CN113559065B - Preparation method and application of inhalation type lung surfactant nano bionic body - Google Patents
Preparation method and application of inhalation type lung surfactant nano bionic body Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a suction-type lung surfactant nano bionic body, which effectively solves the problems of preparation of the suction-type lung surfactant nano bionic body and application of enhancing anti-pulmonary fibrosis treatment and improving curative effect, adopts a nano bionic technology, takes lung surfactant functional components as materials, simulates a multi-membrane structure of a lung surfactant on the surface of an alveolus, utilizes a film dispersion method to prepare the lung surfactant nano bionic body, simultaneously loads an active oxygen scavenger astaxanthin on a lipid layer of the lung surfactant nano bionic body, loads an anti-pulmonary fibrosis drug pirfenidone in an inner core, constructs the suction-type lung surfactant nano bionic body with a nano drug delivery system integrating restoration and treatment, supplies the functional lung surfactant to the damaged alveolus while restoring the function of alveolus II type epithelial cells, quickly and effectively restores the lung function, and enhances the treatment effect of the pirfenidone, the pirfenidone successfully crosses the lung surfactant layer, the efficient transportation of the pirfenidone to lung tissues is improved, and the pulmonary fibrosis treatment is synergistically enhanced.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method and application of an inhalation type lung surfactant nano bionic body.
Background
Pulmonary Fibrosis (PF) is a chronic progressive lung disease with extremely high mortality and an average survival time of 3-5 years after diagnosis. The pathogenesis of PF is not clear, characterized by excessive deposition of extracellular matrix in the pulmonary interstitium and extensive collapse of alveoli, and finally death is caused by respiratory failure. Only Pirfenidone (PFD) and nintedanib are currently approved for PF treatment, and although both drugs can slow the rate of lung capacity decline, they fail to restore lung function. In addition, the lung, which is an organ of the human body in direct contact with the external environment, has a strong defense system against foreign body invasion, wherein the lung surfactant layer is a main barrier for foreign body to enter lung tissue. Therefore, the realization of the pulmonary function recovery and the efficient drug transfer to the pulmonary tissue has important significance for improving the survival rate of PF patients.
The alveoli act as the main sites for gas exchange in the lungs, and their microstructure directly affects lung function. However, during PF pathogenesis, large areas of alveoli collapse due to excessive extracellular matrix deposition, resulting in gas exchange obstruction. Thus, lung function can be restored by reconstructing the alveolar structure. Pulmonary surfactants are the major factors in maintaining the stability of the alveolar structure, and are synthesized and secreted by alveolar Type ii epithelial cells (aecii). In the lung tissue of PF patients, aecii function is impaired due to oxidative stress, and lung surfactant cannot be synthesized. Repair of aecii has become a common means of lung function recovery.
With the rapid development of nanoscience and technology, the nano-biomimetic technology has made great progress in drug delivery systems. At present, the bionic nanoparticles are mainly disguised by endogenous substances to simulate organisms. The bionic technology retains the characteristics of organisms to the maximum extent, but is greatly limited by the difficulty of material selection. The lung surfactant is a layer of lipoprotein complex covering the surface of alveoli and is difficult to obtain directly from the organism. Meanwhile, the lung surfactant exists in lung tissues in various forms, and can be roughly divided into three types according to metabolic processes: tubulose myelin (the direct conversion of newly secreted lung surfactant into a complex structure), a multi-layered structure (which will continue to participate in circulation at the alveolar surface), a single lamellar body (formation of lung surfactant used at the alveolar surface). The multilayer structure of the simulated lung surfactant is an effective technical way for the nano bionic body to enter the circulation of the lung surfactant and realize the high-efficiency transportation of the medicament to lung tissues.
Therefore, a nano bionic technology is adopted, the functional components of the lung surfactant are used as raw materials, the multi-membrane structure of the lung surfactant on the surface of an alveolus is simulated, and the lung surfactant nano bionic bodies (PSBs) are constructed and used as drug delivery carriers for efficiently transporting to lung tissues. Combining action sites of an anti-fibrosis drug Pirfenidone (PFD) and an active oxygen scavenger Astaxanthin (AST) and the circulating characteristics of lung surfactants, encapsulating the PFD in a water-soluble core of PSBs, and loading the AST in a lipid membrane of the PSBs to obtain PSBs/AST @ PFD. After PSBs reach alveoli by inhalation administration, due to the similarity of composition and structure with PS, lipid membranes of PSBs are gradually fused with PS, functional lung surfactant is supplied to alveoli, and AST loaded by lipid membranes is gradually transported to AECII along with the circulation of the lung surfactant and restores the function of AECII. Meanwhile, PFD wrapped in the PSBs inner core is released to the inner side of the lung surfactant layer in situ, successfully crosses the lung surfactant layer, and effectively enters the lung interstitial tissue to exert curative effect. However, no disclosure is made up to now.
