and chronic obstructive pulmonary disease (COPD). Short-acting bronchodilators (Short-acting Beta-agonists, SABA): such as salbutamol, terbutaline, etc., used for emergency relief during acute asthma attacks. Anticholinergic drugs (Anticholinergics): such as ipratropium bromide, tiotropium bromide, etc., used to treat respiratory diseases such as COPD. All of the above inhaled drugs are small molecule drugs, which are limited by airway anatomy and physiological barriers and are difficult to deposit in the lower respiratory tract.

[0004] Patent application CN118286157A discloses an inhalable bionic liposome for the treatment of in situ and metastatic lung cancer, and its preparation method and application, wherein the inhalable bionic liposome at least carries a recombinant plasmid and an epidermal growth factor receptor tyrosine kinase inhibitor; wherein the recombinant plasmid contains m6A regulatory key gene siRNA; and the liposome is a membrane-modified liposome, and the membrane can be a cell membrane, and the cell membrane can express pulmonary surfactant. The invention provides an inhalable liposome based on pulmonary surfactant biomimetic construction, which can effectively promote the liposome loaded with therapeutic drugs to cross the biological barrier, deeply penetrate the lung lesion tissue, and achieve the treatment of brain metastasis lung cancer, so it has good practical application value.

[0005] Patent application CN114765953A discloses a composition and method containing pulmonary surfactant (PS) biomimetic nanoparticles. The composition contains nanoparticles with an average size of 200-400nm, the nanoparticles include a variety of pulmonary surfactant biomimetic molecules, wherein the nanoparticles are negatively charged; and one or more cargo molecules encapsulated by the nanoparticles, wherein the molecular weight of the cargo molecules is less than 1200Da. The composition is usually formulated to be compatible with its intended route of administration, such as inhalation through the nose.

[0006] The circulation of pulmonary surfactant on the alveolar surface is like a natural "channel" that directly accesses lung cells, which can guide drugs to be delivered to the deep lungs via natural transport pathways. Therefore, the use of pulmonary surfactant biomimetic strategies is an effective means to achieve efficient delivery of inhaled drugs to lung tissue. The nanoformulations reported in the above literature and the high-end inhalation nanoformulations amikacin liposome solutions that have been marketed abroad have good drug delivery performance, but these are all indiscriminate and indiscriminate drug delivery, making it difficult to identify lesions, often resulting in drug distribution throughout the lungs, low effective drug utilization, limited therapeutic effects, and obvious side effects.

[0007] Therefore, how to rationally design the inhaled drug carrier according to the microenvironment of lung lesions, while improving its penetration performance, to achieve precise drug release in the lesion site without releasing it in normal lung tissue is an urgent problem to be solved by technical personnel in this field. Summary of the invention

[0008] In view of the defects existing in the above-mentioned prior art, the present invention provides an environment-responsive nanocarrier for inhalation drug administration and a preparation method, a nano-drug combination preparation containing the environment-responsive nanocarrier and a preparation method, and the application of the environment-responsive nanocarrier and the nano-drug combination preparation. The present invention uses alveolar surfactant and biocompatible lipid components to construct an inhalation drug administration nanocarrier with environmental responsive release ability, and provides a safe, reliable, and mass-producible new lung inhalation nanocarrier preparation process, which can achieve effective loading or encapsulation of small molecule drugs and can achieve effective release of drugs under the stimulation of the lung pathological environment.

[0009] The present invention provides an environment-responsive nanocarrier for inhalation administration, wherein the average particle size of the nanocarrier is 30-700 nm; The nanocarrier comprises alveolar surfactant and environmentally responsive lipids; The molar ratio of alveolar surfactant to environmentally responsive lipids is (40-99):(1-60).

[0010] The drug composition of the present invention is simple, the raw materials are easily available, the preparation process is simple and the quality is controllable, and it is easy to mass produce and transform for application. Among them, the nanoscale inhaled drug using the bionic strategy of pulmonary surfactant ensures efficient penetration and delivery to lung tissue, and cooperates with the high responsiveness and selectivity of environmentally responsive lipids to avoid side effects caused by the distribution of drugs throughout the lungs and make up for the defect of insufficient drug concentration in the lesion site. According to the difference in the medication environment, the molar ratio of alveolar surfactant to environmentally responsive lipids can be further optimized to (42-98.5): (1.5-58), and more preferably (50-95): (5-50).

[0011] Preferably, the alveolar surfactant includes at least one of natural alveolar surfactant and artificially synthesized alveolar surfactant; The environmentally responsive lipids include one or more of temperature-responsive lipids, pH-responsive lipids, ROS-responsive lipids, and multi-responsive lipids.

[0012] When exposed to external stimuli such as temperature, pH, inflammation, ultrasound and light, the responsive liposomes show the characteristics of triggering drug release. According to the characteristics of the lesion environment of lung diseases, it is preferred to use multiple responsive lipids, combined with temperature responsive lipids, ROS responsive lipids or pH responsive lipids, so that the carrier can respond to environmental conditions more sensitively, accurately identify lesions and release drugs.

[0013] Preferably, the environmentally responsive lipids include multi-responsive lipids and / or pH-responsive lipids; Multi-responsive lipids include DSPE-PEG2000; The pH-responsive lipids include ionized lipids, and the ionized lipids are selected from at least one of quaternary ammonium lipids, ionizable lipids, and biodegradable lipids.

