Preparation method and application of pulmonary surfactant fusion type nano bionic body

Abstract

The invention relates to a preparation method of a pulmonary surfactant fusion type nano bionic body, which comprises the following steps: mixing dipalmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl-lecithin, cholesterol and dipalmitoyl phosphatidyl glycerol together, adding into chloroform, carrying out vortex oscillation, uniformly mixing to form a lipid solution, adding into an eggplant-shaped bottle filled with chloroform, adding a medicine, uniformly stirring, and carrying out vacuum drying to obtain the pulmonary surfactant fusion type nano bionic body. And after the solvent is completely removed, a layer of uniform white thin film can be obtained on the inner wall of the eggplant-shaped bottle, adding water for dissolving, and carrying out ice-bath ultrasonic detection treatment on the obtained lipid thin film solution by using an ultrasonic cell disruptor, thereby obtaining the product. The method disclosed by the invention is easy to operate, can be used for effectively preparing the lung surfactant fusion type nano biomimetic body, is used for loading a medicine for treating lung diseases to directly reach a lung focus and fully exerting the medicine effect, is good in targeting effect, improves the treatment effect, opens up a new way for treating the lung diseases, and has remarkable economic and social benefits.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to a preparation method and application of a lung surfactant fused nano bionic body. Background Art

[0002] There are many types of cells in normal human lung tissue. Alveolar epithelial cells are the most abundant cells in the lung epithelial cells. There are two types of alveolar epithelial cells: alveolar type I epithelial cells and alveolar type II epithelial cells. Alveolar type I epithelial cells are broad and flat, accounting for 95% of the alveolar surface, 40% of the alveolar epithelium, and 8% of the surrounding lung cells; while alveolar type II epithelial cells are small cubes, accounting for 5% of the alveolar surface, 60% of the alveolar epithelium, and 15% of the surrounding lung cells; in addition, alveolar type II epithelial cells are the progenitor cells of alveolar type I epithelial cells, and play a key role in the main links of alveolar epithelial repair and maintenance of tissue homeostasis. Alveolar type II epithelial cells have multiple functions, such as producing surfactant, regulating lung immune function, proliferating and differentiating into type I epithelial cells to repair alveoli, etc.

[0003] Alveolar type II epithelial cells play an important role in maintaining the normal physiological function of the lungs and in the occurrence and development of lung diseases. They are involved in the occurrence and development of a variety of lung diseases, such as acute lung injury, interstitial lung disease, lung cancer, tuberculosis, chronic obstructive pulmonary disease, etc. Reducing the damage and apoptosis of alveolar type II epithelial cells can alleviate lung damage and delay disease progression. Therefore, alveolar type II epithelial cells are expected to become a target for the treatment of certain lung diseases, and direct delivery of drugs to alveolar type II epithelial cells is expected to become an effective means of treating lung diseases.

[0004] Compared with systemic administration, inhaled drug therapy has obvious advantages in the treatment of lung-related diseases. It can directly act on the lungs, thereby reducing nonspecific distribution of drugs and increasing lung tissue accumulation. The alveoli with a huge specific surface area are the main site for inhaled drugs to be absorbed into the lungs. Alveolar type II epithelial cells are mainly distributed in the alveolar wall, but the area of ​​alveolar type II epithelial cells in the alveoli is relatively small, and it is impossible to directly achieve efficient targeting of alveolar type II epithelial cells by inhalation.

[0005] In addition, a large number of amphiphilic active substances are distributed at the alveolar air-liquid interface. These pulmonary surfactants not only play an important role in reducing alveolar surface tension, but can also quickly adsorb on heterogeneous particle interfaces, thereby preventing foreign particles from approaching alveolar epithelial cells and accelerating the clearance of particles by macrophages, which results in most particles being cleared before reaching alveolar type II epithelial cells. However, most current studies have ignored the clearance effect of the alveolar surface barrier layer. How to avoid the clearance of pulmonary surfactants and deliver drugs directly to alveolar type II epithelial cells is a key issue in the treatment of lung diseases.

