WO2024078443A1 - 一种纳米药物联合制剂及其制备方法和应用 - Google Patents

一种纳米药物联合制剂及其制备方法和应用 Download PDF

Info

Publication number
WO2024078443A1
WO2024078443A1 PCT/CN2023/123526 CN2023123526W WO2024078443A1 WO 2024078443 A1 WO2024078443 A1 WO 2024078443A1 CN 2023123526 W CN2023123526 W CN 2023123526W WO 2024078443 A1 WO2024078443 A1 WO 2024078443A1
Authority
WO
WIPO (PCT)
Prior art keywords
preparation
drug
nano
solution
hours
Prior art date
Application number
PCT/CN2023/123526
Other languages
English (en)
French (fr)
Inventor
吴丽娜
车春利
Original Assignee
哈尔滨医科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 哈尔滨医科大学 filed Critical 哈尔滨医科大学
Publication of WO2024078443A1 publication Critical patent/WO2024078443A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention belongs to the field of biomedicine technology, and specifically relates to a nano-drug combination preparation and a preparation method thereof, and application of the nano-drug preparation in fibrotic diseases.
  • Idiopathic pulmonary fibrosis is a special type of chronic progressive lung disease of unknown etiology, with lesions confined to the lungs.
  • the main pathological feature of IPF is the infiltration of inflammatory cells.
  • a large number of cytokines promote the migration, proliferation and differentiation of fibroblasts into active myofibroblasts, which together promote the increase of collagen and cause excessive proliferation of the extracellular matrix (ECM).
  • ECM extracellular matrix
  • Normal human lung tissue has good ductility and can maintain vital capacity at a normal level. In fibrotic lung tissue, the extracellular matrix is excessively deposited, the rigidity increases, the gas exchange function of the alveoli is greatly damaged, the gas exchange in the lungs is blocked, and the pulmonary ventilation function is seriously impaired.
  • pulmonary interstitial fibrosis can lead to the thickening of the air-blood barrier in the lungs (liquid layer containing pulmonary surfactant, matrix layer, capillary endothelial cell layer, etc.), increase the diffusion distance of air inhaled into the lungs into the blood, reduce the diffusion area, and affect oxygenation due to ventilation-perfusion ratio imbalance, further leading to hypoxia, and eventually causing dyspnea after activity or even at rest.
  • Idiopathic pulmonary fibrosis is called "cancer that is not cancer", and the median survival of patients is only 2-3 years, with a mortality rate higher than that of most tumors. IPF patients have reduced lung compliance, disrupted gas exchange, and progressive loss of lung function, which will eventually lead to respiratory failure and death.
  • Clinical non-drug treatments such as lung transplantation have high requirements for various conditions, and the treatment effects of mechanical ventilation, oxygen inhalation, bronchoalveolar lavage, etc. are not satisfactory.
  • Commonly used drug treatments such as pirfenidone and nintedanib, although both drugs show a trend of reducing mortality and have similar effects on the reduction rate of forced vital capacity over one year, these two drugs have not been shown to significantly increase survival time.
  • the treatment cycle is long, and continuous treatment for 52 weeks can effectively improve the patient's lung function, prolong the patient's progression-free survival, and reduce the patient's risk of death.
  • existing studies have found that after long-term administration, patients will develop tolerance to the drug, reducing the therapeutic effect of the drug.
  • the present invention aims to provide a pulmonary-administered oxygen-carrying nano-drug combination preparation, which, on the one hand, utilizes the encapsulated drug to inhibit the infiltration of inflammatory cells and the generation of inflammatory factors from the source of the disease, and avoids the downstream signal stimulating the proliferation and activation of fibroblasts into myofibroblasts; on the other hand, the oxygen-carrying function of the perfluorocarbon therein is used to improve the lung microenvironment, improve lung compliance, and reduce the expression of vascular endothelial growth factor VEGF mRNA stimulated by hypoxia-inducible factor HIF 1 ⁇ , and reduce the generation of vascular endothelial cells; at the same time, the collagen deposition of the extracellular matrix (EMC) is reduced, and the nano-drug preparation of the present invention is synergistically
  • a multi-pathway combined treatment strategy is adopted to treat idiopathic pulmonary fibrosis, and animal and cell experiments show good effects of improving the progression of pulmonary fibrosis and improving the survival status of animals. There has been no public report on the treatment of IPF using the above strategy.
  • Perfluorocarbons are compounds in which all hydrogen atoms in a hydrocarbon molecule are replaced by fluorine atoms. They have strong chemical stability and extremely high oxygen solubility. Under the same oxygen partial pressure, the oxygen carrying capacity of perfluorocarbons (PFCs) is about 20-25 times that of water and plasma. This high oxygen carrying capacity and good biosafety have led to their widespread development in clinical research for use in blood substitutes, tumor oxygenation, and other purposes. In addition, perfluorocarbons have also been approved by the FDA for the treatment of neonatal respiratory distress.
  • perfluorocarbons into nanoemulsions for oxygen-carrying lung delivery is a direction worthy of research, but since perfluorocarbon compounds are chemically inert and insoluble in water or oil, it is difficult for nanoformulations to encapsulate a high proportion of perfluorocarbons, so their oxygen-carrying use is also affected and greatly reduced.
  • the present invention provides a pulmonary-administered oxygen-carrying nano-drug combination preparation with good oxygen-carrying performance and which can effectively improve the efficacy of IPF and a preparation method thereof.
  • the present invention adopts the following technical solutions:
  • a nano drug combination preparation uses a lipid base as a carrier, can encapsulate a relatively high proportion of perfluorocarbon (PFC), and uses biologically active small molecule proteins and anti-fibrosis drugs as active ingredients.
  • PFC perfluorocarbon
  • the nano-combination preparation is a nano-drug combination preparation for fibrotic diseases.
  • the fibrotic disease includes any one of pulmonary fibrotic disease and liver fibrotic disease.
  • the lipid base mainly includes one or any combination of several of the following: egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoyl lecithin, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, distearoylphosphatidylglycerol, cholesterol, Tween, stearamide, (2,3-dioleoyl-propyl)-trimethylammonium chloride, and glycerol.
  • the perfluorocarbon including 1-bromoperfluoroethane, perfluorohexyl iodide, perfluorooctyl iodide, perfluorohexane, perfluorooctanoic acid, perfluorooctane bromide, perfluorooctane, 1-bromo-3-fluoropropane, 1-bromo-1,1,2,2-tetrafluorobutane, 1-bromo-2,2-difluoroethane, 1H-6-bromoperfluorohexane, 1-bromo-tetrafluorobutane, 1-bromoheptafluorooctane, perfluoro-15-crown-5 ether , potassium perfluorohexane sulfonate, perfluorohexyl iodide, 1-bromoperfluoroethane, 1,6-di
  • the mass volume ratio of the perfluorocarbon (PFC) and the lipid base is 10%-20%.
  • the protein accounts for 0.3%-20% of the mass of the carrier.
  • the anti-fibrotic drug accounts for 2%-30% of the carrier mass.
  • the bioactive small molecule protein is one or more of interleukin-13 (IL-13), interleukin-10 (IL-10), interferon- ⁇ (IFN- ⁇ ), and keratinocyte growth factor (KGF).
  • the anti-fibrosis drug is one or any combination of BIBF1120, pirfenidone, trametinib, nilotinib, resveratrol, curcumin, and remdesivir.
  • the bioactive small molecule protein is interferon- ⁇ (IFN- ⁇ ), and the anti-fibrosis drug is BIBF1120.
  • IFN- ⁇ interferon- ⁇
  • BIBF1120 interferon- ⁇
  • This bioactive small molecule protein is easily soluble in aqueous solution, while the anti-fibrosis drug is insoluble in water, and the anti-fibrosis drug needs to be first fused into the above-mentioned lipid base by dissolving it in an organic solvent.
  • the aqueous solution includes one or any combination of several of sterile ultrapure water, sterile water for injection, sterile sodium chloride solution, drip water, and deionized water.
  • the organic solvent includes one of chloroform, dimethyl sulfoxide, anhydrous ethanol, methanol, isopropane, and tert-butanol, or any combination of several of them.
  • the present invention provides a method for preparing a pulmonary administration oxygen-carrying nano drug combination preparation, comprising the following steps:
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 30-50° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 30-50° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 30-50° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-16 hours to completely remove the organic solvent in the system;
  • step (3) (4) adding 1-10 mL of sterilized ultrapure water and 0.1-2 mL of perfluorocarbon to the film obtained in step (3), performing probe ultrasound at a power of 100-500 W for 2-6 min, and extruding the obtained drug preparation through a 0.22 ⁇ m filter membrane for 5-10 times, and then dialysis purification (4° C., 100 rpm) to remove all unencapsulated drugs;
  • step (4) adding 5% to 15% of a lyophilizing protective agent to the pharmaceutical preparation obtained in step (4), prefreezing at -20 to -80°C for 24 to 48 hours, sublimation drying at -40 to -50°C for 24 to 48 hours, and desorption drying at 25°C for 6 to 10 hours;
  • step (6) Add 1-10 mL of a bioactive small molecule protein solution (100-200 ⁇ g/mL) to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze filter through a 0.2 ⁇ m filter membrane for 10-20 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • a bioactive small molecule protein solution 100-200 ⁇ g/mL
  • the present invention provides a method for preparing a pulmonary administration oxygen-carrying nano drug combination preparation, comprising the following steps:
  • step (1) adding 5% to 15% of a lyophilizing protective agent to the solution obtained in step (1), prefreezing at -20 to -80°C for 24 to 48 hours, sublimation drying at -40 to -50°C for 24 to 48 hours, and desorption drying at 25°C for 6 to 10 hours;
  • step (3) adding 0.5-5 mL of sterile ultrapure water and 0.1-2 mL of perfluorocarbon to the lyophilized powder obtained in step (2) for reconstitution, and then performing probe ultrasound for 2-6 min at a power of 100-500 W.
  • the obtained drug preparation is squeezed through a 0.2 ⁇ m filter membrane for 15-20 times, and then dialyzed and purified (4° C., 100 rpm) to remove all unencapsulated drugs;
  • step (3) incubating the drug preparation obtained in step (3) and 0.5-5 mL of a bioactive small molecule protein solution (200-400 ⁇ g/mL) at 4° C. with shaking for 3-5 h;
  • step (4) The co-incubation solution obtained in step (4) is subjected to ultrafiltration centrifugation (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenation treatment is performed before use).
  • the lyoprotectant includes one or any combination of several of sucrose, trehalose, lactose, mannitol, glycine, sodium chloride, sorbitol, glucose and maltose.
  • the encapsulation rate of anti-fibrosis drugs is greater than 70%, the encapsulation rate of bioactive small molecule proteins is greater than 60%, the particle size of the nano-drug preparation is 130-200nm, and the polydispersity coefficient PDI is less than 0.2.