[0014] The multi-responsive lipid DSPE-PEG2000, also known as phospholipid-polyethylene glycol-azobenzene, is a special compound composed of phospholipids (DSPE), polyethylene glycol (PEG2000) and azobenzene (Azo), which has good temperature responsiveness, ROS responsiveness and light responsiveness. The nanocarrier particles of DSPE-PEG2000 can maintain particle stability and drug protection at 37°C and below, and can show burst release performance when the temperature rises to 42°C and above. The ROS responsiveness of DSPE-TK-PEG2000 enables it to release drugs in the specific physiological environment of the lesion, improve the therapeutic effect and reduce side effects. Based on the light responsiveness of azobenzene, the compound can respond to light stimulation of a specific wavelength to achieve controlled release of drugs. Similar to DSPE-PEG2000 are DSPE-PEG3400, DSPE-PEG5000, etc.

[0015] The pH-responsive lipids include ionized lipids, and the ionized lipids are selected from at least one of quaternary ammonium lipids, ionizable lipids, and biodegradable lipids. When multiple responsive lipids and pH-responsive lipids are used in combination, the ionized lipids in the pH-responsive lipids preferably account for 10-50% of the total mass of the environment-responsive lipids.

[0016] For the specific types of ionized lipids, preferably, the quaternary ammonium lipids include DOTMA; the ionizable lipids are selected from at least one of 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP), DLinDMA, DLin-KC2-DMA, DLin-MC3-DMA, SM-102, ALC-0315, and ALC-0159; and the biodegradable lipids include L319.

[0017] The ROS-responsive lipids include at least one of sulfur-containing polymers, selenium-containing polymers, tellurium-containing polymers, unsaturated lipids, and thioether phosphatidylcholine (SPC).

[0018] Preferably, the alveolar surfactant includes artificially synthesized alveolar surfactant; In terms of percentage, synthetic alveolar surfactant includes the following components: Phospholipids 70-90 % Apolipoprotein 0-15 % Neutral lipids 5-30%; The phospholipids include at least one of phosphatidylcholine and phosphatidylglycerol; The apolipoprotein includes at least one of surfactant protein A (SP-A), B (SP-B), C (SP-C), and D (SP-D); Neutral lipids include cholesterol.

[0019] Optionally, the phospholipids also include secondary phospholipids, the content of secondary phospholipids accounts for 5-10% of the total weight of phospholipids, and the secondary phospholipids are selected from at least one of phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), and lysophosphatidylcholine (LPC).

[0020] In a second aspect, the present invention provides a method for preparing the environmentally responsive nanocarrier, comprising the following steps: Step 1: mixing alveolar surfactant, environmentally responsive liposomes and a first solvent to obtain a carrier mixture; Step 2, evaporating and drying the carrier mixture to obtain a lipid film; Step 3, mixing the lipid film, the second solvent, and an optional stabilizer, and performing ultrasonic hydration treatment; Step 4: Microfluidization high pressure emulsification to obtain nanoemulsion, and dialysis to remove impurities to obtain the environmentally responsive nanocarrier.

[0021] The environmentally responsive nanocarrier of the present invention can be used alone or in combination with other drugs. The above-mentioned preparation method can simply and efficiently obtain the environmentally responsive nanocarrier product.

[0022] In a third aspect, the present invention provides a nano-drug combination preparation, comprising the environmentally responsive nano-carrier and a drug, wherein: The drug is encapsulated in the environmentally responsive nanocarrier; or The drug is loaded on the environmentally responsive nanocarrier.

[0023] When the environmentally responsive nanocarrier is combined with a drug to obtain a nano-drug combination preparation, the combination method with the nanocarrier is reasonably selected according to the physicochemical properties and biological properties of the small molecule drug. For example, when a hydrophobic small molecule drug is used, it is preferred that the drug is dispersed and loaded on the environmentally responsive nanocarrier, but the encapsulation type drug loading method is not excluded. When a hydrophilic small molecule drug is used, it is more preferred that the drug is encapsulated in the environmentally responsive nanocarrier.

[0024] When the encapsulation method is adopted, the present invention also provides a method for preparing the nano-drug combination preparation, comprising the following steps: S1-1, mixing alveolar surfactant, environmentally responsive liposomes and a first solvent to obtain a carrier mixture; S1-2, evaporating and drying the carrier mixture to obtain a lipid film; S1-3, mixing the lipid film, the drug to be encapsulated, the stabilizer and the second solvent, and performing ultrasonic hydration treatment; S1-4, microfluidization high pressure emulsification to obtain nanoemulsion, dialysis to remove impurities, and obtain a nano drug combination preparation in which the drug is encapsulated by a nano carrier.

[0025] In the process of preparing the environmentally responsive nanocarrier, the drug to be encapsulated is added to a solution containing a lipid film, and an encapsulation shell of the environmentally responsive nanocarrier is formed on the surface of the drug to be encapsulated. Preferably, the encapsulation rate of the drug in the environmentally responsive nanocarrier is above 70%.

[0026] When the loading method is adopted, the preparation method of the aforementioned environmentally responsive nanocarrier can be referred to, and the drug to be loaded is added in step 1, so that the drug is loaded between the liposome phospholipid bilayers during the formation of the nanocarrier to achieve uniform drug loading. The preparation process of this method is simple and easy to control, and the drug has good dispersion in the carrier, and the drug loading rate is above 50%. It includes the following steps: S2-1, mixing alveolar surfactant, environmentally responsive liposomes, a drug to be loaded and a first solvent to obtain a mixture; S2-2, evaporating and drying the mixture to obtain a lipid film; S2-3, mixing the lipid film, the stabilizer and the second solvent, and performing ultrasonic hydration treatment; S2-4, microfluidization high pressure emulsification to obtain nanoemulsion, dialysis to remove impurities, and obtain a nano drug combination preparation in which the drug is loaded by a nano carrier.