Disclosure of Invention
In view of the above situation, in order to overcome the defects of the prior art, the present invention aims to provide a method for preparing an inhalation type pulmonary surfactant nano-bionic body and an application thereof, which can effectively solve the problems of preparation of the inhalation type pulmonary surfactant nano-bionic body and medication for enhancing anti-pulmonary fibrosis treatment and improving curative effect.
In order to achieve the above object, the technical solution of the present invention is a method for preparing an inhalation type lung surfactant nano-bionic body, which comprises the steps of using a nano-bionic technology, using functional components of a lung surfactant as materials, simulating a multi-membrane structure of the lung surfactant on the surface of alveoli, preparing lung surfactant nano-bionic bodies (PSBs) by a thin-film dispersion method, simultaneously loading an active oxygen scavenger Astaxanthin (AST) on lipid layers of the lung surfactant nano-bionic bodies, and loading an anti-pulmonary fibrosis drug Pirfenidone (PFD) in an inner core of the lung surfactant nano-bionic bodies, thereby constructing the inhalation type lung surfactant nano-bionic body with a nano-drug delivery system integrating repair and treatment, and specifically comprising the following steps:
(1) respectively dissolving 5-30 mg of dipalmitoyl phosphatidylcholine (DPPC), 2-10 mg of Cholesterol (CHO) and 3-20 mg of lecithin (PC) into 5-30 mL of chloroform, then whirling, shaking and uniformly mixing the prepared solution, and storing at 4 ℃ for later use; weighing 5-10 mg of astaxanthin, dissolving in chloroform, and preparing into an astaxanthin solution with the concentration of 2-3 mg/mL;
(2) DPPC according to the mass ratio: CHO: taking the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) according to the proportion of PC =6:1:3, putting the lipid material solution and the astaxanthin solution into a bottle, carrying out rotary evaporation at the temperature of 43 ℃ and under the pressure of 0.05-0.06Kpa, removing the solvent to obtain a layer of uniform white film on the inner wall of the bottle, adding 2mL of Pirfenidone (PFD) aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube (EP tube);
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube (EP tube) in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection condition is as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 400-500 mu L Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
The particle size of the inhalation type lung surfactant nano bionic body is 150-200 nm.
The inhalation type lung surfactant nano bionic body prepared by the method is applied to preparing drugs for enhancing anti-pulmonary fibrosis treatment and improving curative effect.
The inhalation type lung surfactant nano bionic body prepared by the method is used as a drug delivery system to prepare a drug for realizing integration of treatment and restoration.
The preparation method is simple, and the prepared nano bionic body can repair the function of the alveolar type II epithelial cells and timely supply the functional lung surfactant to damaged alveoli so as to quickly and effectively repair the lung function. On the other hand, the treatment effect of the pirfenidone is enhanced, the pirfenidone successfully crosses the lung surfactant layer, and the efficient transportation of the pirfenidone to lung tissues is improved. The delivery system synergistically enhances the treatment of pulmonary fibrosis, effectively enhances the treatment effect of pirfenidone, and has remarkable economic and social benefits.
Detailed Description
The following examples are provided to explain the present invention in detail.
In specific practice, the invention is illustrated by the following examples.