  • the above-mentioned pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention is used in the treatment of idiopathic pulmonary fibrosis (IPF).
  • IPF idiopathic pulmonary fibrosis
  • the present invention discloses a pulmonary-administered oxygen-carrying nano-drug combined preparation and a preparation method thereof, wherein a chemically inert perfluorocarbon compound, a fat-soluble anti-fibrosis drug and a water-soluble bioactive small molecule protein are co-loaded on the same nano-platform.
  • a multi-pathway combined treatment strategy is adopted, which has the following beneficial effects:
  • the nano drug preparation of the present invention can encapsulate a high proportion of perfluorocarbons and carry a large amount of oxygen into the lungs, thereby improving the hypoxic state of the IPF lung microenvironment, increasing lung compliance, and alleviating the loss of lung function.
  • the nano drug preparation of the present invention has good stability and can be administered to the lungs by atomization inhalation, which is simple and non-invasive. After inhalation, the nano preparation accumulates in the lungs and can quickly reach an effective concentration.
  • the bioactive small molecule protein encapsulated in the nano-drug preparation of the present invention exerts an immunomodulatory and anti-inflammatory effect at the source of the occurrence and development of IPF, and together with the anti-fibrotic drugs encapsulated in the nano-preparation, reduces the expression of TGF- ⁇ , a key stimulator of pulmonary fibrosis, inhibits the proliferation and activation of fibroblasts into myofibroblasts, and achieves the purpose of synergistically inhibiting pulmonary fibrosis.
  • hypoxia-inducible factor HIF-1 ⁇ stimulates the expression of vascular endothelial growth factor VEGF mRNA, and vascular endothelial hyperplasia will lead to further thickening of the gas-blood barrier and increased gas exchange resistance.
  • the oxygen-carrying nano-drug combination preparation of the present invention can improve this situation through oxygen delivery.
  • Collagen is the main component of the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the two drugs encapsulated in the nanodrug preparation of the present invention can reduce the deposition of collagen in the ECM. On the one hand, it further improves the therapeutic effect on pulmonary fibrosis. On the other hand, it can reduce the obstacles for the nanodrug preparation to enter the alveolar tissue after pulmonary administration, thereby improving the effective utilization rate of the nanodrug preparation.
  • FIG1 is a freeze-dried powder and reconstituted appearance of the best formulation ( LPFC-BIBF-IFN ) of the present invention.
  • FIG2 is a transmission electron micrograph of the best preparation ( LPFC-BIBF-IFN ) of the present invention.
  • FIG3 is a particle size distribution diagram of the best formulation ( LPFC-BIBF-IFN ) of the present invention.
  • FIG4 is a characterization of the physical and chemical properties of the nano-drug combination preparation ( LPFC-BIBF-IFN ) before and after atomization in the present invention
  • FIG5 is a graph showing the stability of the best formulation ( LPFC-BIBF-IFN ) of the present invention in biological solution;
  • FIG6 is a graph showing the nanoparticle size, polydispersity coefficient and potential size of other nanoformulations disclosed in the present invention.
  • FIG7 shows the oxygen content in the nanoformulations disclosed in the present invention and other solutions after oxygen is passed;
  • FIG8 is the fluorine signal detection results of the best preparation ( LPFC-BIBF-IFN ) and PFC stock solution in the present invention.
  • FIG9 is a diagram showing the encapsulation efficiency of drugs in the nanoparticle drug preparation of the present invention.
  • FIG10 is the weight changes of mice in each group during the 21-day treatment period
  • FIG11 shows the forced vital capacity parameters of mice in each group
  • FIG12 shows the dynamic compliance of the lungs of mice in each group
  • FIG13 is the Micor-CT imaging results of each group of mice on days 7, 14 and 21 after modeling
  • FIG14 shows the total number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) of mice in each group;
  • FIG15 is the content of inflammatory factors IL-6 and IL-1 ⁇ in bronchoalveolar lavage fluid (BALF) of mice in each group;
  • FIG16 is H&E and Masson staining of lung tissues of mice in each group.
  • FIG17 shows the TGF- ⁇ 1 content in the lung tissues of mice in each group
  • FIG18 is the hydroxyproline content in lung tissues of mice in each group.
  • Figure 19 shows the expression of HIF-1 ⁇ mRNA and VEGF mRNA in BALF of mice in each group
  • FIG. 20 shows the effect of the best preparation ( LPFC-BIBF-IFN ) of the present invention on the activity of human lung fibroblasts stimulated by TGF- ⁇ 1.
  • Example 1 Synthesis and preparation of a pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 15% trehalose solution to the drug preparation obtained in step (4), pre-freezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL solution of the bioactive small molecule protein IFN- ⁇ to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze filter 15 times through a 0.2 ⁇ m filter membrane, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove the free bioactive small molecule protein to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention ( LPFC-BIBF-IFN ) (oxygen treatment is performed before use to obtain L PFC(O2)-BIBF-IFN ).
  • LPFC-BIBF-IFN oxygen treatment is performed before use to obtain L PFC(O2)-BIBF-IFN .
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 15% trehalose solution to the drug preparation obtained in step (4), pre-freezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL bioactive small molecule protein IFN- ⁇ solution to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze and filter through a 0.2 ⁇ m filter membrane 15 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 15% trehalose solution to the drug preparation obtained in step (4), pre-freezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL bioactive small molecule protein IFN- ⁇ solution to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze and filter through a 0.2 ⁇ m filter membrane 15 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 10% trehalose solution to the drug preparation obtained in step (4), pre-freezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL bioactive small molecule protein IFN- ⁇ solution to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze and filter through a 0.2 ⁇ m filter membrane 15 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 5% trehalose solution to the drug preparation obtained in step (4), pre-freezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL bioactive small molecule protein IFN- ⁇ solution to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze and filter through a 0.2 ⁇ m filter membrane 15 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 10% sucrose solution to the drug preparation obtained in step (4), pre-freezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL bioactive small molecule protein IFN- ⁇ solution to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze and filter through a 0.2 ⁇ m filter membrane 15 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • Example 7 Synthesis and preparation of a pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 10% lactose solution to the pharmaceutical preparation obtained in step (4), prefreezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL bioactive small molecule protein IFN- ⁇ solution to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze and filter through a 0.2 ⁇ m filter membrane 15 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • Example 8 Synthesis and preparation of a pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention
  • step (2) The lipid-based solution obtained in step (1) is preliminarily rotary evaporated at 45° C. to form a lipid film; then the drug solution is added to the preliminarily prepared lipid film, and tank ultrasonication is performed at 45° C. for 5-10 minutes to fully embed the drug into the lipid base;
  • step (3) The co-solution obtained in step (2) is subjected to rotary evaporation again at 45° C. to form a film, and then allowed to stand under vacuum at 37° C. for 8-10 hours to completely remove the organic solvent in the system;
  • step (4) adding 10% lactose solution to the pharmaceutical preparation obtained in step (4), prefreezing at -80°C for 24 hours, sublimation drying at -50°C for 48 hours, and desorption drying at 25°C for 10 hours;
  • step (6) Add 2 mL of a 200 ⁇ g/mL bioactive small molecule protein IFN- ⁇ solution to the lyophilized powder obtained in step (5) for reconstitution, and then squeeze and filter through a 0.2 ⁇ m filter membrane 15 times, and ultrafiltration centrifuge (4°C, 14000 rpm, 20 min) to remove free bioactive small molecule proteins to obtain the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • Example 9 Investigating the effects of different types and ratios of the above-mentioned lyophilization protective agents on the results of lyophilized preparations
  • Appearance evaluation Observe the shape, color and looseness of the sample after freeze-drying.
  • a suitable prescription should ensure that the surface of the sample after freeze-drying is smooth and flat, dense and full, without shrinkage, and the powdery solid has no collapse.
  • Reconstitution ability (-) Cannot be completely dissolved even with vigorous shaking (+) Can be completely reconstituted with vigorous shaking, but the dissolution rate is slow (++) Can be completely reconstituted with vigorous shaking (+++) Can be completely dissolved with gentle shaking.
  • step (2) adding 10% sucrose solution (with a volume ratio of 1:1 to tert-butyl alcohol) to the solution obtained in step (1), pre-freezing at -20°C for 24 hours, sublimation drying at -40°C for 24 hours, and desorption drying at 25°C for 6 hours;
  • step (3) Add 2 mL of sterilized ultrapure water and 0.4 mL of perfluorocarbon to the lyophilized powder obtained in step (2) for reconstitution, and then perform probe ultrasound at a power of 450 W for 4 min.
  • the resulting drug preparation is squeezed through a 0.2 ⁇ m filter membrane 20 times, and then dialyzed and purified (4° C., 100 rpm) to remove all unencapsulated drugs; (the perfluorocarbon is perfluorooctane bromide)
  • step (3) incubating the drug preparation obtained in step (3) and 2 mL of a 400 ⁇ g/mL solution of a biologically active small molecule protein IFN- ⁇ at 4° C. with shaking for 3 h;
  • step (4) The co-incubation solution obtained in step (4) is subjected to ultrafiltration centrifugation (4°C, 14000 rpm, 20 min) to remove free IFN- ⁇ , thereby obtaining the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • Example 11 Synthesis and preparation of a pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention
  • step (2) adding 10% sucrose solution (with a volume ratio of 2:1 to tert-butyl alcohol) to the solution obtained in step (1), pre-freezing at -20°C for 24 hours, sublimation drying at -40°C for 24 hours, and desorption drying at 25°C for 6 hours;
  • step (3) Add 2 mL of sterilized ultrapure water and 0.4 mL of perfluorocarbon to the lyophilized powder obtained in step (2) for reconstitution, and then perform probe ultrasound at a power of 450 W for 4 min.
  • the resulting drug preparation is squeezed through a 0.2 ⁇ m filter membrane 20 times, and then dialyzed and purified (4° C., 100 rpm) to remove all unencapsulated drugs; (the perfluorocarbon is perfluorooctane bromide)