[0027] Furthermore, in the method for preparing the nano-drug combination preparation and / or the method for preparing the environmentally responsive nano-carrier, similar process conditions may be used, and at least one of the following conditions may be satisfied: 1) The first solvent is an organic solvent selected from chloroform or a mixed solution of chloroform and methanol in any proportion; 2) the second solvent is one of deionized water, physiological saline, sucrose solution, mannitol solution or phosphate buffer; 3) the stabilizer is selected from at least one of glycerol, olive oil, tea tree oil, oleic acid, Tween 80, Tween 20, Span 20, carboxymethyl cellulose, polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG), and the mass ratio of the stabilizer to the lipid film is (2-30): (16-25); 4) Evaporation and drying treatment, including evaporating the first solvent to dryness at 40-50°C, and then drying in a vacuum drying oven at 30-40°C for 5-15 hours; 5) Ultrasonic hydration treatment, including ultrasonic treatment in a water bath at 25-35°C for 5-15 minutes, preferably 6-12 minutes, and then ultrasonic treatment in an ice bath for 1-8 minutes, preferably 2-6 minutes. The power of the ultrasonic treatment is 150-950W, preferably 200-850W.

[0028] The particle size of the environmentally responsive nanocarrier or nano-drug combination preparation of the present invention can be adjusted by regulating the ultrasonic power and action time. Under the above action conditions, a product with a particle size of 30-700 nm and better comprehensive performance can be obtained.

[0029] Preferably, the microfluidization high pressure emulsification is performed for 10-30 min.

[0030] The present invention also provides the use of the environmentally responsive nanocarrier or nano-drug combination preparation in the preparation of drugs for treating lung diseases.

[0031] The present invention has at least the following beneficial effects: 1. The nanocarrier prepared by the present invention is a nano drug delivery carrier based on alveolar surfactant and environmentally responsive lipids, has good biosafety and stability, and the raw materials are easily obtained, the preparation process is relatively simple, and it can be mass-produced and easily transformed and applied.

[0032] 2. The environmentally responsive nanocarrier and nanodrug combination prepared by the present invention can respond to changes in lung lesions. For example, it is neutrally charged in the neutral environment of normal lungs, thereby reducing the interaction with the respiratory mucosa and diffusing to the distal lung tissues. In the acidic environment of the lesion, it is ionized with a positive charge, thereby facilitating effective internalization by cells and enhancing the effective utilization of the loaded drugs.

[0033] 3. The nanocarriers of the present invention can achieve responsive release in the microenvironment of inflammatory lesions. Compared with existing non-responsive carriers, the combined preparation of environmentally responsive nanocarriers and nanodrugs can adapt well to the pathological environment of the lungs, achieve precise drug release, further improve the effective utilization rate of drugs and have good biocompatibility.

[0034] 4. The nanocarrier and nanodrug combination preparation with intelligent response characteristics prepared by the preparation method of the present invention can overcome the disadvantage of poor water solubility of poorly soluble drugs and greatly improve the bioavailability of the loaded small molecule drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the particle size distribution diagram of the sample of Example 3 of the present invention; Figure 2 This is the potential distribution diagram of the sample of Example 3 of the present invention; Figure 3 This is a comparison chart of particle sizes of samples from Example 3 of the present invention obtained by continuous testing for 28 days; Figure 4 This is a PDI comparison chart of the sample of Example 3 of the present invention tested for 28 consecutive days; Figure 5 This is a comparison diagram of the particle size distribution of the sample in Example 3 of the present invention before and after being atomized by the atomization device; Figure 6 This is a hemolysis experiment diagram of the sample in Example 8 of the present invention; Figure 7 This is a graph showing the effect of different dilution multiples of the sample of Example 8 of the present invention on the activity of BEAS-2B cells; Figure 8 The standard curve of the sample in Example 8 of the present invention established by high performance liquid chromatography; Fig. 9 This is a graph showing the ultraviolet absorption characteristics of the sample of Example 8 of the present invention; Fig.10This is a particle size distribution diagram of the sample of Example 3 of the present invention incubated in H2O2 solutions of different concentrations for 4 hours; Fig.11 This is a fluorescence image showing the effect of the sample in Example 8 of the present invention on NIH-3T3 cell apoptosis; Fig.12 This is a graph showing the surface charge changes of the sample of Example 8 of the present invention after incubation with PBS solutions of different pH values ​​for 2 hours; Fig.13 The samples prepared in Example 3 and Example 8 of the present invention were co-incubated with a PBS solution with a pH of 6.5, and the surface charge changes were compared at different time points; Fig.14 This is a comparison chart of mucus penetration of samples prepared in Example 3 and Example 8 of the present invention; Fig.15 A comparison diagram of the movement trajectories of the samples prepared in Example 3 and Example 8 of the present invention in mucus; Fig.16 This is a comparison diagram of the uptake of the sample of Example 8 of the present invention by human bronchial epithelial cells NIH-3T3; Fig.17 This is a comparison diagram of the uptake of the sample of Example 8 by NIH-3T3 cells before and after atomization measured by flow cytometry; Fig.18 This is a comparison chart of the inhibitory effects of the samples of Example 8 of the present invention on NIH-3T3 cell activation and migration; Fig.19 This is a biodistribution diagram of the sample of Example 8 of the present invention in mice after aerosol inhalation; Fig. 20 This is a comparison of MRI images of lung tissues of mice with pulmonary fibrosis after different treatments in an in vivo animal experiment; Fig.21 This is a comparison of H&E staining of lung tissues of mice in different treatment groups in the in vivo animal experiment. DETAILED DESCRIPTION

[0036] In order to better understand the above technical solution, the above technical solution will be described in detail below in combination with the specification and specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings, and "multiple" generally includes at least two.

[0038] It should also be noted that the term "includes", "comprising" or any other variation thereof is intended to cover non-exclusive inclusion, so that a commodity or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such commodity or device. In the absence of more restrictions, the elements defined by the sentence "comprising a ..." do not exclude the existence of other identical elements in the commodity or device including the elements.

