CN109432047B

CN109432047B - Reverse pulmonary fibrosis nano preparation and preparation method thereof

Info

Publication number: CN109432047B
Application number: CN201811264584.8A
Authority: CN
Inventors: 姜虎林; 常鑫; 邢磊
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2018-10-29
Filing date: 2018-10-29
Publication date: 2021-07-20
Anticipated expiration: 2038-10-29
Also published as: CN109432047A

Abstract

The invention discloses a reverse pulmonary fibrosis nano preparation and a preparation method thereof, wherein the E5 and polypeptide Z modified nano preparation and a carrier thereof are synthesized, and the nano preparation targets receptors of circulating fiber cells CXCR4, CCR2 and CCR7 through E5 so as to reach a pulmonary fibrosis injury part at a fixed point and avoid being wrapped and removed by in vivo macrophages and proteins; the polypeptide Z can specifically target the alveolar epithelial cell II of the high-expression integrin receptor alpha v beta 6, so that the high-efficiency targeted delivery nano preparation is realized. The nano preparation contains antioxidant and/or fibroblast activation inhibitor, and achieves the purpose of reversing pulmonary fibrosis by regulating the oxidative stress level of alveolar epithelial cells II and inhibiting the fibroblast activation through double-channel regulation respectively.

Description

Reverse pulmonary fibrosis nano preparation and preparation method thereof

Technical Field

The invention relates to a circulating fiber cell mediated double-drug loaded and/or single-drug reversed pulmonary fibrosis nano preparation, in particular to preparation and application of a circulating fiber cell targeted double-drug loaded and/or single-drug reversed pulmonary fibrosis nano preparation in a pulmonary fibrosis body.

Background

Pulmonary fibrosis is a progressive interstitial lung disease with extremely high lethality, and the survival period is 3-5 years. In recent years, the air particle (PM 2.5) is too high due to environmental pollution, and alveolar epithelial cells I (AT1) and II (AT2) are damaged. Wherein, the AT1 cell has simple intracellular structure and is mainly responsible for the inhalation of alveolar oxygen and the exhalation of carbon dioxide; AT2 cells are called stem-like cells of lung tissue and have the ability to self-renew. When AT2 is damaged, the proliferation capacity of the alveoli is seriously damaged, after irreversible damage of the function of the alveoli occurs, a large amount of inflammatory factors are secreted, fibroblasts are activated to be transformed into myofibroblasts, the activated myofibroblasts are the main source for collagen secretion AT the damaged part, and the generated collagen has positive feedback regulation on the myofibroblasts in the pathology of pulmonary fibrosis, so that a large amount of collagen AT the damaged part of epithelial cells is accumulated, and the intercellular spaces are extruded and filled by the collagen, thereby forming the pulmonary fibrosis. At present, two oral drugs for resisting pulmonary fibrosis, namely pirfenidone and nintedanib, are approved by the FDA. But only can achieve the purposes of delaying the disease process and not repairing the damaged lung tissue to reverse pulmonary fibrosis.

AT2 alveolar epithelial cell ii plays a crucial role in the development of pulmonary fibrosis. During the development process of pulmonary fibrosis, the injured AT2 recruits a large amount of inflammatory cells to the lung, including macrophages, neutrophils, eosinophils and basophils, and these cells can secrete a large amount of inflammatory factors, including TNF-beta, IL-4, IL-13, IL-1 beta and the like, under the stimulation of the pulmonary fibrosis microenvironment, so that the inflammatory response is further intensified, and recruits a large amount of circulating fibroblasts derived from bone marrow and lung fibroblasts to be transformed into myofibroblasts, and the activated myofibroblasts secrete a large amount of collagen and accumulate, thereby accelerating the progress of fibrosis. Thus, in the treatment of pulmonary fibrosis, repair of damaged AT2 is a key therapeutic target capable of reversing pulmonary fibrotic diseases.

Nanometer preparations are more and more widely concerned in disease treatment, but the nanometer preparations still have the defects of poor targeting and low delivery efficiency in pulmonary disease delivery.

Disclosure of Invention

The purpose is as follows: in order to overcome the defects in the existing pulmonary fibrosis treatment, the invention provides a reverse pulmonary fibrosis nano preparation with efficient targeted delivery.