  • step (3) incubating the drug preparation obtained in step (3) and 2 mL of a 400 ⁇ g/mL solution of a biologically active small molecule protein IFN- ⁇ at 4° C. with shaking for 3 h;
  • step (4) The co-incubation solution obtained in step (4) is subjected to ultrafiltration centrifugation (4°C, 14000 rpm, 20 min) to remove free IFN- ⁇ , thereby obtaining the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • Example 12 Synthesis and preparation of a pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention
  • step (2) adding 10% sucrose solution (with a volume ratio of 1:1 to tert-butyl alcohol) to the solution obtained in step (1), pre-freezing at -20°C for 24 hours, sublimation drying at -40°C for 24 hours, and desorption drying at 25°C for 6 hours;
  • step (3) Add 2 mL of sterilized ultrapure water and 0.4 mL of perfluorocarbon to the lyophilized powder obtained in step (2) for reconstitution, and then perform probe ultrasound at a power of 450 W for 4 min.
  • the resulting drug preparation is squeezed through a 0.2 ⁇ m filter membrane 20 times, and then dialyzed and purified (4° C., 100 rpm) to remove all unencapsulated drugs; (the perfluorocarbon is perfluorooctane bromide)
  • step (3) incubating the drug preparation obtained in step (3) and 2 mL of a 400 ⁇ g/mL solution of a biologically active small molecule protein IFN- ⁇ at 4° C. with shaking for 5 h;
  • step (4) The co-incubation solution obtained in step (4) is subjected to ultrafiltration centrifugation (4°C, 14000 rpm, 20 min) to remove free IFN- ⁇ , thereby obtaining the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention (oxygenated before use).
  • step (2) 2 mL of sterilized ultrapure water and 0.4 mL of perfluorocarbon were added to the lipid film obtained in step (1), and probe ultrasound was performed at a power of 450 W for 4 min.
  • the obtained nanoemulsion was squeezed through a 0.2 ⁇ m filter membrane 20 times to obtain a nanoemulsion ( MPFC ) containing only PFC (the perfluorocarbon was perfluorooctane bromide).
  • step (2) Add 2 mL of sterile ultrapure water and 0.4 mL of perfluorocarbon to the lyophilized powder obtained in step (1) for reconstitution, and then perform probe ultrasound at a power of 450 W for 4 min.
  • the obtained drug preparation is squeezed through a 0.2 ⁇ m filter membrane 20 times, and then dialyzed and purified (4° C., 100 rpm) to remove all unencapsulated drugs, thereby obtaining a nanoemulsion ( LPFC-BIBF ) that simultaneously encapsulates PFC and BIBF 1120; (the perfluorocarbon is perfluorooctane bromide).
  • step (3) 2 mL of sterilized ultrapure water and 0.4 mL of perfluorocarbon were added to the film obtained in step (3), and probe ultrasound was performed at 450 W power for 4 min.
  • the obtained drug preparation was squeezed through a 0.2 ⁇ m filter membrane 20 times, and then a 10% sucrose solution was added thereto.
  • the drug preparation was pre-frozen at -80°C for 24 h, sublimated and dried at -50°C for 48 h, and desorbed and dried at 25°C for 10 h to obtain a freeze-dried powder; (the perfluorocarbon was perfluorooctane bromide)
  • step (3) Add 2 mL of 400 ⁇ g/mL IFN- ⁇ solution to the lyophilized powder obtained in step (2) for reconstitution, and remove free IFN- ⁇ by ultrafiltration centrifugation (4°C, 14000 rpm, 20 min) to obtain a nanoemulsion ( LPFC-IFN ) that simultaneously encapsulates PFC and the bioactive small molecule protein IFN- ⁇ .
  • LPFC-IFN nanoemulsion
  • Example 14 investigates the characterization of the pulmonary administration oxygen-carrying nano drug combination preparation of the present invention (L PFC-BIBF-IFN , taking Example 6 or Example 10 as an example) and other nano preparations ( MPFC , L PFC-BIBF , L PFC-IFN , Example 13)
  • the present invention discloses a pulmonary administration oxygen-carrying nano drug combination preparation ( LPFC-BIBF-IFN )
  • the yellow microemulsion nanoformulation described in 1 was diluted 10 times with deionized water, and the solution was dripped onto a copper mesh. After drying, 1 drop of 2% phosphotungstic acid staining solution was added to the copper mesh and dyed for 30 seconds. The staining solution was aspirated, and after drying again, transmission electron microscopy was used to photograph. The results are shown in Figure 2.
  • the nanoformulation was round and relatively uniform in size, with an average particle size of about 150 nm.
  • the yellow microemulsion nanoformulation described in 1 was diluted 1000 times and characterized by DLS. The results are shown in Figure 3.
  • the particle size is distributed around 150 nm, the polydispersity index PDI is less than 0.2, and the coincidence of the results of three repeated tests is high, indicating that the pulmonary administration oxygen-carrying nanodrug combination preparation ( LPFC-BIBF-IFN ) provided by the present invention not only has a small particle size but also has good dispersibility.
  • Example 13 of the present invention The particle sizes of other nanoformulations disclosed in Example 13 of the present invention are all within 200 nm.
  • the Zeta potentials of MPFC and L PFC-BIBF are both positive, and the Zeta potential of L PFC-IFN is negative, which may be related to the isoelectric point of IFN- ⁇ .
  • the isoelectric point of IFN- ⁇ is about 8-9.
  • F signal detection 19F-MR was used to detect the fluorine signal intensity in equal amounts of PFC stock solution and L PFC- BIBF-IFN (diluted 2 times with water). The results are shown in Figure 8. The left and right figures show the fluorine signal intensity in the PFC stock solution and the 2-fold diluted L PFC-BIBF-IFN , respectively. The signal-to-noise ratio of L PFC-BIBF-IFN accounted for 84.6% of the PFC stock solution.
  • the technical solution disclosed in the present invention is suitable for preparing oxygen-carrying nano drug preparations, and can encapsulate inert perfluorocarbons with a high dose; the nano drug preparation provided by the present invention also has good oxygen carrying capacity, and the dissolved oxygen content after 10 minutes of oxygen ventilation is about 7-8 times that of water and about 2 times that of blood. It lays a good technical foundation for the nano drug preparation of the present invention to improve the hypoxic state of the IPF lung environment and improve lung compliance.
  • BIBF 1120 encapsulation efficiency in nanoformulations Take 50 ⁇ L of each of the L PFC-BIBF and L PFC-BIBF-IFN nanoformulations of the present invention, add 950 ⁇ L of chromatographic grade methanol to break the emulsion, and detect the drug concentration using a high performance liquid chromatograph. Calculate the encapsulation efficiency (EE%) according to the following formula:
  • W pure is the mass of BIBF 1120 in L PFC-BIBF and L PFC-BIBF-IFN nanoformulations; W total is the total dosage of BIBF 1120 when preparing the nanoformulation.
  • C 0 represents the total interferon- ⁇ concentration before preparative ultrafiltration centrifugation
  • C filte represents the interferon- ⁇ concentration in the filtrate
  • the encapsulation efficiency of the anti-fibrosis drug BIBF 1120 in the nanodrug preparation disclosed in the present invention is greater than 70%, and the encapsulation efficiency of IFN- ⁇ is in the range of 50%-70%.
  • the encapsulation efficiency is relatively ideal, indicating that the preparation method disclosed in the present invention is suitable for preparing nanoformulations of the two drugs and similar drugs.
  • Example 15 Investigating the therapeutic effect and influence of the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention on the IPF animal model
  • Group a Control the healthy control group
  • Group b BLM the bleomycin group
  • Group c BIBF 1120 the administration method was intraperitoneal injection of the anti-fibrotic drug BIBF 1120 (35 mg/kg); Group d MPFC(O2) ; Group e L PFC(O2)-BIBF ; Group f L PFC(O2)-BIBF-IFN ; Group g L PFC-BIBF-IFN .
  • the administration method of groups bg was pulmonary administration (50-100uL/mouse, and the dosage of BIBF 1120 and IFN- ⁇ in each group of mice was required to be basically the same).
  • Bleomycin was used for induction. Bleomycin (3.5 mg/mouse) was instilled into the trachea of mice on day 0, and the weight of mice in each group was monitored every other day. Days 1-6 of bleomycin modeling is the transition period from pneumonia to pulmonary fibrosis, followed by the pulmonary fibrosis stage. Therefore, the present invention chooses to start drug administration every other day on the 8th day after modeling, which can more objectively reflect the therapeutic effect on fibrosis.
  • Intraperitoneal injection of BIBF1120 in mice in group c improved the weight of IPF model mice to a certain extent, but in comparison, groups e and f, that is, BIBF1120 combined with oxygen delivery therapy or IFN- ⁇ , had a better effect on maintaining weight, indicating that oxygen delivery or IFN- ⁇ can cooperate with BIBF1120 in anti-fibrosis to varying degrees.
  • Micro-CT imaging examination Micro CT scans of mice were performed on days 7, 14, and 21 after bleomycin modeling, and the scan results of mice in each group are shown in Figure 13. As can be seen from the figure, the ground-glass shadow area of the lungs of mice in the BLM group increased over time, and pulmonary fibrosis was severely diffuse on day 21. From d to f, the degree of pulmonary fibrosis progression of mice in each treatment group gradually decreased, and the degree of pulmonary fibrosis in the L PFC(O2)-BIBF-IFN group was significantly lower, indicating that the fibrosis of the reorganized mice was well improved by treatment.
  • BALF detection On the 21st day of bleomycin modeling, the bronchoalveolar lavage fluid (BALF) of each group of mice was taken to count the total number of cells, and the ELISA kit was used to detect the content of IL-6 and IL-1 ⁇ in BALF. From the total count of inflammatory cells in the alveolar lavage fluid of mice in Figure 14, it can be seen that the pulmonary administration oxygen-carrying nano-drug combined preparation L PFC (O2) -BIBF-IFN provided by the present invention can significantly reduce the total number of inflammatory cells in the BALF of IPF mice, and compared with other groups, the total number of inflammatory cells in the L PFC (O2) -BIBF-IFN group is the least.
  • TGF- ⁇ 1 is the most intensively studied and important cytokine in fibrosis, and plays an important role in promoting the occurrence and development of pulmonary fibrosis.
  • the results of the kit detecting the content of TGF- ⁇ 1 in the lung tissue homogenate of each group of mice are shown in Figure 17. The trend is similar to the results of the inflammatory factor detection, which confirms that the pulmonary administration oxygen-carrying nano-drug combination preparation LPFC (O2)-BIBF-IFN of the present invention can most effectively reduce the content of TGF- ⁇ 1 in the lung tissue of IPF mice.
  • the hydroxyproline content detection of each group of mice is shown in Figure 18. The results of the hydroxyproline content detection show that the pulmonary administration oxygen-carrying nano-drug combination preparation of the present invention has the best anti-fibrosis effect.
  • the pulmonary administration oxygen-carrying nano-drug combination preparation L PFC-BIBF-IFN of the present invention has a good effect in inhibiting the abnormal activation of lung fibroblasts, and the undiluted L PFC-BIBF-IFN has a significant effect in inhibiting the proliferation of lung fibroblasts.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pulmonology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Otolaryngology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