[0006] Similar to biological membranes, pulmonary surfactant is mainly composed of phospholipids and cholesterol. More importantly, after pulmonary surfactant is synthesized and secreted by alveolar type II epithelial cells, nearly 90% of pulmonary surfactant is recycled by alveolar type II epithelial cells. And the renewal rate of pulmonary surfactant is very fast. It is reported that the average renewal time of pulmonary surfactant is less than 11 hours, and the alveolar surface area of ​​an adult is nearly 100m2. Therefore, pulmonary surfactant is like a natural "conveyor belt", which circulates continuously between the alveolar surface and alveolar type II epithelial cells. Therefore, the pulmonary surfactant circulation is a natural channel for the direct transport of adjuvant drugs to type II epithelial cells. So, can we use biomembrane biomimetic technology to construct pulmonary surfactant nano-bionics to reduce the interface difference with pulmonary surfactant, which will be able to avoid the removal of pulmonary surfactant and deliver therapeutic drugs directly to alveolar type II epithelial cells to achieve the treatment of lung diseases, but there has been no public report so far. Summary of the invention

[0007] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of a lung surfactant fusion nano-bionic body, which can effectively solve the problem of preparing a lung surfactant fusion nano-bionic body, loading drugs and directly delivering them to alveolar type II epithelial cells and lung tissue, and realizing the medication problem for the treatment of lung diseases.

[0008] The technical solution provided by the present invention is a method for preparing a lung surfactant-fused nano-bionic body, comprising the following steps:

[0009] (1) Preparation of lipid solution:

[0010] First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 30-50: 25-35: 5-10: 8-15, mix the components together, add them into chloroform with a weight volume of 2-22 times, where the weight volume refers to the solid in mg and the liquid in mL, and then vortex the prepared solution to mix it into a lipid solution, store it at 4°C for later use;

[0011] (2) Preparation of lipid film solution:

[0012] Take the lipid solution prepared in step 1, add 68-110 mg of the lipid material to an eggplant-shaped bottle containing 3-5 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of the lipid material to the drug is 100:1. The rotary evaporator is set to 43°C in a water bath and 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 2-5 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for standby use;

[0013] The drug is monophosphoryl lipid A (MPLA) or other fat-soluble drugs for treating lung diseases;

[0014] (3) Preparation of bionic body:

[0015] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

[0016] The lung surfactant fusion nano-bionics prepared by the above method is used in the preparation of drugs for treating lung diseases.

[0017] The lung disease is acute lung injury, interstitial lung disease, lung cancer, tuberculosis or chronic obstructive pulmonary disease.

[0018] The method of the present invention is easy to operate and can effectively prepare lung surfactant-fused nano-bionics, which are used to load drugs for treating lung diseases to directly reach lung lesions, give full play to the efficacy of the drugs, have good targeting effects, improve the therapeutic effect, open up a new way for the treatment of lung diseases, and have significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a particle size characterization diagram of the lung surfactant fusion nano-bionic body of the present invention;

[0020] Figure 2 This is a potential characterization diagram of the lung surfactant fusion nano-bionic body of the present invention;

[0021] Figure 3 This is a cryo-transmission electron microscopy (TEM) characterization image of the lung surfactant-fused nano-bionic body of the present invention;

[0022] Figure 4 This is a differential scanning calorimeter (DSC) phase transition temperature characterization diagram of the lung surfactant fusion nano-bionic body of the present invention;

[0023] Figure 5This is a distribution behavior diagram of the flow analysis fluorescently labeled lung surfactant fusion nano-bionic body in different cells at different time points of the present invention;

[0024] Figure 6 This is a frozen section scanning analysis of the distribution behavior of the fluorescent lung surfactant fusion nano-bionic body in the lungs of the present invention;

[0025] Figure 7 This is a cryo-transmission electron microscopy (TEM) characterization image of the lung surfactant-fused nano-bionic body loaded with gold nanoparticles of the present invention;

[0026] Figure 8 The biological transmission electron microscope characterization image of the lung surfactant fusion nano-bionic body containing gold nanoparticles of the present invention fused with the lung surfactant;

[0027] Fig. 9 This is a graph showing the detection of IFN-α in serum of different groups of mice according to the present invention;

[0028] Fig.10 This is a graph showing the detection of IFN-α in lung tissue homogenate of different groups of mice of the present invention;

[0029] Fig.11 This is a graph showing IFN-γ detection in serum of different groups of mice of the present invention;

[0030] Fig.12 This is a graph showing IFN-γ detection in lung tissue homogenate of different groups of mice of the present invention;

[0031] Fig.13 This is a graph showing the detection of IL-6 in the serum of different groups of mice of the present invention;

[0032] Fig.14 This is a graph showing the detection of IL-6 in lung tissue homogenate of different groups of mice of the present invention;