[0039] The first part is an environmentally responsive nanocarrier for inhalation administration and a preparation method thereof. The average particle size of the nanocarrier is 30-700 nm and can be used to prepare drugs for treating lung diseases. The nanocarrier comprises alveolar surfactant and environmentally responsive lipids, and the molar ratio of the two is (40-99): (1-60), preferably (42-98.5): (1.5-58), and more preferably (50-95): (5-50).

[0040] (1) Alveolar surfactant, including at least one of natural alveolar surfactant and synthetic alveolar surfactant; preferably synthetic alveolar surfactant, wherein the synthetic alveolar surfactant comprises the following components in percentage: Phospholipids 70-90 % Apolipoprotein 0-15 % Neutral lipids 5-30%; The phospholipids include at least one of phosphatidylcholine and phosphatidylglycerol; Optionally, the phospholipids further include secondary phospholipids, the content of the secondary phospholipids accounts for 5-10% of the total weight of the phospholipids, and is selected from at least one of phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), and lysophosphatidylcholine (LPC); The apolipoprotein includes at least one of surfactant protein A (SP-A), B (SP-B), C (SP-C), and D (SP-D); Neutral lipids include cholesterol.

[0041] (2) Environmentally responsive lipids, including one or more of temperature-responsive lipids, pH-responsive lipids, ROS-responsive lipids, and multi-responsive lipids; preferably, multi-responsive lipids and / or pH-responsive lipids.

[0042] The multi-responsive lipids preferably include DSPE-PEG2000, and DSPE-PEG3400 and DSPE-PEG5000 similar to DSPE-PEG2000 may also be used.

[0043] The pH-responsive lipids include ionized lipids, which are selected from at least one of quaternary ammonium lipids, ionizable lipids, and biodegradable lipids. When multiple responsive lipids and pH-responsive lipids are used in combination, the ionized lipids in the pH-responsive lipids account for 10-50% of the total mass of the environment-responsive lipids. Wherein: Quaternary ammonium lipids include DOTMA; An ionizable lipid selected from at least one of 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP), DLinDMA, DLin-KC2-DMA, DLin-MC3-DMA, SM-102, ALC-0315, and ALC-0159; Biodegradable lipids include L319.

[0044] The ROS-responsive lipids include at least one of sulfur-containing polymers, selenium-containing polymers, tellurium-containing polymers, unsaturated lipids, and thioether phosphatidylcholine (SPC).

[0045] The method for preparing an environmentally responsive nanocarrier comprises the following steps: Step 1: mixing alveolar surfactant, environmentally responsive liposomes and a first solvent to obtain a carrier mixture; Step 2, evaporating and drying the carrier mixture to obtain a lipid film; Step 3, mixing the lipid film, the second solvent, and an optional stabilizer, and performing ultrasonic hydration treatment; Step 4: Microfluidization high pressure emulsification to obtain nanoemulsion, and dialysis to remove impurities to obtain the environmentally responsive nanocarrier.

[0046] The second part is a nano-drug combination preparation containing the aforementioned environmentally responsive nano-carrier and a preparation method thereof, wherein the nano-drug combination preparation also includes a drug, and the nano-drug combination preparation has two drug-carrying forms: (1) The drug is encapsulated in the environmentally responsive nanocarrier, and the encapsulation rate is preferably above 70%. The preparation steps include: S1-1, mixing alveolar surfactant, environmentally responsive liposomes and a first solvent to obtain a carrier mixture; the first solvent is an organic solvent selected from chloroform or a mixed solution of chloroform and methanol in any proportion; S1-2, evaporating and drying the carrier mixture, comprising evaporating the first solvent to dryness at 40-50° C., and then drying in a vacuum drying oven at 30-40° C. for 5-15 hours to obtain a lipid film; S1-3, the lipid film, the drug to be encapsulated, the stabilizer and the second solvent are mixed and subjected to ultrasonic hydration treatment, including ultrasonic treatment in a water bath at 25-35°C for 5-15 minutes, preferably 6-12 minutes, and then ultrasonic treatment in an ice bath for 1-8 minutes, preferably 2-6 minutes. The power of the ultrasonic treatment is preferably 150-950W.

[0047] The second solvent is one of deionized water, physiological saline or phosphate buffer; the stabilizer is at least one of glycerol, olive oil, tea tree oil, oleic acid, Tween 80, Tween 20, Span 20, carboxymethyl cellulose, polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG), and the mass ratio of the stabilizer to the lipid film is (2-30): (16-25); S1-4, microfluidization high pressure emulsification for 10-30 minutes to obtain a nanoemulsion, and dialysis to remove impurities to obtain a nano drug combination preparation in which the drug is encapsulated by a nano carrier.

[0048] The encapsulated nano-drug combination preparation is adopted, and the encapsulated drug can be a hydrophobic small molecule drug and / or a hydrophilic small molecule drug. The hydrophobic small molecule drug is preferred, so as to overcome the problems of difficulty in administration and low efficiency of the hydrophobic small molecule drug.

[0049] (2) The drug is loaded on the environmentally responsive nanocarrier, and the loading rate is preferably above 50%. The process comprises the following steps: S2-1, mixing alveolar surfactant, environmentally responsive liposomes, a drug to be loaded and a first solvent to obtain a mixture; the first solvent is an organic solvent selected from chloroform or a mixed solution of chloroform and methanol in any proportion; S2-2, evaporating and drying the carrier mixture, including evaporating the first solvent to dryness at 40-50° C., and then drying in a vacuum drying oven at 30-40° C. for 5-15 hours to obtain a drug-loaded lipid film; S2-3. Mix the drug-loaded lipid film, stabilizer and second solvent, and perform ultrasonic hydration treatment, including ultrasonic treatment in a water bath at 25-35°C for 5-15 min, and then ultrasonic treatment in an ice bath for 1-8 min. The power of the ultrasonic treatment is 150-950 W, and the conditions of ultrasonic treatment in the water bath and in the ice bath can be the same or different.

