CN110384681A - A kind of nanometer formulation and preparation method thereof for pulmonary fibrosis - Google Patents

Abstract

The invention discloses a nano-preparation for pulmonary fibrosis and a preparation method thereof, that is, a protease-modified nano-preparation with mucin degradation and a carrier thereof. Firstly, the nano-preparation is released responsively by virtue of protease degrading mucin properties, and then the therapeutic drug is efficiently delivered to the injured site of pulmonary fibrosis, so as to realize the efficient targeted delivery of anti-pulmonary fibrosis drugs and achieve the purpose of efficient treatment of pulmonary fibrosis. Aiming at the problem of difficult deep delivery in the currently marketed trachea and inhalation dosage forms, that is, excessive secretion of mucus by goblet cells in the airway makes it difficult for a large amount of drugs to be delivered to the deep alveoli to play a role. Based on the change of mucus hypersecretion in the lungs, a nano-preparation that responds to the release and can efficiently pass through the airway barrier is constructed, and then the therapeutic drug is efficiently delivered to the pulmonary fibrosis injury site deep in the alveoli, and the efficiency of the nano-preparation reaching the deep alveoli is improved.

Description

A nano-preparation for pulmonary fibrosis and its preparation method

technical field

The invention discloses a nano-preparation for pulmonary fibrosis and a preparation method thereof, belonging to the technical field of medicine.

Background technique

Idiopathic pulmonary fibrosis (IPF) is one of the severe and common forms of interstitial lung disease (ILD). ILD is a progressive lung disease that can lead to pulmonary fibrosis at an advanced stage. During the development of the disease, the damaged alveolar epithelial cells will secrete a large amount of inflammatory factors, thereby activating the excessive proliferation of fibroblasts and transforming into myofibroblasts at the same time, resulting in excessive deposition of extracellular matrix (ECM), and damage to the lung parenchyma. Destruction, eventually leading to the death of the patient due to respiratory failure. Although the new anti-fibrotic drug pirfenidone (PFD) has been approved for clinical treatment in Europe and other countries, there are still some problems. Since pirfenidone is usually used as an oral preparation for the treatment of IPF, its bioavailability is low, and a large amount of administration may cause side effects or even toxicity to other organs. Therefore, the strategy of efficiently delivering drugs to the lungs through inhalation provides new ideas and methods for the treatment of IPF.

In inhalation dosage forms, the drug delivery efficiency is generally low due to the limitation of airway physiology. The mucus layer in the airways is the first barrier that must be overcome for tracheal or inhaled drug delivery. The mucus layer is mainly composed of a dense network of fibers of mucin, a negatively charged biomacromolecule containing high-density glycans. In patients with advanced obstructive pulmonary disease and fibrosis, mucus secreted by airway goblet cells acts as part of a clearance system that traps drugs reaching the airways through steric hindrance or adhesion and subsequently clears them from the mucus , leading to the reduction of drugs reaching the deep lungs, resulting in weakened therapeutic effects of drugs on diseases. Even though drugs can pass through the mucus layer, there is still another barrier in the alveoli, the phagocytic system of macrophages in the lipid layer, which is a protective defense barrier of type II alveolar epithelial cells (AT II). A large number of drugs will be phagocytized by macrophages in the lipid layer and cannot play a therapeutic role.

Therefore, for drugs that need to act deep in the lung, these obstacles must be effectively overcome during the delivery process to achieve the desired therapeutic effect.

Contents of the invention

Objective: In order to overcome the deficiencies in the prior art, the present invention provides an anti-fibrosis drug nano-preparation and its preparation method, which improves the efficiency of the nano-preparation reaching the deep alveoli.

Technical solution: In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is:

A nano preparation carrier, the nano preparation carrier includes Y-PEG-MAL and protease X modified with mucin degradation, the protease X modified with mucin degradation is connected to the hydrophilic end of Y-PEG-MAL to form Y -PEG-MAL-X;

Wherein, Y is selected from one or more of PLGA, PLA, PGA, PCL, PC, PS; protease X is selected from one or more of bromelain, papain, acid or alkaline protease, rennet .

Further, the nano-preparation carrier also includes drug V coordinated with metal ion W to form Y-PEG-MAL-X-W-V (PPV), composed of Y-PEG-MAL, protease X modified with mucin degradation and Composition of drug V coordinated with metal ion W;

Wherein W is a metal ion, selected from Mn ²⁺ , Zn ²⁺ , Fe ²⁺ , Fe ³⁺ , Cu ²⁺ , Mg ²⁺ , Ti ⁴⁺ , Zr ⁴⁺ , Co ²⁺ , Al ³⁺ , Ca ² One or more of ⁺ . More preferably, the metal ion W uses Fe ³⁺ as the coordination metal ion.