The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a nanometer preparation carrier for loading anti-pulmonary fibrosis drugs is characterized in that: the nano preparation carrier is X-PEG-MAL, modifies targeting peptide, antibody and chemotactic factor E5 with the targeting of circulating fiber cell receptor Y, and/or modifies polypeptide Z with the targeting of alveolar epithelial cell II; wherein X is a hydrophobic segment and is selected from PLGA, PLA, PGA and PCL; y is a circulating fiber cell high expression receptor, including CXCR4, CCR2, CCR 7; the polypeptide Z comprises cRGD, RGDf (c) and RGD.

Further, the nano-preparation carrier is characterized in that: comprises a carrier PPE modified with E5 only, a carrier PPR modified with polypeptide Z only, and a carrier PPER modified with E5 and polypeptide Z simultaneously.

Further, the nano-preparation carrier is characterized in that: e5 includes a CXCR4 receptor targeting peptide, antibody, chemokine, CCR7 receptor targeting peptide, antibody or chemokine and CCR2 receptor targeting peptide, antibody or chemokine; more preferably, thiol-modified E5 is used. The polypeptide Z comprises cRGD, RGDf (c) and RGD.

In the invention, the anti-pulmonary fibrosis drug is one or more of an antioxidant and an inhibitor, the antioxidant is a drug or bioactive molecule for relieving oxidative stress of a cell level, and the inhibitor is a drug or bioactive molecule for inhibiting fibroblast activation. The loaded anti-pulmonary fibrosis drug can simultaneously comprise an antioxidant and an inhibitor, or one selected from the antioxidant and the inhibitor. The loaded anti-pulmonary fibrosis drugs have certain hydrophobicity, the loading of the anti-pulmonary fibrosis drugs is realized through the hydrophobic section X in the amphiphilic block copolymer, the long circulation of the polymer micelle in vivo is realized through PEG, and the efficient delivery of the lung is realized through the modified E5 specific targeting to the circulating fiber cells in vivo. The polypeptide Z can specifically target the injured AT2, so that the efficient targeted delivery of the anti-pulmonary fibrosis drug to the injured AT2 is realized.

More preferably, the antioxidant is astaxanthin and the inhibitor is trametinib. The reverse pulmonary fibrosis nano preparation is simultaneously loaded with trametinib and astaxanthin. Meanwhile, the lung fibrosis microenvironment is cooperatively regulated by adopting double drugs, and a new way and strategy are provided for the efficient delivery of anti-lung fibrosis drugs and the drug design for reversing lung fibrosis.

The invention also provides a reversed pulmonary fibrosis nano preparation, which is characterized in that: the nano preparation carrier is loaded with anti-pulmonary fibrosis drugs, including PPER/drug, PPE/drug and PPR/drug. The loaded anti-pulmonary fibrosis drug can simultaneously comprise an antioxidant and an inhibitor, or one selected from the antioxidant and the inhibitor. The antioxidant is a drug or bioactive molecule that relieves oxidative stress at the cellular level, and the inhibitor is a drug or bioactive molecule that inhibits CTGF expression.

1) Antioxidant:

antioxidant drugs: radix et rhizoma Rhei, Notoginseng radix, semen Ginkgo, pericarpium Citri Tangerinae, folium Nelumbinis, folium Mori, Polygoni Multiflori radix, Saviae Miltiorrhizae radix, radix astragali, Bulbus Allii, rhizoma Ligustici Chuanxiong, radix Rhodiolae, cornu Cervi Pantotrichum, probucol, indapamide, and verapamil.

Antioxidant active ingredients: vitamin E, carotenoid and its derivatives (astaxanthin, canthaxanthin, lutein, beta-carotene), tuna flavin, canthaxanthin, ginseng total saponin, flavonoid glycosides and terpene lactones, lycium barbarum polysaccharide, dibenzocyclooctene lignans such as schisanhenol, schisandrin B, schisandrin C, gynostemma pentaphyllum, algal polysaccharide, fungus polysaccharide, etc., Butyl Hydroxy Anisole (BHA), dibutyl hydroxy toluene (BHT), Propyl Gallate (PG), tert-butyl hydroquinone (TBHQ);

2) fibroblast activation inhibitor: trametinib, nintedanib, pirfenidone, nilotinib, silybin, curcumin, resveratrol, tanshinone A, berberine, schizandrin, salvianolic acid A, B, luteolin, gallocatechin, ligustrazine and the like.