[0050] The second solvent is one of deionized water, physiological saline or phosphate buffer; the stabilizer is at least one of glycerol, olive oil, tea tree oil, oleic acid, Tween 80, Tween 20, Span 20, carboxymethyl cellulose, polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG), and the mass ratio of the stabilizer to the lipid film is (2-30): (16-25); S2-4, microfluidization high pressure emulsification for 10-30 minutes to obtain a nanoemulsion, and dialysis to remove impurities to obtain a nano drug combination preparation in which the drug is loaded by a nano carrier.

[0051] Example 1 This embodiment provides an environment-responsive nanocarrier with intelligent inhalation drug delivery. The nanocarrier uses DSPE-PEG2000 to have ROS responsiveness and temperature responsiveness. The steps for preparing the nanocarrier are as follows: Step 1: dissolving dipalmitoylphosphatidylcholine DPPC, DSPE-PEG2000 and cholesterol in an organic solvent, wherein the molar ratio of DPPC, DSPE-PEG2000 and cholesterol is 80:10:10, and the total mass is 20 mg, to obtain a carrier mixture; the organic solvent is a mixed solution of chloroform and methanol in a volume ratio of 1:1; Step 2: The carrier mixture is evaporated and dried by a rotary evaporator at 45° C. to obtain a lipid film for later use; Step 3: Mix the lipid film, 20 μL of glycerol and 1 mL of deionized water, hydrate the film by ultrasonic oscillation at 450W for 10 min in a 30°C water bath, and then ultrasonically treat at 275W for 5 min in an ice bath to obtain a suspension solution; Step 4: Treat the suspension solution obtained in step 3 with a high-pressure emulsification device for 20 minutes, and dialysis purification to obtain the environmentally responsive nanocarrier.

[0052] Example 2 This embodiment provides an environment-responsive nano-drug combination preparation with intelligent inhalation administration, in which a hydrophobic small molecule drug is encapsulated in an environment-responsive nano-carrier, which is the same as the nano-carrier in Example 1. The preparation steps of the nano-drug combination preparation are as follows: Step 1: dissolving dipalmitoylphosphatidylcholine DPPC, DSPE-PEG2000 and cholesterol in an organic solvent, wherein the molar ratio of DPPC, DSPE-PEG2000 and cholesterol is 80:10:10, and the total mass is 20 mg, to obtain a carrier mixture; the organic solvent is a mixed solution of chloroform and methanol in a volume ratio of 1:1; Step 2: The carrier mixture is evaporated and dried by a rotary evaporator at 45° C. to obtain a lipid film for later use; Step 3: The lipid film, imatinib, 20 μL of glycerol and 1 mL of deionized water were mixed, and the film was hydrated by ultrasonic vibration at 450W in a 30°C water bath for 10 min, and then ultrasonically treated at 275W in an ice bath for 5 min to obtain a suspension solution; wherein the mass ratio of the drug to the lipid film was 1.4 mg:20 mg; Step 4: The suspension solution obtained in step 3 is treated with a high-pressure emulsification device for 20 minutes, and dialyzed and purified to obtain a nano-drug combination preparation in which the drug is encapsulated by the nano-carrier, and the drug encapsulation rate is about 78%.

[0053] Example 3 The material ratios used in this embodiment are the same as those in Example 2, the main difference being that in the environmentally responsive nano-drug combination preparation of this embodiment, the hydrophobic small molecule drugs are mixed and loaded in the environmentally responsive nano-carrier. The preparation steps of the nano-drug combination preparation are as follows: S2-1, dissolving dipalmitoylphosphatidylcholine DPPC, DSPE-PEG2000, imatinib and cholesterol in an organic solvent, wherein the molar ratio of DPPC, DSPE-PEG2000 and cholesterol is 80:10:10, the total mass of the carrier material is 20 mg, and imatinib is 1.4 mg, to obtain a mixture; the organic solvent is a mixed solution of chloroform and methanol in a volume ratio of 1:1; S2-2, evaporating and drying the organic solvent from the mixture at 45° C. in a rotary evaporator to obtain a lipid film for later use; S2-3, the lipid film, 20 μL of glycerol and 1 mL of deionized water were mixed, and ultrasonically shaken at 450W for 10 min in a 30°C water bath to hydrate the film material, and then ultrasonically treated at 275W for 5 min in an ice bath to obtain a suspension solution; S2-4. The suspension solution obtained in step 3 is treated with a high-pressure emulsification device for 20 minutes, and dialyzed and purified to obtain a nano-drug combination preparation in which the nano-carriers are loaded with drugs, and the loading rate is about 65%.

[0054] Comparative Example 1 Comparative Example 1 differs from Example 3 in that DSPE-PEG2000 is not used, wherein: Step 1: dissolving dipalmitoylphosphatidylcholine DPPC, imatinib and cholesterol in an organic solvent, wherein the molar ratio of DPPC to cholesterol is 85:15, the total mass of the carrier material is 20 mg, and imatinib is 1.4 mg, to obtain a mixture; the organic solvent is a mixed solution of chloroform and methanol in a volume ratio of 1:1; Step 2: The carrier mixture is evaporated and dried at 45° C. on a rotary evaporator to obtain a carrier film for later use; Step 3: Mix the carrier film, 20 μL of glycerol and 1 mL of deionized water, and hydrate the film by ultrasonic oscillation at 450W for 10 min in a 30°C water bath, and then ultrasonically treat at 275W for 5 min in an ice bath to obtain a suspension solution; Step 4: The suspension solution obtained in step 3 was treated with a high-pressure emulsification device for 20 minutes, and the comparative sample 1 containing the drug encapsulated by the nanocarrier was obtained by dialysis purification.