[0033] Fig.15 This is a graph showing the detection of TNF-α in serum of different groups of mice of the present invention;

[0034] Fig.16 This is a graph showing TNF-α detection in lung tissue homogenate of different groups of mice of the present invention;

[0035] Fig.17 This is a graph showing weight changes in different groups of mice of the present invention;

[0036] Fig.18 This is a graph showing the detection of serum IgG in different groups of mice according to the present invention;

[0037] Fig.19 This is a graph showing the detection of IFN-α in serum of different groups of mice according to the present invention;

[0038] Fig. 20This is a graph showing IFN-γ detection in serum of different groups of mice of the present invention;

[0039] Fig.21 This is a graph showing the detection of IL-6 in the serum of different groups of mice of the present invention;

[0040] Fig. 22 This is a graph showing the detection of TNF-α in serum of different groups of mice according to the present invention. DETAILED DESCRIPTION

[0041] The specific implementation modes of the present invention are described in detail below in conjunction with the embodiments.

[0042] The present invention can be specifically implemented by the following examples.

[0043] Example 1

[0044] A method for preparing a lung surfactant-fused nano-bionic body comprises the following steps:

[0045] (1) Preparation of lipid solution:

[0046] First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 40:30:7:12, mix the components together, add them into chloroform with a weight volume of 12 times, where the weight volume refers to the solid in mg and the liquid in mL, and then vortex the prepared solution to mix it into a lipid solution, store it at 4°C for later use;

[0047] (2) Preparation of lipid film solution:

[0048] Take the lipid solution prepared in step 1, add 89 mg of the lipid material solution to an eggplant-shaped bottle containing 4 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of the lipid material to the drug is 100:1. The rotary evaporator water bath temperature is set to 43 ° C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 3 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for standby use;

[0049] (3) Preparation of bionic body:

[0050] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

[0051] Example 2

[0052] A method for preparing a lung surfactant-fused nano-bionic body comprises the following steps: 30:25:5:8

[0053] (1) Preparation of lipid solution:

[0054] First, select lipid materials with a mass ratio of dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoylphosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 30:25:5:8, mix the components together, add them into chloroform with a weight volume twice that of the original, where the weight volume refers to the solid in mg and the liquid in mL, then vortex and shake the prepared solution to mix it into a lipid solution, and store it at 4°C for later use;

[0055] (2) Preparation of lipid film solution:

[0056] Take the lipid solution prepared in step 1, add 68 mg of the lipid material solution to an eggplant-shaped bottle containing 3 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of the lipid material to the drug is 100:1. The rotary evaporator water bath temperature is set to 43 ° C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 2 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for standby use;

[0057] (3) Preparation of bionic body:

[0058] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

[0059] Example 3

[0060] A method for preparing a lung surfactant-fused nano-bionic body comprises the following steps:

[0061] (1) Preparation of lipid solution:

[0062] First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 50:35:10:15, mix the components together, add them into chloroform with a weight volume of 22 times, where the weight volume refers to the solid in mg and the liquid in mL, and then vortex the prepared solution to mix it into a lipid solution, store it at 4°C for later use;

[0063] (2) Preparation of lipid film solution:

[0064] Take the lipid solution prepared in step 1, add every 110 mg of the lipid material solution to an eggplant-shaped bottle containing 5 mL of chloroform, then add the drug to be delivered to the lungs, the mass ratio of the lipid material to the drug is 100:1, and perform rotary evaporation under the conditions of setting the water bath temperature of the rotary evaporator to 43°C and the pressure to 0.05-0.06 Kpa. After the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 5 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for standby use;

[0065] (3) Preparation of bionic body:

[0066] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

[0067] Example 4

[0068] A method for preparing a lung surfactant-fused nano-bionic body comprises the following steps:

[0069] (1) Preparation of lipid solution:

[0070] First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 45:28:8:13, mix the components together, add them into chloroform with a weight volume of 7 times, where the weight volume refers to the solid in mg and the liquid in mL, and then vortex the prepared solution to mix it into a lipid solution, store it at 4°C for later use;

[0071] (2) Preparation of lipid film solution:

[0072] Take the lipid solution prepared in step 1, add 94 mg of the lipid material solution to an eggplant-shaped bottle containing 2 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of the lipid material to the drug is 100:1. The rotary evaporator water bath temperature is set to 43 ° C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 3 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for standby use;