The anti-pulmonary fibrosis drug comprises a drug or a bioactive molecule for relieving oxidative stress at a cellular level and a drug or a bioactive molecule for inhibiting a fibroblast activation inhibitor.

Specifically, the preparation method of the PPER/medicine comprises the following steps:

firstly, mixing an anti-pulmonary fibrosis drug with X-PEG-MAL, and preparing nanoparticles loaded with the anti-pulmonary fibrosis drug by a thin film dispersion method, a direct titration method or a reverse solvent method;

reacting nanoparticles loaded with anti-pulmonary fibrosis drugs with sulfydryl modified E5 and sulfydryl modified polypeptide Z, and covalently modifying E5 and polypeptide Z at the outer ends of the nanoparticles through the reaction of sulfydryl and MAL exposed from the nanoparticle shells; the nanoparticles achieve the target of circulating fiber cells and alveolar epithelial cells II through covalently modified E5 and polypeptide Z.

In the reaction, the mass ratio of the added polymer nanoparticles to the mercapto-modified E5 is 30: 1-12: 1; more preferably 15: 1.

In the reaction, the mass ratio of the added polymer nanoparticles to the thiol-modified polypeptide Z is 50: 1-35: 1; more preferably 40: 1.

The preparation method of PPE/drug is as follows:

reacting nanoparticles loaded with anti-pulmonary fibrosis drugs with sulfydryl-modified E5, and covalently modifying E5 at the outer ends of the nanoparticles through the reaction of sulfydryl and MAL exposed on the nanoparticle shells; the nanoparticles achieve the target of circulating fiber cell targeting through the covalently modified E5.

The preparation method of the PPR/medicine comprises the following steps:

reacting nanoparticles loaded with anti-pulmonary fibrosis drugs with polypeptide Z modified by sulfydryl, and covalently modifying the polypeptide Z at the outer ends of the nanoparticles through the reaction of sulfydryl and MAL exposed on the nanoparticle shells; the nanoparticles achieve the target of alveolar epithelial cell II targeting through polypeptide Z which is covalently modified.

As a control, PP/drug was prepared as follows:

firstly, dissolving an anti-pulmonary fibrosis drug in DMSO (dimethyl sulfoxide), wherein the ratio of X-PEG-MAL to the drug is 100: 1-5: 1, the scheme is 10:1 is more preferable, the encapsulation rate is 82.4%, and the drug loading rate is 7.2%;

and (3) mixing the obtained product with X-PEG-MAL, and preparing the nanoparticles loaded with the anti-pulmonary fibrosis drug by a thin film dispersion method, a direct titration method or a reverse solvent method.

Preferably, the reversal pulmonary fibrosis nano preparation is characterized in that: in X-PEG-MAL, wherein the molecular weight range of X is 1000-50000, and the molecular weight range of PEG is 200-10000. More preferably, PEG with a molecular weight of 2000 is used.

Preferably, the drug loading of the reversal pulmonary fibrosis nano preparation is between 2% and 20%, and the particle size is between 20 nm and 500 nm.

The invention requires the application of the nano preparation carrier and the reversed pulmonary fibrosis nano preparation in preparing the medicine for treating pulmonary fibrosis diseases.

The invention relates to a method for efficiently delivering nano double drugs/single drug by taking in vivo circulating fiber cells as a delivery carrier so as to achieve the purpose of reversing pulmonary fibrosis. Is characterized in that the carrier comprises chemical drug loading components, circulating fiber cell targeting components and damaged AT2 targeting components. The chemical drug loading component is an amphiphilic polymer with maleimide modification AT the tail end, the circulating fiber cell targeting component is sulfhydrylation polypeptide, and the damaged AT2 targeting component is PEG modified with polypeptide Z.

The invention also prepares a preparation PP/medicament without modification of E5 and polypeptide Z, a carrier material PPE/medicament only with modification of E5 and a carrier material PPR/medicament only with modification of polypeptide Z, and the preparation methods of the carrier materials are the same as the method.