[0055] Example 4-Example 6 The components are adjusted based on Example 3, see Table 1: Table 1

[0056] Example 7

[0057] This embodiment provides an environment-responsive nanocarrier with intelligent inhalation drug delivery. The nanocarrier has ROS responsiveness and temperature responsiveness by using DSPE-PEG2000, and has pH responsiveness by using SM-102. The steps for preparing the nanocarrier are as follows: Step 1: dissolving dipalmitoylphosphatidylcholine DPPC, ionizable cationic lipid SM-102, DSPE-PEG2000 and cholesterol in an organic solvent, wherein the molar ratio of DPPC, SM-102, DSPE-PEG2000 and cholesterol is 50:35:5:10, and the total mass is 20 mg, to obtain a carrier mixture; the organic solvent is a mixed solution of chloroform and methanol in a volume ratio of 1:1; Step 2: The carrier mixture is evaporated and dried by a rotary evaporator at 45° C. to obtain a lipid film for later use; Step 3: Mix the lipid film, 20 μL of glycerol and 1 mL of deionized water, hydrate the film by ultrasonic oscillation at 450W for 10 min in a 30°C water bath, and then ultrasonically treat at 275W for 5 min in an ice bath to obtain a suspension solution; Step 4: Treat the suspension solution obtained in step 3 with a high-pressure emulsification device for 20 minutes, and dialysis purification to obtain the environmentally responsive nanocarrier.

[0058] Example 8 This embodiment provides an environment-responsive nano-drug combination preparation with intelligent inhalation administration, in which a hydrophobic small molecule drug is loaded in an environment-responsive nano-carrier, which is the same as the nano-carrier of Example 7. The preparation steps of the nano-drug combination preparation are as follows: Step 1: dissolving dipalmitoylphosphatidylcholine DPPC, imatinib, ionizable cationic lipid SM-102, DSPE-PEG2000 and cholesterol in an organic solvent, wherein the molar ratio of DPPC, SM-102, DSPE-PEG2000 and cholesterol is 50:35:5:10, the total mass is 20 mg, and imatinib is 1.4 mg, to obtain a carrier mixture; the organic solvent is a mixed solution of chloroform and methanol in a volume ratio of 1:1; Step 2: The carrier mixture is evaporated and dried by a rotary evaporator at 45° C. to obtain a lipid film for later use; Step 3: Mix the lipid film, 20 μL of glycerol and 1 mL of deionized water, hydrate the film by ultrasonic oscillation at 450W for 10 min in a 30°C water bath, and then ultrasonically treat at 275W for 5 min in an ice bath to obtain a suspension solution; Step 4: Treat the suspension solution obtained in step 3 with a high-pressure emulsification device for 20 minutes, and dialysis purification to obtain a nano-drug combination preparation in which the drug is encapsulated by the nano-carrier.

[0059] Example 9-Example 10 The components were adjusted based on Example 8, as shown in Table 2: Table 2

[0060] Test methods and results: 1. Particle size distribution and potential distribution test The purified samples of Examples 1-10 and Comparative Example 1 were stored at 4° C. for 24 h, and then their particle size distribution and potential distribution were tested using a Malvern Panalytical particle size analyzer. The test results are shown in Table 3.

[0061] Table 3

[0062] In addition, the test diagrams showing the particle size distribution and potential distribution of the sample of Example 3 are shown in FIG. Figure 1-2 .

[0063] The sample of Example 3 was stored in a 4°C constant temperature refrigerator for 28 days, and the particle size and PDI did not change significantly, indicating that it has good storage stability. Figure 3-4 shown.

[0064] The sample obtained in Example 3 was atomized by a nebulizer and then collected. The particle size changes before and after atomization were detected. The results are as follows: Figure 5 As shown, the atomization operation does not change the size of the nanoparticles.

[0065] 2. Biosafety The sample of Example 8 was co-incubated with red blood cells at a concentration of 10-80 μmol / L, and the damage to red blood cells was investigated by calculating the hemolysis rate. The results are as follows: Figure 6 As shown in Figure 2, the hemolysis rate of each concentration of nanoformulation was less than 5%. Then, the MTT colorimetric method was used to investigate the effects of different concentrations of nanoformulation on the activity of human bronchial epithelial cells. The results are shown in Figure 2. Figure 7 As shown, after co-incubation with the nanoparticles at various concentrations, the calculated cell survival rates were all greater than 80%. The above results indicate that the nanocarriers prepared by the present invention have good biosafety.

[0066] 3. Drug loading performance 3.1 In Example 8, a 140 μg / mL small molecule drug standard solution was prepared and diluted in equal proportions to form a series of standard samples. After detection by high performance liquid chromatography, a standard curve was drawn. The results are as follows: Figure 8 As shown, R 2 The value is greater than 0.99, indicating that the standard curve has good linearity; then, the content of imatinib in the sample of Example 2 is determined by dialysis, the nano drug combination preparation prepared in Example 2 is added to the dialysis cup for 4 hours, 50 μL of the dialyzed nano drug combination preparation is taken, and methanol is quantified to 1 mL. The encapsulation efficiency is calculated according to the following formula: Encapsulation efficiency = (W encapsulation / W administration) × 100%, The results showed that the encapsulation rate could reach over 70%.