本发明提供一种纳米药物联合制剂及其制备方法和应用，属于生物医药技术领域，要解决现有纳米制剂很难同时包载全氟化碳，油溶性小分子药物以及水溶性生物制剂的技术问题。本发明所述纳米药物联合制剂以脂质基为载体，包封全氟化碳(PFC)和活性成分，活性成分为生物活性小分子蛋白质和抗纤维化药物。本发明一方面进一步提高了对肺纤维化的治疗效果，另一方面能减少肺部给药后的纳米药物制剂进入肺泡组织的阻碍，使纳米药物制剂的有效利用率得以提升。

Description

一种纳米药物联合制剂及其制备方法和应用 技术领域
本发明属于生物医药技术领域，具体涉及一种纳米药物联合制剂及其制备方法，和该纳米药物制剂在纤维化疾病中的应用。
背景技术
特发性肺纤维化(Idiopathic pulmonary fibrosis，IPF)是一种病因未明的、特殊类型的慢性进行性肺疾病，病灶局限于肺部。IPF的主要病理特征是炎症细胞浸润，大量细胞因子促进成纤维细胞的迁移、增殖以及分化为活跃的肌成纤维细胞，共同促进胶原蛋白的增多，造成细胞外基质(ECM)过度增生。人正常肺组织具有较好的延展性，可维持肺活量在正常水平。而发生纤维化的肺组织，细胞外基质过度沉积，刚性变大，肺泡的气体交换功能大大受损，肺内气体交换受阻，肺通气功能严重障碍。此外，肺间质纤维化会导致肺内气血屏障(含肺表面活性物质的液体层、基质层、毛细血管内皮细胞层等)增厚，吸入到肺里面的空气进入到血液的弥散距离增加、弥散面积减小，通气血流比失调影响氧合，进一步导致缺氧，最终致使活动后甚至静息状态出现呼吸困难，特发性肺纤维化被称为“不是癌症的癌症”，患者的中位生存期只有2-3年，死亡率高于大多数肿瘤。IPF患者肺顺应性降低，气体交换被破坏，肺功能进行性丧失，最终将导致患者呼吸衰竭和死亡。
临床上的非药物治疗手段如肺移植对各种条件要求较高，而机械通气、吸氧、支气管肺泡灌洗术等治疗效果都不尽人意。常用的药物治疗手段如吡非尼酮和尼达尼布，尽管两种药物都显示出降低死亡率的趋势，对用力肺活量在超过一年的降低率上具有相似的作用，但这两种药物未显示能显著增加生存时间。治疗周期长，连续治疗52周后才能有效改善患者的肺功能，延长患者的无进展生存期，同时降低患者的死亡风险。并且现有的研究发现，在长时间给药后，患者会对药物产生耐受性，降低药物的治疗效果。相较于上述两种药物，其它如激素类药物(甲泼尼龙、地塞米松等)、免疫抑制剂(硫唑嘌呤、环磷酰胺等)、抗氧化剂(乙酰半胱氨酸、虾青素等)等疗效更是十分有限。因此，开发新的疗效好的药物仍是肺纤维化领域亟需解决的重要问题。
肺纤维化药物的开发面临着诸多挑战。因为IPF通常是由多种途径激发的，发病机制极其复杂，病理表现具有时间和空间异质性。关于该疾病信号通路和分子机制的研究虽然很多，但其发病机制尚未被明确阐明。目前用于治疗或处于临床试验阶段的分子药物无法涵盖IPF的众多治疗靶点，而仅针对一个靶点或信号通路的治疗往往效果有限，因此当前IPF研究领域广泛提出的一种观点是“多管齐下”，使用更综合的抗纤维化策略，来应对复杂的纤维化疾病所带来的挑战。
鉴于开发出针对特发性肺纤维化(IPF)的疗效确认、相对安全、价格合理的药物制剂具有重大的社会意义和医学意义，本发明旨在提供一种肺部给药的携氧纳米药物联合制剂，一方面利用其包载药物从疾病源头上抑制炎症细胞浸润和炎症因子的生成，避免下游信号刺激成纤维细胞增殖和活化为肌成纤维细胞；另一方面通过其中全氟化碳的携氧功能改善肺部微环境，提高肺顺应性，并减少缺氧诱导因子HIF 1α刺激血管内皮生长因子VEGFmRNA的表达，减少血管内皮细胞的生成；与此同时，降低细胞外基质(EMC)的胶原沉积，多方面协同促进本发明纳米药物制剂携氧穿透气血屏障，缓解肺部缺氧，提升肺功能。采用多途径联合治疗策略，针对特发性肺纤维化进行治疗，动物和细胞实验显示出良好的改善肺纤维化进展、提高动物生存状态的效果。利用上述策略进行IPF的治疗至今未见有公开报道。
全氟碳化合物(perfluorocarbons，PFCs)，是一种烃分子中的所有氢原子被氟原子取代而成的化合物，具有化学稳定性强、氧溶解度极高等特性，在相同的氧分压下，全氟化碳(PFC)的载氧能力大约是水和血浆的20-25倍，这种高载氧能力和其良好的生物安全性使其在临床研究中被广泛开发用于血液替代品、肿瘤氧合等用途。除此之外，全氟化碳还在新生儿呼吸窘迫的治疗上获得了FDA的批准。将全氟化碳开发成纳米乳用于携氧肺部递送是一个值得研究的方向，但是由于全氟碳类化合物具有较大的化学惰性，既不溶于水也不溶于油，纳米制剂很难包载高比例的全氟化碳，因此其携氧用途也受到影响而大打折扣。
发明内容
有鉴于此，为克服现有技术中存在的上述问题，和特发性肺纤维化(IPF)治疗中存在的缺陷，本发明提供了一种携氧性能好、可有效提高IPF疗效的肺部给药携氧纳米药物联合制剂及其制备方法。
为了解决上述技术问题，本发明采取了以下的技术方案：
一种纳米药物联合制剂，所述纳米药物联合制剂以脂质基为载体，能够包封较高比例的全氟化碳(PFC)，以生物活性小分子蛋白质和抗纤维化药物为活性成分。
进一步地限定，所述纳米联合制剂为用于纤维化疾病的纳米药物联合制剂。
再进一步地限定，所述纤维化疾病包括肺纤维化疾病和肝纤维化疾病中的任一种。
进一步地限定，所述脂质基，主要包括蛋黄卵磷脂、大豆磷脂、氢化大豆卵磷脂、二棕榈酰磷脂酰胆碱、二肉豆蔻磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二油酰基卵磷脂、二棕榈酰磷脂酰甘油、二棕榈酰磷脂酰乙醇胺、二硬脂酰磷脂酰乙醇胺、二硬脂酰磷脂酰甘油、胆固醇、吐温、硬脂酰胺、(2,3-二油酰基-丙基)-三甲基氯化铵、甘油中的一种或其中几种的任意比组合。
进一步地限定，所述全氟化碳(PFC)，包括1-溴全氟乙烷、全氟己基碘烷、全氟辛基碘烷、全氟己烷、全氟辛酸、全氟溴辛烷、全氟辛烷、1-溴-3-氟丙烷、1-溴-1,1,2,2-四氟丁烷、1-溴-2,2-二氟乙烷、1H-6-溴全氟己烷、1-溴-四氟丁烷、1-溴十七氟辛烷、全氟-15-冠-5醚、全氟己烷磺酸钾、全氟己基碘烷、1-溴全氟乙烷、1,6-二碘代十二氟己全氟己基磺酸、1,1,2,2,3,3-六氟丙烷、1,1,1,3-四氟丙烷、1,1,1,3,3-五氟丙烷、1-氟己烷、1,1,2,2,3,3,4,4-八氟丁烷、1,1,1,2,2-五氟丙烷中的一种或其中几种的任意比组合。
进一步地限定，所述全氟化碳(PFC)和脂质基的质量体积比为10％-20％。
进一步地限定，蛋白质占载体质量的0.3％-20％。
进一步地限定，抗纤维化药物占载体质量的2％-30％。
进一步地限定，所述生物活性小分子蛋白质为白介素-13(IL-13)、白介素-10(IL-10)、干扰素-γ(IFN-γ)、角化细胞生长因子(KGF)中的一种或几种。所述抗纤维化药物为BIBF1120、吡非尼酮、曲美替尼、尼罗替尼、白藜芦醇、姜黄素、瑞德西韦中的一种或其中几种的任意比组合。
更优选的，所述生物活性小分子蛋白质为干扰素-γ(IFN-γ)，所述抗纤维化药物为BIBF1120。此生物活性小分子蛋白质易溶于水溶液，而该抗纤维化药物不溶于水，需要先通过有机溶剂溶解的方法将该抗纤维化药物先一步融合在上述脂质基中。
进一步地限定，所述水溶液，包括灭菌的超纯水、灭菌注射用水、灭菌氯化钠溶液、滴滴水、去离子水中的一种或其中几种的任意比组合。
进一步地限定，所述有机溶剂，包括氯仿、二甲基亚砜、无水乙醇、甲醇、异丙烷、叔丁醇中的一种或其中几种的任意比组合。
本发明提供了一种肺部给药携氧纳米药物联合制剂的制备方法，包括以下步骤：
(1)将10-30mg脂质基与10-30mL的氯仿-甲醇共溶剂(体积比＝2-5:0.5-1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将抗纤维化药物与二甲基亚砜混合(质量体积比＝1:2-4mg/mL)，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在30-50℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，30-50℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在30-50℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-16h，完全去除体系中的有机溶剂；
(4)将1-10mL灭菌的超纯水、0.1-2mL全氟化碳加入步骤(3)得到的薄膜中，100-500W功率进行探头超声2-6min，所得药物制剂经0.22μm滤膜挤压5-10次后，透析纯化(4℃，100rpm)除尽未包封的药物；
(5)将步骤(4)所得的药物制剂中加入5％-15％的冻干保护剂，-20～-80℃预冻24-48h，-40～-50℃升华干燥24-48h，25℃解析干燥6-10h；
(6)在步骤(5)得到的冻干粉中加入1-10mL的生物活性小分子蛋白质溶液(100-200μg/mL)进行重构，后经0.2μm滤膜挤压过滤10-20次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
本发明提供了一种肺部给药携氧纳米药物联合制剂的制备方法，包括以下步骤：
(1)将10-30mg脂质基、0.68-3mg抗纤维化药物与5-30mL的叔丁醇混合，涡旋震荡均匀；
(2)在步骤(1)得到的溶液中加入5％-15％的冻干保护剂，-20～-80℃预冻24-48h，-40～-50℃升华干燥24-48h，25℃解析干燥6-10h；
(3)在步骤(2)得到的冻干粉中加入0.5-5mL灭菌的超纯水、0.1-2mL全氟化碳进行重构，然后用100-500W功率进行探头超声2-6min，所得药物制剂经0.2μm滤膜挤压15-20次后，透析纯化(4℃，100rpm)除尽未包封的药物；