On the other hand, the present invention also provides a nano-preparation, wherein the nano-preparation carrier is used to load the hydrophobic anti-pulmonary fibrosis drug Z. More specifically, the nano preparations include:

Z-Y-PEG-MAL-X (PPZ), formed by Y-PEG-MAL, modified mucin-degrading protease X carrier carrying drug Z;

Z-Y-PEG-MAL-X-W-V (PPZV), formed by Y-PEG-MAL, protease X modified with mucin degradation and drug V entrapped drug Z coordinated by metal ion W.

The hydrophobic anti-pulmonary fibrosis drug Z is one or more of anti-inflammatory factor secretion or anti-fibrosis drug/drug for alleviating cell oxidative stress. Drugs or biologically active molecules, drugs for alleviating cellular oxidative stress are drugs or biologically active molecules that reduce the production of singlet oxygen, superoxide, and peroxides in cells. The hydrophobic anti-pulmonary fibrosis drug Z is selected from imatinib, gefitinib, icotinib, dasatinib, sunitinib, sorafenib, nilotinib, trametinib Naproxen, ibuprofen, naproxen, diclofenac, naproxen, ibuprofen, nimesulide, rofecoxib, celecoxib, nintedanib, pirfenidone, cortisone, and prednisone One or more of pine and cyclosporine.

The loaded anti-pulmonary fibrosis drugs can include multiple or one of anti-collagen production and lowering the secretion of inflammatory factors, and the antioxidant drugs in the complex can simultaneously include multiple or one of the drugs that reduce singlet oxygen and superoxide secretion. Antioxidants. The loaded anti-pulmonary fibrosis drug has a certain hydrophobicity, and the anti-pulmonary fibrosis drug with a certain hydrophobicity is loaded through the hydrophobic segment Y in the amphiphilic block copolymer, and the long circulation of the polymer micelles in the body is realized by PEG; The antioxidant drug in the complex has a certain degree of hydrophilicity, which can reduce mucus secretion by inhibiting the oxidative stress of goblet cells, thereby improving the effective delivery of nanoparticles. The modified protease X has the effect of degrading mucin, and achieves the purpose of efficient delivery to the lungs by avoiding being cleared by mucus. Through the deep delivery of antioxidant drugs into the lungs, anti-pulmonary fibrosis drugs can achieve the purpose of treating pulmonary fibrosis by inhibiting the expression of collagen and/or inhibiting the secretion of inflammatory factors at the site of lung injury.

More preferably, the drug V is metformin, and the hydrophobic anti-pulmonary fibrosis drug Z is pirfenidone. The anti-pulmonary fibrosis nano-preparation is loaded with pirfenidone through a hydrophobic block, and metformin is adsorbed through Fe ³⁺ coordination as a treatment system for pulmonary fibrosis. By synergistically regulating the pulmonary fibrosis microenvironment with dual drugs, it provides a new therapeutic strategy for the efficient delivery of anti-pulmonary fibrosis drugs.

Wherein, the loaded hydrophobic anti-pulmonary fibrosis drug may simultaneously include multiple drugs or active molecules that reduce the expression of inflammatory factors or anti-collagen production, or one selected from them. Antioxidants are drugs or biologically active molecules that relieve oxidative stress at the cellular level and reduce singlet oxygen and superoxide.

1) Antioxidants: Antioxidant active ingredients: metformin, glutathione, α-lipoic acid, carotenoids, ergothioneine;

2) Anti-collagen or inflammatory factor production drugs: imatinib, gefitinib, icotinib, dasatinib, sunitinib, sorafenib, nilotinib, trametinib, Ibuprofen, naproxen, diclofenac, naproxen, ibuprofen, nimesulide, rofecoxib, celecoxib, nintedanib, pirfenidone, cortisone, and prednisone , Cyclosporine.

The anti-pulmonary fibrosis drugs include alleviating oxidative stress at the cell level, reducing intracellular singlet oxygen or superoxide, and inhibiting the expression of inflammatory factors or collagen.