[0073] (3) Preparation of bionic body:

[0074] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

[0075] Example 5

[0076] A method for preparing a lung surfactant-fused nano-bionic body comprises the following steps:

[0077] (1) Preparation of lipid solution:

[0078] First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 35:32:7:11, mix the components together, add them into chloroform with a weight volume of 17 times, where the weight volume refers to the solid in mg and the liquid in mL, and then vortex the prepared solution to mix it into a lipid solution, store it at 4°C for later use;

[0079] (2) Preparation of lipid film solution:

[0080] Take the lipid solution prepared in step 1, add 85 mg of the lipid material solution to an eggplant-shaped bottle containing 3 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of the lipid material to the drug is 100:1. The rotary evaporator water bath temperature is set to 43 ° C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 4 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for standby use;

[0081] (3) Preparation of bionic body:

[0082] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

[0083] The lung surfactant-fused nano-bionics prepared by the method described in Examples 1-3 have uniform particle sizes ranging from 100 to 200 nm and a potential of -43.22±0.5 mV.

[0084] The lung surfactant-fused nano-bionics prepared by the method described in Examples 1-3 were observed to be spherical in shape using a transmission electron microscope, and were substantially uniform in size.

[0085] The above are only embodiments, which are used to illustrate the implementation mode of the present invention, rather than to limit the scope of protection of the present invention. Any technical solutions that are essentially the same as those of the present application and are made by using equivalent and equivalent replacement means shall fall within the scope of protection of the present invention. Any required amount of lung surfactant fusion nano-bionics can be made according to the proportion relationship given above.

[0086] The method of the present application is easy to operate, and the obtained product is of good quality. It can effectively prepare lung surfactant fusion nano-bionics, which are used to carry drugs for treating lung diseases directly to lung lesions, give full play to the efficacy of drugs, have good targeting effect, improve the treatment effect, provide technical support for the treatment of lung diseases, and have achieved very good beneficial technical effects through experiments. The relevant information is as follows (taking Example 1 as an example):

[0087] Experiment 1: Characterization of the Lung Surfactant-Integrated Nanobiomimetic

[0088] The lung surfactant fusion nano-bionics prepared according to the method described in the example has a uniform particle size, with an average particle size of about 100 nm (see Figure 1 ), the potential is -43.22±0.5mV (see Figure 2 ).

[0089] The pulmonary surfactant-fused nano-bionics prepared according to the method described in the example were observed to be spherical in shape with substantially uniform size (see Figure 3 ).

[0090] The pulmonary surfactant fusion nano-bionics prepared according to the method described in the example is temperature sensitive as determined by DSC, and its phase transition temperature is 36.58°C (see Figure 4 ).

[0091] Experiment 2: Targeting experiment of alveolar type II epithelial cells with lung surfactant-fused nano-bionics

[0092] The experimental materials are: lung surfactant fusion nano-bionics loaded with cell membrane far-infrared fluorescent probe (DiD), and the preparation method of the bionics is:

[0093] (1) Preparation of lipid solution:

[0094] First, a lipid material with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 30:30:10:15 was selected. According to the above mass ratio, 30 mg of dipalmitoylphosphatidylcholine (DPPC), 30 mg of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 10 mg of cholesterol (CHO) and 15 mg of dipalmitoylphosphatidylglycerol (DPPG) were selected and dissolved in 10 mL of chloroform respectively, and then the prepared solution was vortexed and mixed to form a lipid solution, which was stored at 4° C. for standby use;

[0095] (2) Preparation of lipid film solution:

[0096] The lipid solution prepared by dissolving 85 mg of lipid material in 40 mL of chloroform in step (1) was added to an eggplant-shaped bottle containing 5 mL of chloroform, and then the cell membrane far-infrared fluorescent probe (DiD) was added, the mass ratio of the lipid material to the cell membrane far-infrared fluorescent probe (DiD) was 200:1, and the rotary evaporator was set at a water bath temperature of 43° C. and a pressure of 0.05-0.06 KPa for rotary evaporation. After the solvent in the eggplant-shaped bottle was completely removed, a uniform white film was obtained on the inner wall of the eggplant-shaped bottle, 5 mL of water was added to dissolve it, and the obtained lipid film solution was placed in an EP tube for standby use;

[0097] (3) Preparation of biomimetic liposomes:

[0098] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell crusher. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, that is, a lung surfactant fusion nano-bionic body loaded with a cell membrane far-infrared fluorescent probe (DiD).