(4)将步骤(3)所得的药物制剂和0.5-5mL生物活性小分子蛋白质溶液(200-400μg/mL)在4℃振摇条件下共孵育3-5h；
(5)将步骤(4)得到的共孵育溶液进行超滤离心(4℃，14000rpm，20min)，去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
进一步地限定，所述冻干保护剂，包括蔗糖、海藻糖、乳糖、甘露醇、甘氨酸、氯化钠、山梨醇、葡萄糖、麦芽糖中的一种或其中几种的任意比组合。
优选地，所述肺部给药携氧纳米药物联合制剂中，包载抗纤维化药物的包封率大于70％，包载生物活性小分子蛋白质包封率大于60％，纳米药物制剂的粒径大小为130-200nm，多分散系数PDI小于0.2。
根据本发明的另一方面，本发明上述肺部给药携氧纳米药物联合制剂在特发性肺纤维化(IPF)治疗中的应用。
本发明公开了一种肺部给药的携氧纳米药物联合制剂及其制备方法，一种化学惰性全氟碳化合物和一种脂溶性抗纤维化药物及一种水溶性生物活性小分子蛋白质共负载于同一纳米平台。采取了多途径联合治疗的策略，具有如下有益效果：
(1)本发明纳米药物制剂能够包载高比例的全氟化碳，携载大量的氧气进入肺部，进而改善IPF肺部微环境的缺氧状态，提高肺顺应性，缓解肺功能的丧失。
(2)本发明的纳米药物制剂具有良好的稳定性，可通过雾化吸入方式进行肺部给药，该方式简单、无创。纳米制剂经吸入后在肺内聚集，可快速达到起效浓度。
(3)本发明纳米药物制剂包载的生物活性小分子蛋白质从IPF发生发展的源头上发挥免疫调节抗炎作用，并和纳米制剂中包载的抗纤维化药物共同降低肺纤维化关键刺激因子TGF-β的表达，抑制成纤维细胞向肌成纤维细胞的增生、活化，达到协同抑制肺纤维化的目的。
(4)缺氧状态下，缺氧诱导因子HIF 1α会刺激血管内皮生长因子VEGF mRNA的表达，而血管内皮增生会导致气血屏障进一步增厚，气体交换阻力增大，本发明的携氧纳米药物联合制剂可通过氧气递送改善这一情况。
(5)胶原蛋白是细胞外基质(ECM)的主要成分，本发明纳米药物制剂包载的两种药物均能减少ECM中胶原蛋白的沉积，一方面进一步提高了对肺纤维化的治疗效果，另一方面能减少肺部给药后的纳米药物制剂进入肺泡组织的阻碍，使纳米药物制剂的有效利用率得以提升。
附图说明
图1是本发明中最佳制剂(LPFC-BIBF-IFN)的冻干粉末和重构后的外观图；
图2是本发明中最佳制剂(LPFC-BIBF-IFN)的透射电镜图；
图3是本发明中最佳制剂(LPFC-BIBF-IFN)的粒径分布图；
图4是本发明中纳米药物联合制剂(LPFC-BIBF-IFN)雾化前后理化性质表征；
图5是本发明中最佳制剂(LPFC-BIBF-IFN)在生物溶液中的稳定性；
图6是本发明公开的其它纳米制剂的纳米粒径、多分散系数和电位大小；
图7是通氧气后本发明公开的纳米制剂和其它溶液中的氧含量；
图8是本发明中最佳制剂(LPFC-BIBF-IFN)和PFC原液的氟信号检测结果；
图9是本发明中纳米药物制剂中药物的包封率；
图10是各组小鼠在21天治疗周期内的体重变化；
图11为各组小鼠的用力肺活量参数；
图12为各组小鼠的肺动态顺应性；
图13是各组小鼠建模后第7、14和21天的Micor-CT影像检查结果；
图14是各组小鼠支气管肺泡灌洗液(BALF)中的炎症细胞总数；
图15是各组小鼠支气管肺泡灌洗液(BALF)中的炎症因子IL-6和IL-1β的含量；
图16是各组小鼠的肺组织免疫组化H&E和Masson染色；
图17是各组小鼠的肺组织中TGF-β1含量；
图18是各组小鼠肺组织中羟脯氨酸含量；
图19是各组小鼠BALF中HIF-1αmRNA和VEGF mRNA的表达；
图20是本发明中最佳制剂(LPFC-BIBF-IFN)TGF-β1刺激的人肺成纤维细胞活性的影响。
具体实施方式
下面结合附图和具体实施例对本公开内容作更进一步的说明。这些实施例仅用于举例说明目的，本公开内容的范围并不受实施例的限制。
实施例1本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将23mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.4mL全氟化碳入步骤(3)得到的薄膜中，200W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(5)将步骤(4)所得的药物制剂中加入15％的海藻糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(LPFC-BIBF-IFN)(在使用前进行通氧处理得到LPFC(O2)-BIBF-IFN)。
实施例2本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(3)得到的薄膜中，200W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(5)将步骤(4)所得的药物制剂中加入15％的海藻糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例3本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(3)得到的薄膜中，450W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(5)将步骤(4)所得的药物制剂中加入15％的海藻糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例4本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(3)得到的薄膜中，450W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(5)将步骤(4)所得的药物制剂中加入10％的海藻糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例5本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(3)得到的薄膜中，450W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(5)将步骤(4)所得的药物制剂中加入5％的海藻糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例6本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(3)得到的薄膜中，450W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(5)将步骤(4)所得的药物制剂中加入10％的蔗糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例7本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(3)得到的薄膜中，450W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟冠醚)
(5)将步骤(4)所得的药物制剂中加入10％的乳糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例8本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀，得脂质基溶液，4℃保存备用；将3mg抗纤维化药物BIBF 1120与6mL二甲基亚砜混合，涡旋震荡均匀，得药物溶液；
(2)将步骤(1)得到的脂质基溶液在45℃条件下，初步旋转蒸发成脂膜；然后药物溶液加入此初步制备的脂膜中，45℃条件下槽式超声5-10min，将药物充分嵌合到脂质基上；
(3)将步骤(2)得到的共溶液在45℃条件下，再次旋转蒸发成膜，然后在真空、37℃条件下静置8-10h，完全去除体系中的有机溶剂；
(4)将2mL灭菌的超纯水、0.01mL全氟化碳加入步骤(3)得到的薄膜中，450W功率进行探头超声4min，所得药物制剂经0.22μm滤膜挤压10次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟冠醚)
(5)将步骤(4)所得的药物制剂中加入10％的乳糖溶液，-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h；
(6)在步骤(5)得到的冻干粉中加入2mL浓度为200μg/mL生物活性小分子蛋白质IFN-γ溶液进行重构，后经0.2μm滤膜挤压过滤15次，超滤离心(4℃，14000rpm，20min)去除游离的生物活性小分子蛋白质，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例9考察上述冻干保护剂不同种类和比例对冻干制剂的结果影响
实验方法：
(1)外观评价：观察冻干后的样品形状、颜色和疏松状态。合适的处方应该冻干后样品表面平整光洁，致密饱满，无皱缩，粉状固体无塌陷。
(2)重构能力评价：观察振摇力度、重构难易程度、复溶时间和速度。冻干后的纳米药物制剂加入原体积的溶液，手动振摇使其分散。合适的处方应分散时间短，复溶速度快，轻微手摇后即可得到外观均一澄清的纳米药物制剂。
(3)粒径和多分散系数表征：冻干后的纳米药物制剂加入原体积的水化介质，手动振摇使其分散，用粒度仪测定其粒径多分散系数PDI。适宜处方的粒径和PDI的测量在200nm以内较为理想。
冻干保护剂不同种类和比例对冻干制剂的影响结果见表1；
表1冻干保护剂不同种类和比例对冻干制剂的影响结果