Has the advantages that: compared with the prior art, the double-drug/single-drug encapsulated nano preparation taking circulating fiber cells as delivery carriers provided by the invention has the following advantages: the X-PEG-MAL (X is a hydrophobic segment, and X is PLGA, PLA, PGA, PCL) in the carrier can well realize the entrapment of fat-soluble drugs, the PEG in the carrier can prolong the circulation time of the carrier in blood, the modified E5 in the carrier can well realize the specific targeting of in vivo circulating fibroblasts, and the polypeptide Z in the carrier can specifically realize the specific targeting of the damaged AT 2. The application of the carrier can realize the high-efficiency lung delivery of nano-drugs, inhibit the key protein CTGF through specific targeting damaged and repaired AT2, and effectively inhibit the activation of fiber cells, thereby achieving the purpose of reversing pulmonary fibrosis. The E5 and polypeptide Z modified nano preparation provided by the invention can specifically target in vivo circulating fibroblasts by using E5, and can specifically target damaged AT2 by using polypeptide Z, so that efficient pulmonary fibrosis resistant double-drug/single-drug delivery is realized, and the aim of reversing pulmonary fibrosis is fulfilled. The invention innovatively utilizes in vivo circulating fibroblasts as a delivery carrier for the first time to realize the high-efficiency lung delivery of the anti-pulmonary fibrosis drug, takes the damaged AT2 as a treatment target and specifically targets the double-drug/single-drug nano-drug preparation, and provides a new way for the high-efficiency delivery and reversal treatment of the pulmonary fibrosis drug preparation. In the invention, the aim of reversing pulmonary fibrosis is fulfilled by innovating and utilizing the circulating fiber cell target which is proliferated in vivo in a large amount as a delivery system for the first time and simultaneously loading the double-drug/single-drug nano-drug preparation by E5 and the polypeptide Z. At present, the delivery by using the hydrophobic block loaded with the hydrophobic drug in the amphiphilic block copolymer is one of common methods, PLGA (polylactic acid-polyglycolic acid), PLA (polylactic acid), PGA (polyglycolide), and PCL (polycaprolactone) are common hydrophobic blocks with good biocompatibility, are often used as hydrophobic cores in the amphiphilic block copolymer, and have good affinity for most hydrophobic drugs.

In the invention, E5 and X-PEG (X is a hydrophobic segment, and X is PLGA, PLA, PGA, PCL) nanoparticles modified by polypeptide Z are utilized, the loading of double drugs/single drugs with certain hydrophobicity is realized through the hydrophobic segment in an amphiphilic block copolymer, or the nanoparticles loaded with hydrophilic drugs can be prepared through a reverse solvent method, the long circulation of a polymer micelle in a body is realized through PEG, and the modified E5 specifically targets circulating fiber cells in the body, so that the efficient delivery of the lung is realized. The polypeptide Z can specifically target the injured AT2, so that the efficient targeted delivery of the anti-fibrosis double drug/single drug to the injured AT2 is realized.

Drawings

FIG. 1 is a schematic flow diagram of the preparation of a nanoformulation according to the present invention;

FIG. 2 shows the optimum ratio of the components of the nano-formulation prepared in the present invention;

FIG. 3 is a nano-formulation particle size distribution of the best formulation of the invention (PPER/drug);

FIG. 4 is a transmission electron micrograph of the best formulation of the invention (PPER/drug) of the nano-formulation;

fig. 5 is a transmission electron micrograph of a nano-formulation of the present invention (PPER/drug) (R = cRGD);

fig. 6 is a transmission electron micrograph of a nano-formulation of the present invention (PPER/drug) (R = RGD);

FIG. 7 is an uptake experiment of the nano-formulation prepared by the present invention AT a cell level, which proves that RGDf (c) can specifically target AT2 and increase the uptake of the nano-formulation;

FIG. 8 is a diagram of the nano-carrier prepared by the invention, which examines different carriers PPER, PPE, PPR, PP AT the in vivo level, and realizes the accurate positioning of nano-preparation by using circulating fiber cells as the lung delivery capability of a delivery system and specifically targeting injured AT 2;

FIG. 9 shows the accumulation change of the nano-preparation in the lung in the in vivo nano-carrier prepared by the invention at different time points by using circulating fiber cells as a delivery system;

fig. 10 is a photograph of a living body of a small animal showing accumulation of the nanopreparation in the lung of a mouse with pulmonary fibrosis in which the nanopreparation coated with a fluorescent Dye (DiR) prepared according to the present invention was molded for 3 weeks and a quantitative analysis of different organs.

Fig. 11 is an analysis of the effect of different nano-preparations in reversing pulmonary fibrosis under the in vivo level examination of the nano-carrier prepared by the invention.

Detailed Description

The invention will be further described with reference to the following drawings and specific embodiments.