[0099] Healthy Kunming mice were used as model animals. Fluorescently labeled nano-bionics were administered into the animals by aerosol administration. The lungs of the mice were taken out at 1h, 4h, 8h, 12h, and 24h. The content of nano-bionics in alveolar type II epithelial cells (AT2), alveolar macrophages (AM), and interstitial macrophages (IM) in the lung tissues was analyzed by flow cytometry. The targeting of the pulmonary surfactant-fused nano-bionics was analyzed from the results. The results showed that the pulmonary surfactant-fused nano-bionics can efficiently target alveolar type II epithelial cells (see Figure 5 ).

[0100] Healthy Kunming mice were used as model animals. Fluorescently labeled nano-bionics were administered into the animals by aerosol administration. The lungs of the mice were taken out at 4 hours, and the co-localization of DiD fluorescently labeled nano-bionics and SFTPC labeled alveolar type II epithelial cells was observed by lung sectioning. The results showed that the pulmonary surfactant fusion nano-bionics and alveolar type II epithelial cells were highly co-localized (see Figure 6 ).

[0101] Experiment 3: Experiment on the fusion of lung surfactant-fused nano-bionics and lung surfactant

[0102] The experimental materials are: lung surfactant fusion nano-bionics loaded with gold nanoparticles, and the preparation method of the bionics is:

[0103] (1) Preparation of lipid solution:

[0104] First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 50:35:8:11, and dissolve each lipid component in chloroform, 50 mg of dipalmitoylphosphatidylcholine (DPPC), 35 mg of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 8 mg of cholesterol (CHO) and 11 mg of dipalmitoylphosphatidylglycerol (DPPG) in 30 mL of chloroform, and then vortex the prepared solution to mix it into a lipid solution, and store it at 4°C for later use;

[0105] (2) Preparation of lipid film solution:

[0106] The lipid solution prepared by dissolving 104 mg of lipid material in 120 mL of chloroform in step (1) was added to an eggplant-shaped bottle containing 3 mL of chloroform, and the rotary evaporator was set at a water bath temperature of 43° C. and a pressure of 0.05-0.06 KPa for rotary evaporation. After the solvent in the eggplant-shaped bottle was completely removed, a uniform white film was obtained on the inner wall of the eggplant-shaped bottle. 3 mL of the gold nanoparticles hydrated with the aqueous solution were added, and the mass ratio of the lipid material to the gold nanoparticles was 200:1. The obtained lipid film solution was placed in an EP tube for standby use;

[0107] (3) Preparation of biomimetic liposomes:

[0108] The lipid film solution in the EP tube in step (2) is subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions are: working for 5 seconds, resting for 6 seconds, total working time for 5 minutes, and power of 100W, so as to form a single-layer vesicle-like nano-bionic body, that is, a lung surfactant fusion nano-bionic body encapsulating gold nanoparticles.

[0109] The nano-bionic bodies loaded with gold nanoparticles are spherical in shape and are basically uniform in size, with an average particle size of about 150 nm (see Figure 7 ).

[0110] The gold nanoparticles were injected into the lungs of mice by microperfusion. The lungs of mice were taken out 4 hours later, and the distribution of gold nanoparticles in the lung tissue was observed by transmission electron microscopy. The results showed that the gold nanoparticles in the lung surfactant-fused nanobionics were fused with the lung surfactant and were diffusely distributed (see Figure 8 ).

[0111] Experiment 4: Drug targeting ability and efficacy experiment of lung surfactant-fused nano-bionics

[0112] The lung surfactant fusion nano-bionics prepared according to the method described in Example 1 were used as healthy BALB / c mice as model animals. The nano-bionics containing MPLA drugs were administered into the animals by aerosol administration. After 4 hours, the mouse serum and mouse lungs were collected to detect TNF-α, IL-6, IFN-α, and IFN-γ in the serum and lung homogenate to determine the immune activation of the lungs. The results showed that the lung surfactant fusion nano-bionics containing MPLA showed a stronger immune activation effect than free MPLA (see Figure 9-16 ).

[0113] Experiment 5: In vivo safety study of lung surfactant-fused nano-bionics

[0114] Comparison of the body weight curves, blood biochemical analysis, and serum inflammatory cytokine detection of normal mice and mice that inhaled the pulmonary surfactant-fused nano-bionics showed that the pulmonary surfactant-fused nano-bionics had good biosafety (see Figure 17-22 ).