外观：(-)冻干粉末塌陷、脱离瓶壁(+)表面平整、略有皱缩(++)表面平整、饱满(+++)表面平滑、饱满、粉饼状；
重构能力：(-)用力振摇也无法完全溶解(+)用力振摇可完全复溶，但溶解速度较慢(++)用力振摇可完全复溶(+++)轻轻摇晃即可完全溶解。
从上表结果可知，15％的海藻糖和10％的蔗糖作为冻干保护剂效果都相对较好。考虑到成本和准别时间，之后的实施例中采用10％蔗糖溶液作为冻干保护剂进行实验。
实施例10本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将8mg蛋黄卵磷脂、15mg二棕榈酰磷脂酰胆碱、5mg胆固醇、3mg抗纤维化药物BIBF 1120与8mL的叔丁醇混合，涡旋震荡均匀；
(2)在步骤(1)得到的溶液中加入10％的蔗糖溶液(和叔丁醇的体积比为1:1)，-20℃预冻24h，-40℃升华干燥24h，25℃解析干燥6h；
(3)在步骤(2)得到的冻干粉中加入2mL灭菌的超纯水、0.4mL全氟化碳进行重构，然后用450W功率进行探头超声4min，所得药物制剂经0.2μm滤膜挤压20次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(4)将步骤(3)所得的药物制剂和2mL浓度为400μg/mL的生物活性小分子蛋白质IFN-γ溶液在4℃振摇条件下共孵育3h；
(5)将步骤(4)得到的共孵育溶液进行超滤离心(4℃，14000rpm，20min)，去除游离的IFN-γ，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例11本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将8mg蛋黄卵磷脂、15mg二棕榈酰磷脂酰胆碱、5mg胆固醇、3mg抗纤维化药物BIBF 1120与8mL的叔丁醇混合，涡旋震荡均匀；
(2)在步骤(1)得到的溶液中加入10％的蔗糖溶液(和叔丁醇的体积比为2:1)，-20℃预冻24h，-40℃升华干燥24h，25℃解析干燥6h；
(3)在步骤(2)得到的冻干粉中加入2mL灭菌的超纯水、0.4mL全氟化碳进行重构，然后用450W功率进行探头超声4min，所得药物制剂经0.2μm滤膜挤压20次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(4)将步骤(3)所得的药物制剂和2mL浓度为400μg/mL的生物活性小分子蛋白质IFN-γ溶液在4℃振摇条件下共孵育3h；
(5)将步骤(4)得到的共孵育溶液进行超滤离心(4℃，14000rpm，20min)，去除游离的IFN-γ，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例12本发明的一种肺部给药的携氧纳米药物联合制剂的合成及制备
(1)将8mg蛋黄卵磷脂、15mg二棕榈酰磷脂酰胆碱、5mg胆固醇、3mg抗纤维化药物BIBF 1120与8mL的叔丁醇混合，涡旋震荡均匀；
(2)在步骤(1)得到的溶液中加入10％的蔗糖溶液(和叔丁醇的体积比为1:1)，-20℃预冻24h，-40℃升华干燥24h，25℃解析干燥6h；
(3)在步骤(2)得到的冻干粉中加入2mL灭菌的超纯水、0.4mL全氟化碳进行重构，然后用450W功率进行探头超声4min，所得药物制剂经0.2μm滤膜挤压20次后，透析纯化(4℃，100rpm)除尽未包封的药物；(全氟化碳为全氟溴辛烷)
(4)将步骤(3)所得的药物制剂和2mL浓度为400μg/mL的生物活性小分子蛋白质IFN-γ溶液在4℃振摇条件下共孵育5h；
(5)将步骤(4)得到的共孵育溶液进行超滤离心(4℃，14000rpm，20min)，去除游离的IFN-γ，得到本发明的肺部给药携氧纳米药物联合制剂(在使用前进行通氧处理)。
实施例13本发明公开的实施例实验中使用的纳米制剂的制备和表征
1.制备单独包载PFC的纳米乳(MPFC)
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀后，45℃真空负压条件下旋转蒸发成脂膜，放入真空干燥箱过夜以进一步去除有机溶剂；
(2)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(1)得到的脂膜中，450W功率进行探头超声4min，所得纳米乳经0.2μm滤膜挤压20次，即得到单独包载PFC的纳米乳(MPFC)；(全氟化碳为全氟溴辛烷)。
2.制备同时包载PFC和抗纤维化药物BIBF 1120的纳米制剂(LPFC-BIBF)
(1)将8mg蛋黄卵磷脂、15mg二棕榈酰磷脂酰胆碱、5mg胆固醇、3mg抗纤维化药物BIBF 1120与8mL的叔丁醇混合，涡旋震荡均匀后加入10％的蔗糖溶液(和叔丁醇的体积比为1:1)，-20℃预冻24h，-40℃升华干燥24h，25℃解析干燥6h；
(2)在步骤(1)得到的冻干粉中加入2mL灭菌的超纯水、0.4mL全氟化碳进行重构，然后用450W功率进行探头超声4min，所得药物制剂经0.2μm滤膜挤压20次后，透析纯化(4℃，100rpm)除尽未包封的药物，即得到同时包载PFC和BIBF 1120的纳米乳(LPFC-BIBF)；(全氟化碳为全氟溴辛烷)。
3.制备同时包载PFC和生物活性小分子蛋白质IFN-γ的纳米乳(LPFC-IFN)
(1)将10mg蛋黄卵磷脂、13mg二棕榈酰磷脂酰胆碱、5mg胆固醇与12mL的氯仿-甲醇共溶剂(体积比＝2:1)混合，涡旋震荡均匀后，45℃真空负压条件下旋转蒸发成脂膜，放入真空干燥箱过夜以进一步去除有机溶剂；
(2)将2mL灭菌的超纯水、0.4mL全氟化碳加入步骤(3)得到的薄膜中，450W功率进行探头超声4min，所得药物制剂经0.2μm滤膜挤压20次后，向其中加入10％的蔗糖溶液，经过-80℃预冻24h，-50℃升华干燥48h，25℃解析干燥10h后得到冻干粉末；(全氟化碳为全氟溴辛烷)
(3)在步骤(2)得到的冻干粉中加入2mL浓度为400μg/mL IFN-γ溶液进行重构，超滤离心(4℃，14000rpm，20min)去除游离的IFN-γ，即得到同时包载PFC和生物活性小分子蛋白质IFN-γ的纳米乳(LPFC-IFN)。
实施例14考察本发明的肺部给药携氧纳米药物联合制剂(LPFC-BIBF-IFN，以实施例6或实施例10为例)和其它纳米制剂(MPFC、LPFC-BIBF、LPFC-IFN，实施例13)的表征情况
1本发明公开的肺部给药携氧纳米药物联合制剂(LPFC-BIBF-IFN)
(1)外观观察
冻干后呈淡黄色、饱满、平滑均匀的粉饼装，轻轻敲碎后呈现质地均匀的粉末状，如图1A所示。加溶液重构后呈黄色微乳状，无沉淀无分层，如图1B所示。
(2)透射电镜(TEM)表现
将1中所述的黄色微乳状纳米制剂用去离子水稀释10倍后，吸取溶液滴加到铜网上，烘干后滴加1滴2％磷钨酸染色液至铜网上染色30s，吸去染色液，再次烘干后进行透射电镜拍摄，结果如图2所示，纳米制剂呈圆形，大小较为均一，平均粒径在150nm左右。
(3)DLS表征
将1中所述的黄色微乳状纳米制剂进行1000倍稀释后，进行DLS表征，结果如图3所示，粒径在150nm左右分布，多分散系数PDI小于0.2，三次重复检测结果重合度较高，说明本发明提供的肺部给药携氧纳米药物联合制剂(LPFC-BIBF-IFN)不仅粒径小，分散性也较好。
(4)雾化前后理化性质表征
将500μL纳米制剂置入振动筛孔雾化器，另一端连接1.5mL的EP管。启动振动筛空雾化器的开关开始雾化，5min后收集雾化后的纳米制剂。结果如图4所示，LPFC-BIBF-IFN雾化后粒径略微增大，PDI呈不同程度增加，但大小都在0.3左右。包封率测量结果显示，雾化前后LPFC-BIBF-IFN中的两种药物含量均无明显差别。说明本发明提供的肺部给药纳米药物联合制剂(LPFC-BIBF-IFN)雾化前后理化性质基本保持一致，能够在体内动物实验中较准确的反映本发明纳米药物制剂的在体疗效。
(5)在不同溶液中的稳定性表征
取适量LPFC-BIBF-IFN，分别加入10-20倍体积的灭菌注射用水、0.9％的NaCl溶液、10％胎牛血清、人造模拟肺液中，涡旋混合均匀。在混合后的第1h、2h、4h、8h、12h分别进行混合溶液的DLS表征，图5为表征结果，可见本发明提供的肺部给药纳米药物联合制剂(LPFC-BIBF-IFN)在各溶液中均保持一定的良好稳定性，具有良好的生物相容性和生物应用能力。
2本发明实施例13中公开的其它纳米制剂的DLS表征