Example 1 synthesis and preparation of Nanometric formulation ingredients, as shown in FIG. 1:

preparation of PLGA-PEG-MAL nanoparticles entrapping double drugs/single drug

The polymer nanoparticles loaded with anti-pulmonary fibrosis drugs or fluorescent dyes (used as a model for replacing drugs and used for preparation tracking) can be prepared by adopting a film dispersion method, a direct titration method and a reverse solvent method. The invention preferably adopts a direct titration method to prepare PLGA-PEG-MAL nanoparticles coated with double drugs/single drugs (trametinib and/or astaxanthin) of anti-fibrosis drugs. The preparation method comprises the following steps:

100 mg PLGA-PEG-MAL and 10 mg double/single medicines are weighed and respectively dissolved in 2 mL DMSO solutions, and ultrasonic dissolution is carried out. The DMSO solution was added dropwise to 100 mL of purified water with stirring, and after stirring at room temperature for 2 hours, the mixture was centrifuged at 2500 rpm/min for 20 minutes to remove the unbound free drug. The nanoparticles were concentrated to a volume of 500 μ L using an ultrafiltration tube of molecular weight 10000.

Secondly, preparation of polymer nanoparticles modified by E5 and RGDf (c)

E5 includes a CXCR4 receptor targeting peptide, antibody, chemokine, CCR7 receptor targeting peptide, antibody or chemokine and CCR2 receptor targeting peptide, antibody or chemokine; more preferably, thiol-modified E5 is used. The polypeptide Z comprises cRGD, RGDf (c) and RGD.

The anti-pulmonary fibrosis drug comprises:

antioxidant drugs and active ingredients:

fibroblast activation inhibitor: trametinib, nintedanib, pirfenidone, nilotinib, silybin, curcumin, resveratrol, tanshinone A, berberine, schizandrin, salvianolic acid A, B, luteolin, gallocatechin, ligustrazine and the like.

1. Preparation of polymer nanoparticles modified by circulating fibroblast receptor CXCR4 targeting peptide and polypeptide Z

Modification of E5: carrying out a reaction on PLGA-PEG-MAL nanoparticles loaded with a drug and 6.5 mg of thiolated polypeptide in a pure water solution in a shaking table at 4 ℃ overnight, wherein E5 is a target polypeptide of a circulating fiber cell receptor CXCR 4;

modification of polypeptide Z: PLGA-PEG-MAL nanoparticles carrying drugs and thiolated polypeptide RGDf (c) 2.5 mg are reacted in a pure water solution in a shaking table at 4 ℃ overnight; resuspend three times by centrifugation at 10000 rpm/min, collect the prepared nanoparticles, remove unreacted thiolated E5 and Z.

The drug-loading rate of PP/(trametinib and/or astaxanthin), PPE/(trametinib and/or astaxanthin) and PPR/(trametinib and/or astaxanthin) prepared by the method is 2-10%, and the particle size is 50-500 nm. The optimal preparation (PPER/(trametinib and/or astaxanthin)) of the invention has the nanometer preparation particle size distribution, and the transmission electron microscope images are shown in fig. 3 and 4, the nanometer preparation particle size distribution is uniform, the shape is uniform, and the electron microscope images show that the core-shell structure is obvious.

2. Preparation of polymer nanoparticles modified by circulating fibroblast receptor CCR7 (receptor of CCR 7) and polypeptide Z

Modification of E5: carrying out a reaction on PLGA-PEG-MAL nanoparticles loaded with a drug and 6.5 mg of a thiolated antibody in a pure water solution in a shaking table at 4 ℃ overnight, wherein E5 is a target antibody of a circulating fiber cell receptor CCR 7;

The drug-loading rate of PP/(trametinib and/or astaxanthin), PPE/(trametinib and/or astaxanthin) and PPR/(trametinib and/or astaxanthin) prepared by the method is 2-10%, and the particle size is 50-500 nm. The nanometer preparation of the optimal preparation (PPER/(trametinib and/or astaxanthin)) has uniform particle size distribution and uniform shape.

3. Preparation of polymer nanoparticles modified by circulating fibroblast receptor CCR2 targeted chemokine and polypeptide Z

Modification of E5: the PLGA-PEG-MAL nano-particle loaded with the drug and 6.5 mg of thiolated chemokine are reacted in a pure water solution in a shaking table at 4 ℃ overnight, wherein E5 is a target antibody of a circulating fiber cell receptor CCR 7;

modification of polypeptide Z: carrying out a reaction on PLGA-PEG-MAL nanoparticles loaded with a drug and 2.5 mg of thiolated polypeptide Z in a pure water solution in a shaking table at 4 ℃ overnight; resuspend three times by centrifugation at 10000 rpm/min, collect the prepared nanoparticles, remove unreacted thiolated E5 and Z.