Example 1
The invention relates to a preparation method of an inhalation type lung surfactant nano bionic body, which comprises the following steps:
(1) respectively dissolving 17.5mg of dipalmitoyl phosphatidylcholine (DPPC), 6mg of Cholesterol (CHO) and 11.5mg of lecithin (PC) into 27.5mL of chloroform, then whirling, shaking and mixing the prepared solution uniformly, and storing at 4 ℃ for later use; weighing 7.5mg of astaxanthin, dissolving in chloroform, and preparing into an astaxanthin solution with the concentration of 2.5 mg/mL;
(2) DPPC according to the mass ratio: CHO: taking the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) according to the proportion of PC =6:1:3, placing the solutions into a bottle, performing rotary evaporation at the temperature of 43 ℃ and under the pressure of 0.055Kpa, removing the solvent to obtain a layer of uniform white film on the inner wall of the bottle, adding 2mL of Pirfenidone (PFD) aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube (EP tube);
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube (EP tube) in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection condition is as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 450 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
Example 2
The invention relates to a preparation method of an inhalation type lung surfactant nano bionic body drug loading system, which comprises the following steps:
(1) respectively dissolving 20.3mg of dipalmitoyl phosphatidylcholine (DPPC), 5.8mg of Cholesterol (CHO) and 12.4mg of lecithin (PC) into 5mL of chloroform, then whirling, shaking and mixing the prepared solution uniformly, and storing at 4 ℃ for later use; weighing 5.2mg of astaxanthin, dissolving in chloroform, and preparing into an astaxanthin solution with the concentration of 2.6 mg/mL;
(2) DPPC according to mass ratio: CHO: putting the corresponding lipid material solution prepared in the step (1) and the astaxanthin solution into an eggplant-shaped bottle according to the proportion of PC =6:1:3, performing rotary evaporation in a water bath of a rotary evaporator under the conditions of 43 ℃ and 0.05-0.06Kpa, removing the solvent to obtain a uniform white film on the inner wall of the eggplant-shaped bottle, adding 2mL of Pirfenidone (PFD) aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube (EP tube);
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube (EP tube) in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection condition is as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 400 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
Example 3
The invention relates to a preparation method of an inhalation type lung surfactant nano bionic body drug loading system, which comprises the following steps:
(1) respectively dissolving three lipid materials of Dipalmitoylphosphatidylcholine (DPPC) 18.7mg, Cholesterol (CHO) 5.1mg and lecithin (PC) 11.3mg in chloroform 5mL, vortexing, shaking, mixing, and storing at 4 deg.C; weighing 4.8mg of astaxanthin, dissolving in chloroform, and preparing into an astaxanthin solution with the concentration of 2.4 mg/mL;
(2) DPPC according to the mass ratio: CHO: putting the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) into a solanaceous bottle according to the proportion of PC =6:1:3, carrying out rotary evaporation in a water bath of a rotary evaporator at the temperature of 43 ℃ and under the pressure of 0.05-0.06Kpa, removing the solvent, obtaining a layer of uniform white film on the inner wall of the solanaceous bottle, adding 2mL of Pirfenidone (PFD) aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube (EP tube);
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube (EP tube) in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection condition is as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 400 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
Example 4
The invention relates to a preparation method of an inhalation type lung surfactant nano bionic body drug loading system, which comprises the following steps:
(1) respectively dissolving 19.8mg of Dipalmitoylphosphatidylcholine (DPPC), 5.7mg of Cholesterol (CHO) and 11.2mg of lecithin (PC) in 5mL of chloroform, vortexing, shaking, and storing at 4 deg.C; weighing 5mg of astaxanthin, dissolving the astaxanthin in chloroform, and preparing an astaxanthin solution with the concentration of 2 mg/mL;
(2) DPPC according to the mass ratio: CHO: putting the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) into a solanaceous bottle according to the proportion of PC =6:1:3, carrying out rotary evaporation in a water bath of a rotary evaporator at the temperature of 43 ℃ and under the pressure of 0.05-0.06Kpa, removing the solvent, obtaining a layer of uniform white film on the inner wall of the solanaceous bottle, adding 2mL of Pirfenidone (PFD) aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube (EP tube);
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube (EP tube) in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection condition is as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 500 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
The particle size of the inhalation type lung surfactant nano bionic body is 150-200 nm.
The inhalation type lung surfactant nano bionic body prepared by the methods of the embodiments 1 to 4 is applied to the preparation of drugs for enhancing the anti-pulmonary fibrosis treatment and improving the curative effect.
The inhalation type lung surfactant nano bionic body prepared by the methods of the embodiments 1 to 4 is used as a medicine carrying system to prepare a medicine for realizing the integration of treatment and repair.