Specifically, the preparation method of Z-Y-PEG-MAL-X-W-V (PPZV):

Step (1) First mix the hydrophobic anti-pulmonary fibrosis drug Z and Y-PEG-MAL, and prepare anti-pulmonary fibrosis drug-loaded nanoparticles Z-Y-PEG-MAL by film dispersion method, direct titration method or reverse solvent method (PPZ);

Step (2) Mix metal ion W with drug V and protease X in a certain proportion to form metal ion W-mediated complex X-W-V, and combine nanoparticle Z-Y-PEG-MAL (PPZ) loaded with anti-pulmonary fibrosis drug with metal ion W Mediate complex X-W-V through the MAL reaction exposed by the sulfhydryl group on the protease X and the shell of the nanoparticle, and covalently modify the coordination compound containing the metal ion W at the outer end of the nanoparticle to form Z-Y-PEG-MAL-X-W-V; contribute to Nanoparticles efficiently penetrate the over-secreted mucus layer in the airway; the responsively released drug V can relieve the oxidative stress of goblet cells, reduce mucus secretion, and improve the delivery efficiency of nanoparticles; the drug delivered to the deep layer can play an anti-inflammatory role. The role of fibrosis in order to achieve the purpose of fibrosis treatment.

During the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to protease X is 5:1-10:1; more preferably 5:1.

During the reaction, the mass ratio of Y-PEG-MAL polymer nanoparticles added to anti-pulmonary fibrosis drug Z is 20:1-5:1; more preferably 10:1.

During the reaction, the mass ratio of Fe ³⁺ added to drug V is 40:1-10:1; more preferably 30:1.

Specifically, the preparation method of Z-Y-PEG-MAL-X (PPZ) is as follows:

Step (1) First mix the hydrophobic anti-pulmonary fibrosis drug Z and Y-PEG-MAL, and prepare anti-pulmonary fibrosis drug-loaded nanoparticles Z-Y-PEG-MAL by film dispersion method, direct titration method or reverse solvent method (PPZ);

Step (2) The nanoparticle Z-Y-PEG-MAL (PPZ) loaded with anti-pulmonary fibrosis drug was grafted with protease X, and the sulfhydryl group on the protease X reacted with the MAL exposed on the nanoparticle shell, and the outer surface of the nanoparticle Protease X is covalently modified at the end; the covalently modified protease X of the nanoparticle helps the nanoparticle to efficiently penetrate the over-secreted mucus layer in the airway; the drug delivered to the deep layer can exert anti-fibrosis effect to achieve fibrosis purpose of treatment.

During the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to protease X is 5:1-10:1; more preferably 5:1.

During the reaction, the mass ratio of Y-PEG-MAL polymer nanoparticles added to anti-pulmonary fibrosis drug Z is 20:1-5:1; more preferably 10:1.

The preparation method of Y-PEG-MAL-X-W-V (PPV) is as follows:

Step (1) First prepare nanoparticle PP by Y-PEG-MAL by film dispersion method, direct titration method or reverse solvent method;

Step (2) Metal ion W is mixed with drug V and protease X in a certain proportion to form metal ion W-mediated complex X-W-V, and nanoparticle PP and metal ion W-mediated complex X-W-V pass through the sulfhydryl group on protease X and nanoparticles The exposed MAL reaction of the outer shell covalently modifies the coordination compound containing metal ion W on the outer end of the nanoparticle to form Y-PEG-MAL-X-W-V; it helps the nanoparticle to efficiently penetrate the over-secreted mucus layer in the airway; Responsively released drug V could alleviate the oxidative stress of goblet cells, reduce mucus secretion, and improve the delivery efficiency of nanoparticles.

During the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to protease X is 5:1-10:1; more preferably 5:1.

During the reaction, the mass ratio of metal ion W and drug V added is 40:1-10:1; more preferably 30:1.

As a control, the preparation method of PP/drug is as follows:

Step (1) First, dissolve the anti-pulmonary fibrosis drug (pirfenidone) in DMSO, the ratio of Y-PEG-MAL to the drug is 100:1~5:1, the more preferred solution is 10:1, and the encapsulation rate is 71.6%, and the drug loading is 5.4%.

Step (2) mix with Y-PEG-MAL, and prepare nanoparticles loaded with anti-pulmonary fibrosis drugs by film dispersion method, direct titration method or reverse solvent method.

As a preferred solution, the anti-pulmonary fibrosis drug nano-preparation is Y-PEG-MAL, wherein the molecular weight range of Y 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 amount of the anti-pulmonary fibrosis drug nano-preparation is 5-25%, and the particle size is 20 nm-500 nm.

The present invention requires the application of the nano-preparation carrier and anti-pulmonary fibrosis drug nano-preparation in the preparation of drugs for treating pulmonary fibrosis.