方法同上，结果如图6，本发明实施例13中公开的其它纳米制剂粒径均在200nm以内，MPFC和LPFC-BIBF的Zeta电位均为正值，LPFC-IFN的Zeta电位为负，这可能和IFN-γ的等电点有关，IFN-γ的等电点约为8-9，当外界环境pH值大于物质的等电点时，物质电荷表现为负。
3本发明的肺部给药携氧纳米药物联合制剂(LPFC-BIBF-IFN)和其它纳米制剂(MPFC、LPFC-BIBF、LPFC-IFN)对PFC的包载能力和对药物包封率的检测
(1)PFC包载能力检测
1)溶氧量比较：分别取适量等体积的PFC原液、水、人血、LPFC-BIBF-IFN、MPFC、LPFC-BIBF和LPFC-IFN，通氧气10min后，用便携式溶氧仪测定以上纳米制剂的含氧量(单位mg/L)。
结果如图7所示，通氧气10min后的LPFC-BIBF-IFN含氧量在30-33mg/L范围内，MPFC含氧量为34.2mg/L，LPFC-BIBF含氧量为32.8mg/L，LPFC-IFN含氧量为29.8mg/L，PFC原液含氧量为48.6mg/L，而水和人血的含氧量分别为7.8mg/L和23.2mg/L。
2)F信号检测：用19F-MR检测等量的PFC原液和LPFC-BIBF-IFN(加水稀释2倍)中氟信号强度，结果如图8所示，左右图分别为PFC原液和稀释2倍的LPFC-BIBF-IFN中氟信号强度表现，LPFC-BIBF-IFN的信噪比占PFC原液的84.6％。
以上实验结果说明本发明公开的技术方案适用于制备携氧纳米药物制剂，能包载剂量较高的惰性全氟化碳；本发明提供的纳米药物制剂还拥有良好的携氧能力，通氧气10min后的溶氧量大约是水的7-8倍，血液的2倍左右。为本发明的纳米药物制剂用于改善IPF肺部环境的乏氧状态、提高肺顺应性奠定了良好的技术基础。
(2)药物包封率的检测
1)纳米制剂中BIBF 1120包封率检测：分别取本发明的LPFC-BIBF和LPFC-BIBF-IFN纳米制剂各50μL，加950μL色谱级甲醇破乳，用高效液相色谱仪检测药物浓度。按下式计算包封率(EE％)：
EE％＝(Wpure/Wtotal)×100％
式中Wpure是LPFC-BIBF和LPFC-BIBF-IFN纳米制剂中BIBF 1120的质量；Wtotal是制备纳米制剂时BIBF 1120总的投药量。
2)纳米制剂中IFN-γ包封率检测：取LPFC-IFN和LPFC-BIBF-IFN纳米制剂超滤离心后的滤液，用ELISA试剂盒检测滤液中干扰素的含量，按照下式计算包封率(EE％)：
EE％＝(C0-Cfilter)/C0×100％
式中C0代表制备超滤离心前总的干扰素-γ浓度，Cfilte代表滤液中干扰素-γ的浓度。
结果如图9所示，本发明公开的纳米药物制剂中抗纤维化药物BIBF 1120的包封率大于70％，IFN-γ的包封率在50％-70％范围内，包封效率均较为理想，说明本发明公开的制备方法适于制备两种药物及其相似药物的纳米制剂。
实施例15考察本发明的肺部给药携氧纳米药物联合制剂对IPF动物模型的治疗效果和影响
1动物分组：a组Control，为健康对照组；b组BLM，为博来霉素组；c组BIBF 1120，给药方式为腹腔注射抗纤维化药物BIBF 1120(35mg/kg)；d组MPFC(O2)；e组LPFC(O2)-BIBF；f组LPFC(O2)-BIBF-IFN；g组LPFC-BIBF-IFN，b-g组的给药方式均为肺部给药(50-100uL/只，要求每组鼠的BIBF 1120和IFN-γ的给药量基本一致)。
2动物模型建立和其它实验：采用博来霉素诱导，第0天给小鼠气管内滴注博来霉素(3.5mg/只)，随后隔天监测各组小鼠体重。博来霉素建模1-6天是肺炎向肺纤维化过渡期，之后是肺纤维化期，因此本发明选择在建模后第8天开始隔天给药治疗，能比较客观地反映对纤维化的治疗效果。
1)体重监测：从第0天开始，隔天监测小鼠体重，对比各个组的小鼠体重情况。结果如图10所示，比较各组小鼠体重变化趋势，可以看出LPFC(O2)-BIBF-IFN组小鼠质量较其它治疗组，21天下降趋势明显缓慢，说明该组对维持小鼠生存质量有最大的优势。MPFC组小鼠体重下降趋势和BLM组相似，说明单独的PFC携氧对IPF并无良好的治疗效果。c组小鼠腹腔注射BIBF1120，对IPF模型小鼠的体重有一定的改善，但是相比之下e和f组，即BIBF1120联合携氧治疗或IFN-γ对体重的维持效果更好，说明携氧或IFN-γ能在不同程度上协同BIBF1120抗纤维化。
2)肺功能检测：在博来霉素建模第14天时，使用小动物肺功能检测系统检测各组小鼠的用力肺活量(FVC)和肺动态顺应性。结果如图11和12所示。从图可以看出联合IFN-γ后，LPFC(O2)-BIBF-IFN组肺功能较LPFC(O2)-BIBF组得到了具有差异性的提高。另外，分析各组与BLM组比较结果可知，本发明的肺部给药携氧纳米药物联合制剂携氧和不携氧均能改善小鼠肺功能，但是LPFC(O2)-BIBF-IFN组的FVC和肺动态顺应性可达到与Control对照组相当的水平，从而证明了本发明提供的肺部给药携氧纳米药物制剂可以更有效地改善IPF小鼠的肺功能。
3)Micro-CT影像学检查：在博来霉素建模后第7、14和21天行小鼠Micro CT扫描，各组小鼠的扫描结果如图13所示。由图可知，BLM组小鼠随时间进展肺部磨玻璃影面积增大，第21天肺纤维化严重弥漫。从d到f各治疗组的小鼠肺部纤维化进展程度逐渐下降，LPFC(O2)-BIBF-IFN组小鼠肺纤维化程度明显较低，说明改组小鼠纤维化得到了较好的治疗改善。c组单独的PFC携氧治疗组效果较其它携氧组较差，f组携氧纳米药物联合制剂较不携氧的g组肺部阴影更少，均表明要在针对纤维化治疗的基础之上，联合携氧才能达到治疗目的。
4)BALF检测：在博来霉素建模第21天时，取各组小鼠支气管肺泡灌洗液(BALF)，进行细胞总数计数，并用ELISA试剂盒检测BALF中IL-6、IL-1β的含量。从图14小鼠肺泡灌洗液中炎症细胞总计数结果可知，本发明提供的肺部给药携氧纳米药物联合制剂LPFC(O2)-BIBF-IFN能够显著降低IPF小鼠BALF中的炎症细胞总数，且较其他组而言，LPFC(O2)-BIBF-IFN组的炎症细胞总数最少。试剂盒检测BLAF中IL-6、IL-1β含量结果见图15，结合图14炎症细胞总计数可知，IFN-γ的存在和炎症细胞及炎症因子数量的减少呈现一定相关性，从而说明本发明中纳米制剂联合IFN-γ的纳米药物制剂可以发挥免疫调节作用，减少IPF小鼠中炎症的发生，从纤维化疾病发生的源头上减轻疾病进展。
5)免疫组化：取各组小鼠的肺组织，切片进行H&E和Masson染色，通过肺组织结构和蓝色染色的胶原蛋白颜色、分布和面积来分析肺纤维化的程度。结果如图16所示，LPFC(O2)-BIBF-IFN治疗组胶原蛋白水平达到与正常肺组织相当水平，且该组较其他治疗组的肺泡结构完整清晰，炎症细胞浸润较少，间质无明显增厚。
6)肺组织TGF-β1和羟脯氨酸含量检测：TGF-β1是纤维化中研究最深入、最重要的一种细胞因子，在肺纤维化的发生发展过程中具有重要的促进作用。试剂盒检测各组小鼠肺组织匀浆中TGF-β1含量的结果如图17所示，趋势和炎症因子检测结果较为相似，证实本发明的肺部给药携氧纳米药物联合制剂LPFC(O2)-BIBF-IFN可以最有效地降低IPF小鼠肺组织中TGF-β1的含量。各组小鼠的羟脯氨酸含量检测见图18，羟脯氨酸含量检测结果表明本发明的肺部给药携氧纳米药物联合制剂抗纤维化效果为最佳。
7)PCR和ELISA检测各组小鼠肺组织中的HIF-1αmRNA和VEGF mRNA的表达(结果见图19)，比较含有和不含抗纤维化药物BIBF 1120的纳米药物联合制剂，以及载氧前后的纳米制剂治疗组的结果，发现携氧的纳米制剂可以降低HIF-1α刺激VEGF mRNA的表达，而BIBF1120可以协同增进这一效果。
实施例16考察本发明的肺部给药携氧纳米药物联合制剂对肺纤维化相关细胞的影响
LPFC-BIBF-IFN对肺成纤维细胞增殖抑制实验：TGF-β1(10ng/mL)刺激肺成纤维细胞48小时后，肺成纤维细胞增殖率明显增加，说明TGF-β1可以在体外刺激诱导肺成纤维细胞增殖。将用本发明的肺部给药纳米药物联合制剂LPFC-BIBF-IFN分别稀释5倍、10倍后，和TGF-β1刺激的肺成纤维细胞共孵育后48小时后，用MTT检测细胞增值情况。
结果：以TGF-β1未刺激组作为对照，实验结果如图20所示，本发明的肺部给药携氧纳米药物联合制剂LPFC-BIBF-IFN抑制肺成纤维异常活化效果较好，未稀释的LPFC-BIBF-IFN抑制肺成纤维细胞增殖效果显著。
上述详细说明是针对本发明其中之一可行实施例的具体说明，该实施例并非用以限制本发明的专利范围，凡未脱离本发明所为的等效实施或变更，均应包含于本发明技术方案的范围内。