It can be seen from the above that, according to the technical scheme of the present invention, a method for preparing inhalation type pulmonary surfactant nano-bionic body is provided, wherein a nano-bionic technology is adopted, functional components of a pulmonary surfactant are used as materials, a multi-membrane structure of the pulmonary surfactant on the surface of an alveolus is simulated, a thin-film dispersion method is used to prepare pulmonary surfactant nano-bionic body (PSBs), an active oxygen scavenger Astaxanthin (AST) is loaded on a lipid layer of the lung surfactant nano-bionic body, an anti-pulmonary fibrosis drug Pirfenidone (PFD) is loaded in an inner core of the lung surfactant nano-body, a nano-drug delivery system with integrated repair and treatment is constructed, pulmonary fibrosis treatment is synergistically enhanced, and experiments show that very good beneficial technical effects are obtained, and relevant experimental data are as follows (taking example 1):
experiment 1: toxicity test of PSBs/AST @ PFD nano-drug delivery system on human umbilical vein endothelial cells
In vitro cytotoxicity test, human umbilical vein endothelial cells are used as study objects, a blank control group, a PFD group and a PSBs/AST @ PFD group are arranged, PFDs with different concentrations are used for treating the cells, the cells are continuously cultured for 24h, and the toxicity of the cells on the vascular endothelial cells is inspected through a CCK-8 method. The result shows that the PSBs/AST @ PFD nano-carrier has little influence on the activity of vascular endothelial cells and can be safely fused with pulmonary surfactant on the surface of alveoli to play a role.
Experiment 2: PSBs nano bionic body lung distribution form experiment
The pulmonary distribution forms of the three nano-carriers are examined by an inhalation administration mode by taking bleomycin induced pulmonary fibrosis mice as an administration animal model. Three groups were set up for the experiment: 1. a single membrane nanovesicle group (UVs @ DiO); 2. a set of cationic nanovesicles (PVs @ DiO); 3. nanoantibiont groups (PSBs @ DiO). Each group of mice was administered with nebulization using a commercially available nebulizer at an administration rate of 15 mg/kg. After the administration is finished for 4h, dissecting each group of mice in the dark to take lung tissues, gently clamping the lung tissues by using forceps, cleaning the lung tissues by using normal saline, putting the lung tissues into an Ep tube coated by tinfoil paper, and storing and delivering the lung tissues into a refrigerator at the temperature of-80 ℃. The results showed that PSBs @ DiO showed strong lung fluorescence intensity and high lung uptake compared to the other two groups, indicating that PSBs are more likely to be distributed in lung tissue by the lung surfactant cycle.
Experiment 3: examination experiment for capability of PSBs nano bionic body in transferring PFD into lung
The bleomycin-induced pulmonary fibrosis mice are used as an administration animal model, and the infiltration capacity of PSBs carrying anti-fibrosis drug PFD to enter the lung is examined in an inhalation administration mode. Three groups were set up for the experiment: 1. a single membrane nanovesicle set (UVs @ RB); 2. a set of cationic nanovesicles (PVs @ RB); 3. nanoantibiont group (PSBs @ RB). Each group of mice was administered with nebulization using a commercially available nebulizer at an administration rate of 15 mg/kg. After the administration is finished for 4h, dissecting each group of mice in the dark to take lung tissues, gently clamping the lung tissues by using forceps, cleaning the lung tissues by using normal saline, putting the lung tissues into an Ep tube coated by tinfoil paper, and storing and delivering the lung tissues into a refrigerator at the temperature of-80 ℃. The results show that PSBs @ RB carried RB with strong lung fluorescence intensity compared to the other two groups. Thus, PSBs enhance the efficient transport of PFD to lung tissue by fusing with pulmonary surfactant at the alveolar surface and by circulating through the pulmonary surfactant, allowing PFD to cross the pulmonary surfactant layer barrier.
Experiment 4: PSBs/AST @ PFD nano-drug delivery system in-vivo anti-pulmonary fibrosis experiment
After the mice are anesthetized, bleomycin is dripped into the oropharynx to induce the mouse pulmonary fibrosis animal model. The administration treatment was carried out 10 days after the model was established. The experimental groups were as follows: 1) a blank healthy mouse group (Normal); 2) bleomycin model group (BLM); 3) pirfenidone group (PFD); 4) the group of single membrane vesicles (UVs/AST @ PFD); 5) a set of cationic nanovesicles (PVs/AST @ PFD); 6) nanoantibiotic groups (PSBs/AST @ PFD). The administration was 1 time per day for 14 consecutive times. After the treatment, lung tissues of each group of mice are subjected to H & E staining and Masson staining, and the sections are placed under a microscope to observe the alveolar morphology and the collagen deposition amount of each group and photographed. Experimental results show that the space thickness and alveolar cavity size of PSBs/AST @ PFD groups of alveoli are close to normal, and the collagen deposition amount is reduced. PSBs/AST @ PFD has good anti-fibrosis effect and improves survival rate.