[0115] The above results indicate that the lung surfactant-fused nano-bionics of the present invention is temperature sensitive, can fuse with lung surfactant, can efficiently target alveolar type II epithelial cells, and has good biosafety.

[0116] The present invention has carried out the same experiments on other embodiments according to the above experimental method, and has obtained the same or similar results, which will not be listed one by one here.

[0117] In summary, the present invention provides a lung surfactant fusion type nano-bionic body, which is composed of a variety of phospholipids and cholesterol with the same composition as lung surfactant, and can load the therapeutic fat-soluble drug monophosphoryl lipid A (MPLA). Based on the composition, fusion and circulation characteristics of lung surfactant, the present invention continuously regulates the ratio between different lipids based on the composition of lung surfactant, and prepares a multifunctional lung surfactant fusion type nano-bionic body with temperature sensitivity that can be fused with lung surfactant, and with the help of lung surfactant circulation, efficiently targets alveolar type II epithelial cells and lung tissue. The method is stable and reliable, and the product has a good targeting effect. Compared with the prior art, it has the following advantages and beneficial effects: the lung surfactant fusion type nano-bionic body of the present invention can be fused with lung surfactant, and can efficiently target alveolar type II epithelial cells with the help of surfactant circulation. While delivering drugs to alveolar type II epithelial cells, it also has good biosafety, gives full play to the role of drugs, provides technical support for the treatment of lung diseases, improves the efficacy, and has significant social and economic benefits.

Claims

1. A method for preparing a lung surfactant-fused nano-bionic body, comprising the following steps: (1) Preparation of lipid solution: First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 30-50: 25-35: 5-10: 8-15, mix the components together, add them into chloroform with a weight volume of 2-22 times, where the weight volume refers to the solid in mg and the liquid in mL, then vortex and shake the prepared solution to mix it into a lipid solution, and store it at 4°C for later use; (2) Preparation of lipid film solution: Take the lipid solution prepared in step 1, add 68-110 mg of the lipid material to an eggplant-shaped bottle containing 3-5 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of lipid material to drug is 100:

1. The rotary evaporator water bath is set to 43°C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 2-5 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for standby use; The drug is monophosphoryl lipid A (MPLA); (3) Preparation of bionic body: The lipid film solution in the EP tube in step (2) was subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions were: working for 5 s, resting for 6 s, total working time for 5 min, and power of 100 W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

2. The method for preparing the lung surfactant-fused nano-bionic body according to claim 1, characterized in that: The following steps are involved: (1) Preparation of lipid solution: First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 40:30:7:12, mix the components together, add them into chloroform with a weight volume of 12 times, where the weight volume refers to the solid in mg and the liquid in mL, then vortex and shake the prepared solution to mix it into a lipid solution, store it at 4°C for later use; (2) Preparation of lipid film solution: Take the lipid solution prepared in step 1, add 89 mg of the lipid material solution to an eggplant-shaped bottle containing 4 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of lipid material to drug is 100:

1. The rotary evaporator water bath is set to 43°C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 3 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for later use; (3) Preparation of bionic body: The lipid film solution in the EP tube in step (2) was subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions were: working for 5 s, resting for 6 s, total working time for 5 min, and power of 100 W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

3. The method for preparing the lung surfactant fusion nano-bionic body according to claim 1, characterized in that: The following steps are involved: (1) Preparation of lipid solution: First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 30:25:5:8, mix the components together, add them into chloroform with a weight volume twice that of the original, where the weight volume refers to the solid in mg and the liquid in mL, then vortex and shake the prepared solution to mix it into a lipid solution, store it at 4°C for later use; (2) Preparation of lipid film solution: Take the lipid solution prepared in step 1, add 68 mg of the lipid material into an eggplant-shaped bottle containing 3 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of lipid material to drug is 100:

1. The rotary evaporator water bath is set to 43°C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 2 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for later use; (3) Preparation of bionic body: The lipid film solution in the EP tube in step (2) was subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions were: working for 5 s, resting for 6 s, total working time for 5 min, and power of 100 W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