[0067] 3.2 Weigh the small molecule drug, dilute it with methanol to an appropriate concentration, use the methanol solution as a blank control, and use a UV spectrophotometer to perform full wavelength scanning in the range of 200-700nm. Determine the characteristic absorption wavelength of imatinib based on the position of the characteristic peak. Then use a UV spectrophotometer to check the UV absorption characteristics of the blank lipid samples of Example 7 and Example 8 under the same conditions. The results are as follows: Fig. 9 As shown, Im is the UV absorption graph of imatinib, LPS is the UV absorption graph of blank lipids, and LPMS is the UV absorption graph of the sample. The characteristic absorption wavelength of imatinib is about 269nm, and the characteristic absorption peak of imatinib can be seen in the absorption characteristic graph of LPMS, which preliminarily indicates that the drug loading is successful.

[0068] 4. Responsiveness The samples prepared in Example 3 were incubated in 0 mM, 1 mM and 10 mM H2O2 solutions for 4 h and the changes in particle size distribution were detected by DLS. Fig.10As shown: After incubation with 0 mM H2O2 solution, the particle size of the nanoformulation did not change significantly and remained normally distributed. After incubation with 1 mM and 10 mM H2O2 solutions, the particle size changed to varying degrees, and the particle size distribution diagram showed multiple peaks, indicating that the nanoformulation can be decomposed and destroyed in the presence of ROS.

[0069] 5. In vitro anti-fibrosis performance TGF-β1 was used to induce proliferation and activation of NIH-3T3 cells. The nanomedicine combination preparation prepared in Example 8 was co-incubated with the cells. The cells were stained using the Tunel apoptosis staining kit, and the fluorescence changes were observed under a confocal microscope. The results are as follows: Fig.11 As shown, compared with the blank control group, the green fluorescence signal representing cell apoptosis was slightly weakened after TGF-β1 induction, and the green fluorescence was significantly enhanced after the nanoformulation treatment, indicating that the nanoformulation prepared by the present invention can inhibit the activation of fibroblasts and promote their apoptosis.

[0070] 6. Surface charge changes in solutions with different pH values The nano drug combination prepared in Example 8 was incubated with PBS solutions of pH = 4.5, 5.5, 6.5, 6.8 and 7.5 for 1 hour, and then a suitable amount was taken out and its surface charge was measured using a Malvern Panalytical particle size analyzer. The results are shown in FIG. Fig.12 As shown in Figure 2, as the pH value decreases, the surface charge of the nanoformulation gradually increases. The nano-drug combination preparations prepared in Examples 3 and 8 were co-incubated with the nanoformulation at a pH value of 6.5, and appropriate amounts were taken out at different time points to detect changes in their surface charge. Fig.13 As shown, as time goes by, the surface charge of the nano drug combination formulation of Example 8 (SM) gradually increases, while the surface charge of the nano drug combination formulation of Example 3 (NSM) does not change significantly. The above results show that the nano formulation of Example 8 of the present invention can effectively respond to changes in pH value.

[0071] 7. Mucus penetration characteristics A layer of mucus was spread in the upper chamber of the Transwell device, and the samples of Example 3 and Example 8 were dispersed on the mucus, respectively. Aqueous medium was added to the lower chamber, and the nanoformulations that penetrated into the lower layer were scanned in 3D using a confocal microscope. The results are shown in FIG. Fig.14 As shown in the figure, the sample of Example 8 (LPMS) penetrated the mucus more than the sample of Example 3 (LPM) to reach the lower layer. Then, the two nano-drug combination preparations were dispersed in an aqueous medium, and their movement was observed using an inverted fluorescence microscope and recorded using a digital camera. The movement trajectory was then analyzed using software. The results are shown in the figure. Fig.15As shown, both nano-drug combination preparations exhibit irregular Brownian motion. In the same period of time, the movement distance of the sample in Example 8 (LPMS) is greater.

[0072] 8. Uptake of Nanodrug Combination Formulations by NIH-3T3 Cells After the sample of Example 8 was co-incubated with cells, flow cytometry was used to detect the internalization of the probe by cells at different time points. Fig.16 As shown, the uptake of the nanomedicine combination formulation by NIH-3T3 cells increased in a time-dependent manner. Fig.17 The results showed that the cellular uptake of the nanodrug combination preparation was not affected before or after atomization.

[0073] 9. Effects of nano-drug combination preparations on activation and migration of NIH-3T3 cells TGF-β1 was used to induce proliferation and activation of NIH-3T3 cells, and the sample of Example 8 was co-incubated with the cells, and then Masson staining was performed. The results are shown in Figure 18. Compared with the control group, after TGF-β1 induction, cell proliferation and activation, increased density, and increased blue collagen, while each medication group reduced cell proliferation and activation to varying degrees, among which Example 8 (LPMS) had the most significant effect.

[0074] 10. Distribution of nano-drug combination preparations in mice After the mice were aerosolized and inhaled the nano-drug combination preparation prepared in Example 8 for 1 hour, they were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney, muscle and small intestine were removed and imaged using an IVIS optical imager. Fig.19 As mentioned above, the inhaled nanoformulations were mainly distributed in the lungs, and no obvious fluorescence signals were observed in other tissues.

[0075] 11. Anti-fibrosis properties in vivo The experimental mice were divided into a blank control group (Control), a bleomycin BLM modeling group (BLM), an imatinib single-drug oral gavage group (Imatinib), the sample of Example 3 (LPM) and the sample of Example 8 (LPMS).

[0076] Control group: no treatment; BLM group: tracheal instillation of BLM, nebulized saline; Single-drug group: tracheal instillation of BLM, intraperitoneal injection and nebulized inhalation of the same dose of imatinib solution; Example 3: BLM was instilled into the trachea and the nanoformulation was inhaled by atomization; Example 8: BLM was instilled into the trachea and the nanoformulation was inhaled by atomization.