The invention not only carries out the four repeated experiments, but also carries out the same experiments on the products given in the embodiments 2-4, and all obtains the same and similar results, which are not listed one by one, and the experiments show that the method is stable and reliable, the product effect is good, and compared with the prior art, the invention has the following beneficial technical effects:
(1) rapidly repairing lung function: the method adopts the drugs of 'internal and external combination', and supplies functional pulmonary surfactant to damaged alveoli in time while repairing the functions of damaged alveoli II type epithelial cells by AST neutralizing oxidative stress products carried by a nano bionic liposome layer, thereby realizing the rapid double repair of the lung functions;
(2) has the function of accurate targeted delivery of the drug: AST and PFD are respectively encapsulated in PSBs lipid layers and cores, after the AST reaches alveoli through inhalation administration, the AST is gradually transported to alveoli II type epithelial cells along with the circulation of pulmonary surfactant, and PFD in the cores is released in situ to the inner side of pulmonary surfactant layers and directly enters pulmonary interstitial tissues to play a curative effect, so that the anti-fibrosis effect of the PFD is improved;
(3) based on the highly bionic lung surfactant and simulating the multi-membrane microstructure of the lung surfactant on the surface of the alveolus, the lung surfactant participates in the circulation of the lung surfactant, and the high-efficiency transport of the drug to lung tissues is enhanced.
(4) The medicine can realize the integration of treatment and repair, provides a new technical support (medicine) for treating pulmonary fibrosis, develops a new way for treating pulmonary fibrosis, can improve the survival time by more than 5 years by 1-2 times, is a great innovation for treating pulmonary fibrosis, and has obvious economic and social benefits.
Claims (7)
1. A preparation method of an inhalation type lung surfactant nano bionic body is characterized in that a nano bionic technology is adopted, a lung surfactant functional component is used as a material, a multi-membrane structure of a lung surfactant on the surface of an alveolus is simulated, the lung surfactant nano bionic body is prepared by a thin film dispersion method, an active oxygen scavenger astaxanthin is loaded on a lipid layer of the lung surfactant nano bionic body, an anti-pulmonary fibrosis drug pirfenidone is loaded in an inner core, and the inhalation type lung surfactant nano bionic body with a repair and treatment integrated nano-drug delivery system is constructed, and the preparation method specifically comprises the following steps:
(1) respectively dissolving 5-30 mg of dipalmitoyl phosphatidylcholine, 2-10 mg of cholesterol and 3-20 mg of lecithin in 5-30 mL of chloroform, then whirling, shaking and uniformly mixing the prepared solution, and storing at 4 ℃ for later use; weighing 5-10 mg of astaxanthin, dissolving in chloroform, and preparing into an astaxanthin solution with the concentration of 2-3 mg/mL;
(2) dipalmitoyl phosphatidylcholine: cholesterol: putting the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) into a bottle according to the ratio of lecithin =6:1:3, performing rotary evaporation at the temperature of 43 ℃ and under the pressure of 0.05-0.06Kpa, removing the solvent to obtain a layer of uniform white film on the inner wall of the bottle, adding 2mL of pirfenidone aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube;
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection conditions are as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 400-500 mu L Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
2. The method of preparing an inhaled lung surfactant nanobiomimetic as in claim 1, comprising the steps of:
(1) respectively dissolving three lipid materials of 17.5mg of dipalmitoyl phosphatidylcholine, 6mg of cholesterol and 11.5mg of lecithin in 27.5mL of chloroform, then whirling, shaking and uniformly mixing the prepared solution, and storing at 4 ℃ for later use; weighing 7.5mg of astaxanthin, dissolving in chloroform, and preparing into an astaxanthin solution with the concentration of 2.5 mg/mL;
(2) dipalmitoyl phosphatidylcholine: cholesterol: putting the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) into a bottle according to the ratio of lecithin =6:1:3, performing rotary evaporation at 43 ℃ under the pressure of 0.055Kpa, removing the solvent to obtain a uniform white film on the inner wall of the bottle, adding 2mL of pirfenidone aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube;