Claims (12)

  1. 一种纳米药物联合制剂，其特征在于，所述纳米药物联合制剂以脂质基为载体，包封全氟化碳(PFC)及活性成分，活性成分为生物活性小分子蛋白质和抗纤维化药物。
  2. 根据权利要求1所述的纳米药物联合制剂，其特征在于，所述纳米联合制剂为用于纤维化疾病的纳米药物联合制剂。
  3. 根据权利要求2所述的纳米药物联合制剂，其特征在于，所述纤维化疾病包括肺纤维化疾病和肝纤维化疾病中的任一种。
  4. 根据权利要求1所述纳米药物联合制剂，其特征在于，所述脂质基为蛋黄卵磷脂、大豆磷脂、氢化大豆卵磷脂、二棕榈酰磷脂酰胆碱、二肉豆蔻磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二油酰基卵磷脂、二棕榈酰磷脂酰甘油、二棕榈酰磷脂酰乙醇胺、二硬脂酰磷脂酰乙醇胺、二硬脂酰磷脂酰甘油、胆固醇、吐温、硬脂酰胺、(2,3-二油酰基-丙基)-三甲基氯化铵、甘油中的一种或其中几种的任意比组合。
  5. 根据权利要求1所述纳米药物联合制剂，其特征在于，所述全氟化碳(PFC)为1-溴全氟乙烷、全氟己基碘烷、全氟辛基碘烷、全氟己烷、全氟辛酸、全氟溴辛烷、全氟辛烷、1-溴-3-氟丙烷、1-溴-1,1,2,2-四氟丁烷、1-溴-2,2-二氟乙烷、1H-6-溴全氟己烷、1-溴-四氟丁烷、1-溴十七氟辛烷、全氟-15-冠-5醚、全氟己烷磺酸钾、全氟己基碘烷、1-溴全氟乙烷、1,6-二碘代十二氟己全氟己基磺酸、全氟三丁胺、1,1,2,2,3,3-六氟丙烷、1,1,1,3-四氟丙烷、1,1,1,3,3-五氟丙烷、1-氟己烷、1,1,2,2,3,3,4,4-八氟丁烷、1,1,1,2,2-五氟丙烷中的一种或其中几种的任意比组合。
  6. 根据权利要求1所述纳米药物联合制剂，其特征在于，所述生物活性小分子蛋白质为白介素-13(IL-13)、白介素-10(IL-10)、干扰素-γ(IFN-γ)、角化细胞生长因子(KGF)中的一种或其中几种的任意比组合；所述抗纤维化药物为BIBF1120、吡非尼酮、曲美替尼、尼罗替尼、白藜芦醇、姜黄素、瑞德西韦中的一种或其中几种的任意比组合；所述抗纤维化药物为BIBF 1120。
  7. 根据权利要求1所述纳米药物联合制剂，其特征在于，全氟化碳(PFC)和脂质基的质量体积比为10％-20％(w/v)，蛋白质占载体质量的0.3％-20％，抗纤维化药物占载体质量的2％-30％。
  8. 一种肺部给药携氧纳米药物联合制剂的制备方法，其特征在于，包括以下步骤：
    (1)将10-30mg脂质基与10-30mL的有机溶剂混合，涡旋震荡均匀，得脂质基溶液，将抗纤维化药物与有机溶剂，涡旋震荡均匀，得药物溶液；
    (2)将步骤(1)得到的脂质基溶液在30-50℃下，旋转蒸发成脂膜，然后加入药物溶液，在30-50℃下槽式超声5-10min；
    (3)将步骤(2)得到的共溶液在30-50℃下，旋转蒸发成膜，然后在真空、37℃下静置8-16h；
    (4)将1-10mL水溶液、0.1-2mL全氟化碳加入步骤(3)得到的薄膜中，100-500W功率进行探头超声2-6min，再经0.22μm滤膜挤压5-10次后，在4℃，100rpm下透析纯化；
    (5)将步骤(4)所得的药物制剂中加入5％-15％的冻干保护剂，-20～-80℃预冻24-48h，-40～-50℃升华干燥24-48h，25℃解析干燥6-10h；
    (6)在步骤(5)得到的冻干粉中加入1-10mL的生物活性小分子蛋白质溶液(100-200μg/mL)进行重构，后经0.2μm滤膜挤压过滤10-20次，在4℃，14000rpm下超滤离心20min去除游离的生物活性小分子蛋白质，得到所述的纳米药物联合制剂。
  9. 一种肺部给药携氧纳米药物联合制剂的制备方法，其特征在于，包括以下步骤：
    (1)将10-30mg脂质基、0.68-3mg抗纤维化药物与5-30mL的有机溶剂混合，涡旋震荡均匀；
    (2)在步骤(1)得到的溶液中加入5％-15％的冻干保护剂，-20～-80℃预冻24-48h，-40～-50℃升华干燥24-48h，25℃解析干燥6-10h；
    (3)在步骤(2)得到的冻干粉中加入0.5-5mL水溶液、0.1-2mL全氟化碳进行重构，然后用100-500W功率进行探头超声2-6min，再经0.2μm滤膜挤压15～20次后，在4℃，100rpm条件下透析纯化；
    (4)将步骤(3)所得的药物制剂和0.5-5mL生物活性小分子蛋白质溶液(200-400μg/mL)在4℃振摇下共孵育3-5h；
    (5)将步骤(4)得到的共孵育溶液在4℃，14000rpm下进行超滤离心20min，得到所述的纳米药物联合制剂。
  10. 根据权利要求8或9所述制备方法，其特征在于，所述冻干保护剂为蔗糖、海藻糖、乳糖、甘露醇、甘氨酸、氯化钠、山梨醇、葡萄糖、麦芽糖中的一种或其中几种的任意比组合。
  11. 根据权利要求8或9所述制备方法，其特征在于，所述水溶液为灭菌的超纯水、灭菌注射用水、灭菌氯化钠溶液、滴滴水、去离子水中的一种或其中几种的任意比组合。
  12. 根据权利要求8或9所述制备方法，其特征在于，所述有机溶剂为氯仿、二甲基亚砜、无水乙醇、甲醇、异丙烷、叔丁醇中的一种或其中几种的任意比组合。
PCT/CN2023/123526 2022-10-10 2023-10-09 一种纳米药物联合制剂及其制备方法和应用 WO2024078443A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211231990.0A CN115708867B (zh) 2022-10-10 2022-10-10 一种用于特发性肺纤维化的肺部给药携氧纳米药物联合制剂及其制备方法
CN202211231990.0 2022-10-10

Publications (1)

Publication Number Publication Date
WO2024078443A1 true WO2024078443A1 (zh) 2024-04-18

Family

ID=85230956

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/123526 WO2024078443A1 (zh) 2022-10-10 2023-10-09 一种纳米药物联合制剂及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN115708867B (zh)
WO (1) WO2024078443A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115708867B (zh) * 2022-10-10 2024-06-04 哈尔滨医科大学 一种用于特发性肺纤维化的肺部给药携氧纳米药物联合制剂及其制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108114273A (zh) * 2018-02-02 2018-06-05 南京大学 一种全氟化碳白蛋白纳米粒及其制备方法与应用
CN109620818A (zh) * 2018-01-19 2019-04-16 中国药科大学 用于肺部递送药物增强肺部药物蓄积量的脂质纳米乳剂
US20190201556A1 (en) * 2016-05-16 2019-07-04 Mtm Research, Llc Fluorochemical targeted therapies
CN113559065A (zh) * 2021-07-28 2021-10-29 郑州大学 一种吸入式肺表面活性剂纳米仿生体的制备方法及其应用
CN113631179A (zh) * 2019-01-25 2021-11-09 努梅迪公司 用于治疗特发性肺纤维化的方法
CN115708867A (zh) * 2022-10-10 2023-02-24 哈尔滨医科大学 一种用于特发性肺纤维化的肺部给药携氧纳米药物联合制剂及其制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110339168B (zh) * 2019-07-26 2020-05-22 中国药科大学 一种负载抗肺纤维化药物和免疫调节剂的纳米制剂及其制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190201556A1 (en) * 2016-05-16 2019-07-04 Mtm Research, Llc Fluorochemical targeted therapies
CN109620818A (zh) * 2018-01-19 2019-04-16 中国药科大学 用于肺部递送药物增强肺部药物蓄积量的脂质纳米乳剂
CN108114273A (zh) * 2018-02-02 2018-06-05 南京大学 一种全氟化碳白蛋白纳米粒及其制备方法与应用
CN113631179A (zh) * 2019-01-25 2021-11-09 努梅迪公司 用于治疗特发性肺纤维化的方法
CN113559065A (zh) * 2021-07-28 2021-10-29 郑州大学 一种吸入式肺表面活性剂纳米仿生体的制备方法及其应用
CN115708867A (zh) * 2022-10-10 2023-02-24 哈尔滨医科大学 一种用于特发性肺纤维化的肺部给药携氧纳米药物联合制剂及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHANG CJ ET AL.: "Overcoming interferon (IFN)-γ resistance ameliorates transforming growth factor (TGF)-β-mediated lung fibroblast-to-myofibroblast transition and bleomycin-induced pulmonary fibrosis", BIOCHEMICAL PHARMACOLOGY, vol. 183, 4 December 2020 (2020-12-04), XP086419000, ISSN: 0006-2952, DOI: 10.1016/j.bcp.2020.114356 *
LING DING: "Perfluorocarbon Nanoemulsions Enhance Therapeutic siRNA Delivery in the Treatment of Pulmonary Fibrosis", ADVANCED SCIENCE, JOHN WILEY & SONS, INC, GERMANY, vol. 9, no. 8, 1 March 2022 (2022-03-01), Germany, pages 2103676, XP093159274, ISSN: 2198-3844, DOI: 10.1002/advs.202103676 *
TUONG N. VU: "Interferon-γ enhances the antifibrotic effects of pirfenidone by attenuating IPF lung fibroblast activation and differentiation", RESPIRATORY RESEARCH, BIOMED CENTRAL LTD., GB, vol. 20, no. 1, 1 December 2019 (2019-12-01), GB , pages 206, XP093159270, ISSN: 1465-993X, DOI: 10.1186/s12931-019-1171-2 *

Also Published As

Publication number Publication date
CN115708867B (zh) 2024-06-04
CN115708867A (zh) 2023-02-24

Similar Documents

Publication Publication Date Title
WO2010083778A1 (zh) 注射用肺靶向脂质体药物组合物
KR970007187B1 (ko) 개선된 암포테리신 b 리포좀 제조법
JP6357481B2 (ja) プロスタサイクリン化合物およびプロスタサイクリン化合物を使用する方法
WO2024078443A1 (zh) 一种纳米药物联合制剂及其制备方法和应用
KR102060210B1 (ko) 안용 스테로이드의 합병증을 감소시키기 위한 약학 조성물
KR20190035776A (ko) 5-콜레스텐-3, 25-디올, 3-술페이트 (25hc3s) 또는 이의 약학적으로 허용 가능한 염, 및 적어도 하나의 시클릭 올리고당을 포함하는 조성물
JP2021522163A (ja) 肺疾患の処置に使用するための吸入可能なリポソーム徐放性組成物
WO2021196659A1 (zh) 糖基聚醚类化合物脂质体及其制备方法和药物
US20200370025A1 (en) Acute Respiratory Distress Syndrome Therapeutic Agent
JP2006199589A (ja) 生理活性タンパク質またはペプチドを含有するナノ粒子およびその製造方法、ならびに当該ナノ粒子からなる外用剤
CA3034875C (en) Combination therapies for the treatment of hepatocellular carcinoma
WO2009062299A1 (en) Gel-stabilized liposome compositions, methods for their preparation and uses thereof
JP2016523884A (ja) 創傷治癒のための脂質ナノ粒子
WO2022068241A1 (zh) 一种雷帕霉素制剂及其制备方法
WO2013176223A1 (ja) 炎症性疾患治療用医薬組成物
CN110464835B (zh) 一种胰岛素柔性微粒及其制剂
KR20190011750A (ko) 엘라스타제 억제 활성을 갖는 베타-헤어핀 펩티드 모방체 및 이의 에어로졸 투여 형태
Fu et al. Synergistic chemotherapy/PTT/oxygen enrichment by multifunctional liposomal polydopamine nanoparticles for rheumatoid arthritis treatment
KR20210126515A (ko) 니클로사마이드를 포함하는 폐고혈압의 예방 및 치료용 조성물
TWI607766B (zh) 核酸、醫用奈米粒子組以及醫藥組合物
CN115569115A (zh) 一种同时包载全氟化碳和二甲双胍的脂质纳米制剂及其制备方法与应用
TW201946613A (zh) 用於治療增生性失調的劑量方案
RU2175556C2 (ru) Способы и композиции для легочной доставки инсулина
KR20020097033A (ko) 당뇨병 환자에게서 공복시 당 및 체중 증가를 감소시키는방법
CN113288871B (zh) 用于调控表观遗传与免疫检查点的药物组合物脂质体制剂

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23876645

Country of ref document: EP

Kind code of ref document: A1