Preparation of polymer nanoparticles modified by E5 and cRGD or RGD

Modification of E5: PLGA-PEG-MAL nanoparticles carrying the drug and sulfhydrylated E56.5 mg are reacted in a pure water solution in a shaking table at 4 ℃ overnight;

modification of polypeptide cRGD or RGD: carrying out a reaction on PLGA-PEG-MAL nanoparticles loaded with a medicament and thiolated polypeptide cRGD or RGD 2.5 mg in a pure water solution in a shaking table at 4 ℃ overnight; and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted sulfhydrylated E5 and cRGD or RGD.

The drug-loading rate of PP/(trametinib and/or astaxanthin), PPE/(trametinib and/or astaxanthin) and PPR/(trametinib and/or astaxanthin) prepared by the method is 2-10%, and the particle size is 50-500 nm. The optimal preparation (PPER/(trametinib and/or astaxanthin)) of the invention has the nanometer preparation particle size distribution, and the transmission electron microscope images are shown in fig. 5 and 6, the nanometer preparation particle size distribution is uniform, the shape is uniform, and the electron microscope images show that the core-shell structure is obvious.

Example 2 uptake analysis of Nanopropreparations at A549 cell level and quantitative analysis by flow cytometry

PP/coumarin 6, PPE/coumarin 6, PPR/coumarin 6 and PPER/coumarin 6 nanoformulations were prepared as described in example 1. A549 cells were cultured at 5X 10⁵Perml/mL in 24-well plates at 37 ℃ 5% C0₂After the cells grow in a cell culture box in an adherent manner for 24 hours, the culture medium is sucked, serum-free diluted TGF-beta (5 ng/mL) is added to stimulate the cells for 24 hours, then free coumarin 6, PP/coumarin 6, PPR/coumarin 6, PPC/coumarin 6 and PPCR/coumarin 6 diluted by the serum-free culture medium are respectively added, the concentration of the coumarin 6 in each hole is 1 mu g/mL, the concentration of the coumarin 6 in each hole is 500 mu L, and each hole is provided with three auxiliary holes. The culture was continued for 4h, washed three times with PBS,

(1) the laser confocal LSM-700 is used for shooting the condition that the cells take the nanoparticles and the free coumarin 6;

(2) pancreatin digestion, 1500 rpm centrifugal precipitation of cells, with serum free medium heavy suspension of cells, using flow cytometry on the uptake of coumarin 6 quantitative detection.

The cellular uptake data measured in this example are shown in fig. 7, and the a549 cells evaluated AT the in vivo level belong to adenocarcinoma alveolar basal epithelial cells, which can simulate the uptake of nanoparticles by injured alveolar epithelial cells AT2 in vivo. After 24h of TGF-beta (5 ng/mL), alveolar epithelial cells are damaged, integrin receptors alpha v beta 6 are highly expressed on the surface of cell membranes, and polypeptides cRGD, RGDf (c) and RGD can be specifically combined with the alpha v beta 6. The confocal result shows that the uptake of PPR/coumarin 6 is obviously higher than that of PP/coumarin 6 and coumarin 6, and the combination of cRGD, RGDf (c) and RGD is favorable for increasing the specific uptake of A549 cells to the nano preparation; in A549 cells which are not stimulated by TGF-beta, the integrin receptor alpha v beta 6 on the surface of a cell membrane is not highly expressed, and the result shows that the uptake of the nanoparticles and the free coumarin 6 is not obviously different.

Example 4 pulmonary specific delivery assay of fluorescent dye coated (DiR) nanoformulations in pulmonary fibrosis mice

PP/DiR, PPE/DiR, PPR/DiR and PPER/DiR nanoformulations were prepared as described in example 1.