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection conditions are as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 450 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
3. The method of preparing an inhaled lung surfactant nanobiomimetic as in claim 1, comprising the steps of:
(1) respectively dissolving three lipid materials of dipalmitoyl phosphatidylcholine 20.3mg, cholesterol 5.8mg and lecithin 12.4mg in chloroform 5mL, then whirling, shaking and mixing the prepared solution uniformly, and storing at 4 ℃ for later use; weighing 5.2mg of astaxanthin, dissolving in chloroform, and preparing into an astaxanthin solution with the concentration of 2.6 mg/mL;
(2) dipalmitoyl phosphatidylcholine: cholesterol: putting the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) into a eggplant-shaped bottle according to the ratio of lecithin =6:1:3, performing rotary evaporation in a water bath of a rotary evaporator at the temperature of 43 ℃ and under the pressure of 0.05-0.06Kpa, removing the solvent to obtain a uniform white film on the inner wall of the eggplant-shaped bottle, adding 2mL of pirfenidone aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube;
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection conditions are as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 400 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
4. The method of preparing an inhaled lung surfactant nanobiomimetic as in claim 1, comprising the steps of:
(1) respectively dissolving three lipid materials of dipalmitoyl phosphatidylcholine 18.7mg, cholesterol 5.1mg and lecithin 11.3mg in chloroform 5mL, then whirling, shaking and mixing the prepared solution uniformly, and storing at 4 ℃ for later use; weighing 4.8mg of astaxanthin, dissolving in chloroform, and preparing into 2.4mg/mL astaxanthin solution;
(2) dipalmitoyl phosphatidylcholine: cholesterol: putting the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) into a eggplant-shaped bottle according to the ratio of lecithin =6:1:3, performing rotary evaporation in a water bath of a rotary evaporator at the temperature of 43 ℃ and under the pressure of 0.05-0.06Kpa, removing the solvent to obtain a uniform white film on the inner wall of the eggplant-shaped bottle, adding 2mL of pirfenidone aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube;
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection conditions are as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 400 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
5. The method of preparing an inhaled lung surfactant nanobiomimetic as in claim 1, comprising the steps of:
(1) dissolving 19.8mg of dipalmitoylphosphatidylcholine, 5.7mg of cholesterol and 11.2mg of lecithin in 5mL of chloroform respectively, then whirling, shaking and mixing the prepared solution uniformly, and storing at 4 ℃ for later use; weighing 5mg of astaxanthin, dissolving the astaxanthin in chloroform, and preparing an astaxanthin solution with the concentration of 2 mg/mL;
(2) dipalmitoyl phosphatidylcholine in a mass ratio of: cholesterol: putting the corresponding lipid material solution and the astaxanthin solution prepared in the step (1) into a eggplant-shaped bottle according to the ratio of lecithin =6:1:3, performing rotary evaporation in a water bath of a rotary evaporator at the temperature of 43 ℃ and under the pressure of 0.05-0.06Kpa, removing the solvent to obtain a uniform white film on the inner wall of the eggplant-shaped bottle, adding 2mL of pirfenidone aqueous solution with the concentration of 2mg/mL for hydration, and transferring the obtained solution to a centrifuge tube;
(3) carrying out ice-bath ultra-detection treatment on the solution in the centrifugal tube in the step (2) by using an ultrasonic cell crusher, wherein the ultra-detection conditions are as follows: working for 5s, pausing for 6s, and forming a unilamellar vesicle by the total working time of 2min and the power of 100W;
(4) adding 500 mu L of Ca with the concentration of 2mg/mL into the unilamellar vesicle solution obtained in the step (3)2+And (3) carrying out vortex on the solution for 5min, carrying out ice bath ultra-detection for 2min, and alternately carrying out the vortex and the ice bath ultra-detection for 1h at the power of 100W to obtain the lung surfactant nano-bionic body.
6. The method for the preparation of an inhaled lung surfactant nanobiomimetic according to any of claims 1-5, wherein the particle size of said inhaled lung surfactant nanobiomimetic is 150-200 nm.
7. Use of an inhaled lung surfactant biomimetic produced by the process of any of claims 1-6 for the manufacture of a medicament for enhancing the therapeutic effect of anti-pulmonary fibrosis.
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