4. The method for preparing the lung surfactant-fused nano-bionic body according to claim 1, characterized in that: The following steps are involved: (1) Preparation of lipid solution: First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 50:35:10:15, mix the components together, add them into chloroform with a weight volume of 22 times, where the weight volume refers to the solid in mg and the liquid in mL, then vortex and shake the prepared solution to mix it into a lipid solution, store it at 4°C for later use; (2) Preparation of lipid film solution: Take the lipid solution prepared in step 1, add every 110 mg of the lipid material solution to an eggplant-shaped bottle containing 5 mL of chloroform, then add the drug to be delivered to the lungs, the mass ratio of the lipid material to the drug is 100:1, and perform rotary evaporation under the conditions of setting the water bath temperature of the rotary evaporator to 43°C and the pressure to 0.05-0.06 Kpa. After the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle, add 5 mL of water to dissolve, and place the obtained lipid film solution into an EP tube for later use; (3) Preparation of bionic body: The lipid film solution in the EP tube in step (2) was subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions were: working for 5 s, resting for 6 s, total working time for 5 min, and power of 100 W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

5. The method for preparing the lung surfactant fusion type nano-bionic body according to claim 1, characterized in that: The following steps are involved: (1) Preparation of lipid solution: First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 45:28:8:13, mix the components together, add them into chloroform with a weight volume of 7 times, where the weight volume refers to the solid in mg and the liquid in mL, then vortex and shake the prepared solution to mix it into a lipid solution, store it at 4°C for later use; (2) Preparation of lipid film solution: Take the lipid solution prepared in step 1, add 94 mg of the lipid material solution to an eggplant-shaped bottle containing 2 mL of chloroform, then add the drug to be delivered to the lungs, the mass ratio of lipid material to drug is 100:1, set the water bath temperature of the rotary evaporator to 43°C and the pressure to 0.05-0.06 Kpa for rotary evaporation, and remove the solvent in the eggplant-shaped bottle. A uniform white film will be obtained on the inner wall of the eggplant-shaped bottle, add 3 mL of water to dissolve, and place the obtained lipid film solution into an EP tube for later use; (3) Preparation of bionic body: The lipid film solution in the EP tube in step (2) was subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions were: working for 5 s, resting for 6 s, total working time for 5 min, and power of 100 W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

6. The method for preparing the lung surfactant-fused nano-bionic body according to claim 1, characterized in that: The following steps are involved: (1) Preparation of lipid solution: First, select lipid materials with a mass ratio of: dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC): cholesterol (CHO): dipalmitoylphosphatidylglycerol (DPPG) = 35:32:7:11, mix the components together, add them into chloroform with a weight volume of 17 times, where the weight volume refers to the solid in mg and the liquid in mL, then vortex and shake the prepared solution to mix it into a lipid solution, store it at 4°C for later use; (2) Preparation of lipid film solution: Take the lipid solution prepared in step 1, add 85 mg of the lipid material into an eggplant-shaped bottle containing 3 mL of chloroform, and then add the drug to be delivered to the lungs. The mass ratio of lipid material to drug is 100:

1. The rotary evaporator water bath is set to 43°C and the pressure is 0.05-0.06 Kpa for rotary evaporation. When the solvent in the eggplant-shaped bottle is completely removed, a uniform white film will be obtained on the inner wall of the eggplant-shaped bottle. Add 4 mL of water to dissolve it, and place the obtained lipid film solution into an EP tube for later use; (3) Preparation of bionic body: The lipid film solution in the EP tube in step (2) was subjected to ice bath ultrasonic treatment using an ultrasonic cell disruptor. The ultrasonic conditions were: working for 5 s, resting for 6 s, total working time for 5 min, and power of 100 W, so as to form a single-layer vesicle-like nano-bionic body, namely, a lung surfactant fusion nano-bionic body.

7. A pulmonary surfactant-fused nano-bionic body prepared according to any one of claims 1 to 6, characterized in that: The lung surfactant fusion nano-bionics has a uniform particle size of 100-200nm and a potential of -43.22±0.5 mV.

8. A pulmonary surfactant-fused nano-bionic body prepared according to any one of claims 1 to 6, characterized in that: The lung surfactant fusion type nano-bionic body is observed to be spherical in shape by transmission electron microscopy, and its size is basically uniform.

9. Use of the lung surfactant-fused nano-bionics prepared by any one of the methods described in claims 1 to 6 in preparing drugs for treating lung diseases.

10. The use of the lung surfactant fusion nano-bionics according to claim 9 in preparing medicines for treating lung diseases, characterized in that: The lung disease is acute lung injury, interstitial lung disease, lung cancer, tuberculosis or chronic obstructive pulmonary disease.

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