Firstly, a molding test of a pulmonary fibrosis model is carried out by adopting male C57BL/6 mice with the age of 6-8 weeks, and the lungs of the mice are directly molded by using a tracheal intubation method during molding. And (3) during molding, bleomycin hydrochloride is used as a mouse pulmonary fibrosis inducer, the concentration is 2USP/Kg, and after three weeks, the nanoparticle tracer experiment is continued to be carried out after the mouse pulmonary fibrosis is molded. For judging the model forming time of the mouse pulmonary fibrosis model, a large number of documents report that the mouse pulmonary fibrosis is in a very rapid development stage at the beginning of three weeks, and the proliferation of circulating fibroblasts in vivo is most obvious. Mice molded for three weeks were randomly assigned to 4 groups of 4 mice each, and free DiR, PP/DiR, PPE/DiR, PPR/DiR and PPER/DiR were injected via tail vein, respectively, with the injected DiR at 1. mu.g/g mouse body weight as a standard. Mice were imaged in vivo with a small animal in vivo imager at 1h, 4h, 8h, 12h, 24h after tail vein injection.

The imaging picture of the lung accumulation of the pulmonary fibrosis mice formed by the fluorescent Dye (DiR) -coated nano preparation prepared in this example after the bleomycin hydrochloride molding and the quantitative analysis thereof are shown in fig. 8; the accumulation degree of the nano preparation in the lung is as follows: PPER/DiR > PPE/DiR > PPR/DiR > PP/DiR, it was demonstrated that modification of E5 and polypeptide Z (cRGD, RGDf (c), RGD) is advantageous for targeted delivery of the nanoformulation to the lung and high accumulation.

Example 5 fluorescent Dye (DiI) -coated nano-formulations in pulmonary fibrosis mice, nanoparticles adhesion to circulating fibroblasts in vivo, pulmonary accumulation time and delivery mechanism.

PP/DiI, PPE/DiI, PPR/DiI and PPER/DiI nanoformulations were prepared as described in example 1.

Firstly, a molding test of a pulmonary fibrosis model is carried out by adopting male C57BL/6 mice with the age of 6-8 weeks, and the lungs of the mice are directly molded by using a tracheal intubation method during molding. And (3) when the model is made, bleomycin hydrochloride is used as a mouse pulmonary fibrosis inducer, the concentration is 2USP/Kg, and after three weeks, the nanoparticle delivery mechanism experiment is continued to be carried out after the mouse pulmonary fibrosis is formed. For judging the model forming time of the mouse pulmonary fibrosis model, a large number of documents report that the mouse pulmonary fibrosis is in a very rapid development stage at the beginning of three weeks, and the proliferation of circulating fibroblasts in vivo is most obvious. Mice molded for three weeks were randomly assigned into 4 groups of 3 mice each, and free DiI, PP/DiI, PPE/DiI, PPR/DiI and PPER/DiI were injected via tail vein, respectively, with the injected DiI at 1. mu.g/g mouse body weight as a standard. And taking the lung tissue of the mouse for carrying out lung tissue immunostaining experiments 30min, 1h, 2h and 4h after tail vein injection. The results are shown in FIGS. 9 and 10. Wherein DAPI is a nucleus blue mark, alpha-SMA is a circulating fiber cell membrane protein green mark, and DiI is a nanoparticle with a red mark. The results show that the attachment of the nanoparticle to the circulating fiber cells with red and green markers is obvious, and the accumulation of red fluorescence in lung tissues is increased along with the increase of the administration time. The nanoparticles can be efficiently delivered to lung tissues through a circulating fiber cell mediated nanoparticle delivery system, so that the aim of targeted delivery is fulfilled.

Example 6 efficacy of nano-formulation PPATER in treatment of over-reversed pulmonary fibrosis.

The preparations of the nanopreparations PP/A, PP/T, PP/AT, PPR/AT, PPE/A, PPE/T, PPER/AT and PPER/A, PPER/T were carried out as described in example 1. A represents an antioxidant such as astaxanthin, and T represents a fibroblast activation inhibitor such as trametinib.