[0077] After the above grouping and treatment, the lung tissues of mice were scanned by MRI and stained with H&E on the 21st day of treatment. Figure 20-21 As shown, MRI images showed sheet-like high signal shadows in the lung tissue, and H&E staining showed that the normal structure of the mouse lung tissue disappeared and the alveolar septum thickened. After treatment, the above-mentioned lesions were alleviated to varying degrees, among which the effect of aerosol inhalation of Example 8 (LPMS) was the most significant.

[0078] In summary, the environmentally responsive nanocarrier and its nano-drug combination preparation for inhalation administration prepared by the present invention have good stability and biosafety, can achieve sustained drug release under the stimulation of environmental factors such as temperature, pH value, ROS, etc., and show good anti-fibrosis effects both in vivo and in vitro. The environmentally responsive nanocarrier and its nano-drug combination preparation of the present invention have broad application prospects in the preparation of drugs for the treatment of lung diseases.

[0079] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the present invention is intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention.

Claims

1. An environmentally responsive nanocarrier for inhalation administration, characterized in that: The average particle size of the nanocarrier is 30-700 nm; The nanocarrier comprises alveolar surfactant and environmentally responsive lipids; The molar ratio of alveolar surfactant to environmentally responsive lipids is (40-99):(1-60).

2. The environmentally responsive nanocarrier according to claim 1, wherein: The alveolar surfactant includes at least one of natural alveolar surfactant and artificially synthesized alveolar surfactant; The environmentally responsive lipids include one or more of temperature-responsive lipids, pH-responsive lipids, ROS-responsive lipids, and multi-responsive lipids.

3. The environmentally responsive nanocarrier according to claim 2, wherein: The environmentally responsive lipids include multi-responsive lipids and / or pH-responsive lipids; Multi-responsive lipids include DSPE-PEG2000; The pH-responsive lipids include ionized lipids, and the ionized lipids are selected from at least one of quaternary ammonium lipids, ionizable lipids, and biodegradable lipids.

4. The environmentally responsive nanocarrier according to any one of claims 1 to 3, characterized in that: Alveolar surfactant includes artificially synthesized alveolar surfactant; In terms of percentage, synthetic alveolar surfactant includes the following components: Phospholipids 70-90 % Apolipoprotein 0-15 % Neutral lipids 5-30%; The phospholipids include at least one of phosphatidylcholine and phosphatidylglycerol; The apolipoprotein includes at least one of surfactant protein A (SP-A), B (SP-B), C (SP-C), and D (SP-D); Neutral lipids include cholesterol.

5. A method for preparing the environmentally responsive nanocarrier according to any one of claims 1 to 4, characterized in that: The steps include: Step 1: mixing alveolar surfactant, environmentally responsive lipid and a first solvent to obtain a carrier mixture; Step 2, evaporating and drying the carrier mixture to obtain a lipid film; Step 3, mixing the lipid film, the second solvent, and an optional stabilizer, and performing ultrasonic hydration treatment; Step 4: Microfluidization high pressure emulsification to obtain nanoemulsion, and dialysis to remove impurities to obtain the environmentally responsive nanocarrier.

6. A nano-drug combination preparation, comprising the environmentally responsive nano-carrier according to any one of claims 1 to 4, characterized in that: Also included are medicines, which: The drug is encapsulated in the environmentally responsive nanocarrier; or The drug is loaded on the environmentally responsive nanocarrier.

7. A method for preparing the nano-drug combination preparation as claimed in claim 6, characterized in that: The steps include: S1-1, mixing alveolar surfactant, environmentally responsive liposomes and a first solvent to obtain a carrier mixture; S1-2, evaporating and drying the carrier mixture to obtain a lipid film; S1-3, mixing the lipid film, the drug to be encapsulated, the stabilizer and the second solvent, and performing ultrasonic hydration treatment; S1-4, microfluidization high pressure emulsification to obtain nanoemulsion, dialysis to remove impurities, and obtain a nano drug combination preparation in which the drug is encapsulated by a nano carrier.

8. A method for preparing the nano-drug combination preparation as claimed in claim 6, characterized in that: The steps include: S2-1, mixing alveolar surfactant, environmentally responsive liposomes, a drug to be loaded and a first solvent to obtain a mixture; S2-2, evaporating and drying the mixture to obtain a lipid film; S2-3, mixing the lipid film, the stabilizer and the second solvent, and performing ultrasonic hydration treatment; S2-4, microfluidization high pressure emulsification to obtain nanoemulsion, dialysis to remove impurities, and obtain a nano drug combination preparation in which the drug is loaded by a nano carrier.

9. The preparation method according to claim 5, 7 or 8, characterized in that: At least one of the following must be met: 1) The first solvent is an organic solvent selected from chloroform or a mixed solution of chloroform and methanol in any proportion; 2) the second solvent is one of deionized water, physiological saline, sucrose solution, mannitol solution or phosphate buffer; 3) the stabilizer is selected from at least one of glycerol, olive oil, tea tree oil, oleic acid, Tween 80, Tween 20, Span 20, carboxymethyl cellulose, polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG), and the mass ratio of the stabilizer to the lipid film is (2-30): (16-25); 4) Evaporation and drying treatment, including evaporating the first solvent to dryness at 40-50°C, and then drying in a vacuum drying oven at 30-40°C for 5-15 hours; 5) Ultrasonic hydration treatment, including ultrasonic treatment in a 25-35°C water bath for 5-15 min, and then ultrasonic treatment in an ice bath for 1-8 min.

10. Use of the environmentally responsive nanocarrier according to any one of claims 1 to 4, the environmentally responsive nanocarrier obtained by the preparation method of claim 5, the nano-drug combination preparation according to claim 6, or the nano-drug combination preparation obtained by the preparation method of claim 7 or 8 in the preparation of drugs for treating lung diseases.

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