Firstly, a molding test of a pulmonary fibrosis model is carried out by adopting male C57BL/6 mice with the age of 6-8 weeks, and the lungs of the mice are directly molded by using a tracheal intubation method during molding. And (3) when the model is made, bleomycin hydrochloride is used as a mouse pulmonary fibrosis inducer, the concentration is 2USP/Kg, and after three weeks, the effect analysis of the nano preparation in the treatment of the over-reversed pulmonary fibrosis is continuously carried out after the mouse pulmonary fibrosis is shaped. Mice molded for three weeks were randomly assigned to 8 groups of 5 mice each, and were injected with free PP/A, PP/T, PP/AT, PPR/AT, PPE/A, PPE/T, PPER/AT, PPER/A, PPER/T and saline, respectively, via the tail vein, using an injected TRA of 2 μ g/g mouse body weight as a standard. After 4 weeks of treatment, the final preparations PPE/AT, PPE/A, PPE/T, and PPER/AT, PPER/A, PPER/T groups showed significantly reduced levels of pulmonary fibrosis compared to the BLM group as analyzed by H & E staining for reversal of pulmonary fibrosis effects for different nano-formulations as shown in FIG. 11, wherein the PPER/AT group and the PPE/AT group showed particularly good effects, better than the PPR/AT group, the PP/AT group, the PPE/T group and the PP/A group. More importantly, lung sections of the final preparation group PPER/AT and the single-target PPE/AT group are consistent with those of the normal group, clear alveolar structures can be seen, and the fact that efficient lung targeting and double-drug combined treatment can be achieved through a nano preparation delivery system for mediating circulating fibroblasts can effectively reverse pulmonary fibrosis.

PLGA (polylactic acid-polyglycolic acid), PLA (polylactic acid), PGA (polyglycolide) and PCL (polycaprolactone) are common hydrophobic blocks with good biocompatibility, are often used as hydrophobic cores in amphiphilic block copolymers, have better affinity to most of drugs with certain hydrophobicity, or can be used for preparing nanoparticles loaded with hydrophilic drugs by a reverse solvent method. In the above embodiment, PLA-PEG-MAL, PGA-PEG-MAL, and PCL-PEG-MAL block copolymers are used to replace PLGA-PEG-MAL block copolymers, and a drug with certain hydrophobicity can be loaded as well, or nanoparticles loaded with a hydrophilic drug can be prepared by a reverse solvent method, so as to form a polymer micelle, which is clear to those skilled in the art.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. A nanometer preparation carrier for loading anti-pulmonary fibrosis drugs is characterized in that: the nano preparation carrier is X-PEG-MAL, and E5 and polypeptide Z with alveolar epithelial cell II targeting are simultaneously modified, wherein E5 is targeting peptide, antibody and chemotactic factor with circulating fiber cell receptor Y targeting; wherein X is a hydrophobic segment and is selected from PLGA, PLA, PGA and PCL; y is a circulating fiber cell high expression receptor selected from CXCR4, CCR2 and CCR 7; the polypeptide Z is selected from cRGD, RGDf (c) and RGD.

2. The Nanopropreparational vehicle according to claim 1, wherein: e5 is selected from the group consisting of CXCR4 receptor targeting peptides, antibodies, chemokines, CCR7 receptor targeting peptides, antibodies or chemokines and CCR2 receptor targeting peptides, antibodies or chemokines.

3. The Nanopropreparational vehicle according to claim 1, wherein: the anti-pulmonary fibrosis drug is one or more of an antioxidant and an inhibitor, the antioxidant is a drug or bioactive molecule for relieving oxidative stress of a cell level, and the inhibitor is a drug or bioactive molecule for inhibiting fibroblast activation.

4. A reverse pulmonary fibrosis nano-preparation is characterized in that: the nanoformulation carrier of any one of claims 1-3 loaded with an anti-pulmonary fibrosis drug.

5. The reverse pulmonary fibrosis nanoformulation of claim 4, wherein: the anti-pulmonary fibrosis drug is one or more of an antioxidant and an inhibitor, the antioxidant is a drug or bioactive molecule for relieving oxidative stress of a cell level, and the inhibitor is a drug or bioactive molecule for inhibiting fibroblast activation.

6. A reversed pulmonary fibrosis nanoformulation according to claim 4 or 5, wherein: the preparation method comprises the following steps:

7. The reverse pulmonary fibrosis nanoformulation of claim 6, wherein: in X-PEG-MAL, wherein the molecular weight range of X is 1000-50000, and the molecular weight range of PEG is 200-10000.

8. The reverse pulmonary fibrosis nanoformulation of claim 4, wherein: the drug loading of the reversal pulmonary fibrosis nano preparation is between 2 and 20 percent, and the particle size is between 20 nm and 500 nm.

9. Use of the nanoformulation carrier of any one of claims 1-3, the reverse pulmonary fibrosis nanoformulation of claim 4 in the preparation of a medicament for the treatment of pulmonary fibrosis diseases.