The invention provides an enzyme-mediated response type high-efficiency delivery of therapeutic drugs to achieve the purpose of treating pulmonary fibrosis. The characteristic is that the carrier includes chemical drug loading components and airway responsive degradation and effective delivery components. The chemical drug loading component is an amphiphilic polymer with a maleimide modification at the end, and the responsive degraded mucin component is a sulfhydrylated polypeptide.

The present invention has a mucin-degrading protease-modified nano-preparation Y-PEG-MAL, wherein Y is selected from PLGA, PLA, PGA, PCL, PC and PS. The loading of anti-fibrosis drugs with certain hydrophobicity is realized through the hydrophobic segment in the amphiphilic block copolymer, and the loading of antioxidant drugs is realized through coordination. Overcoming the mucus layer barrier in the airway through mucin-degrading proteases further improves the efficiency of nano-preparations reaching the deep alveoli, providing a new therapeutic strategy for the efficient delivery of anti-pulmonary fibrosis drugs.

Beneficial effects: the present invention provides an enzyme-mediated response-type high-efficiency delivery of therapeutic drugs to achieve the purpose of treating pulmonary fibrosis. Compared with the prior art, it has the following advantages: Y-PEG-MAL in the carrier, wherein Y is Hydrophobic segment, can realize the entrapment of fat-soluble drugs very well, the PEG in the carrier can prolong the circulation time of the carrier in the blood, the metal ion W in the modified carrier in the carrier is in the mucus slightly acidic protease X in the mucin high Under the condition of expression, drug V can be released responsively, and the delivery efficiency of nano-preparation can be improved by regulating the oxidative stress of goblet cells to reduce mucus secretion, and the drugs expressed by anti-fibrosis or anti-inflammatory factors in the deep lung can be passed Reducing the expression of inflammatory factors or inhibiting collagen secretion can achieve the purpose of treating pulmonary fibrosis. The application of this carrier can realize the efficient pulmonary delivery of nano-drugs, and the dual responsiveness of enzyme response and acid response can realize the efficient release of anti-oxidative stress drugs, and the nanoparticles reaching the deep alveoli can effectively play a role in resisting the progress of pulmonary fibrosis . The present invention utilizes the highly secreted mucin microenvironment in the trachea of pulmonary fibrosis to efficiently deliver the therapeutic drug to the injury site of pulmonary fibrosis through responsive release nano-preparation, and at the same time utilizes the anti-pulmonary fibrosis drug Z to inhibit the progression of fibrosis, and provides anti-pulmonary Efficient delivery of fibrosis drugs and drug design for effective treatment of pulmonary fibrosis provide a new approach and strategy. The present invention modifies the nano-carrier Y-PEG-MAL with protease X to carry the drug Z, and the nano-preparation and the carrier of the metal ion W to coordinate the drug V. Firstly, the mucus secretion in the trachea is significantly increased during the occurrence of pulmonary fibrosis. , the nano-preparation reaches the mucus layer through the tracheal administration, and the therapeutic drug is efficiently delivered to the damaged site of pulmonary fibrosis through the responsive release of the nano-preparation, avoiding being cleared by the mucus in the trachea. The metal ion WV dissociates from the nano-preparation PPZ under slightly acidic mucus conditions, releasing the drug V, which improves the delivery efficiency of the nano-preparation by regulating goblet cell oxidative stress to reduce mucus secretion, and releases the nano-preparation PPZ exposed to protease X. , under the condition of high expression of mucin in the airway mucus layer, it can quickly pass through the airway mucus layer to reach the deep lung, and improve the therapeutic effect of anti-fibrosis drugs. At present, the use of hydrophobic blocks in amphiphilic block copolymers to load hydrophobic drugs for delivery is one of the common methods, PLGA (polylactic acid-polyglycolic acid), PLA (polylactic acid), PGA (polyglycolide) , PCL (polycaprolactone) is a common hydrophobic block with good biocompatibility, often used as a hydrophobic core in amphiphilic block copolymers, and has a good affinity for most hydrophobic drugs. At the same time, this article uses the coordination method to entrap antioxidants. The complexes have good stability under normal physiological conditions, and can achieve the purpose of responsive release under the slightly acidic conditions of the mucus layer.

In the present invention, Y-PEG (X is a hydrophobic segment, X is PLGA, PLA, PGA, PCL) nanoparticles modified by protease X is used to achieve a certain hydrophobicity through the hydrophobic segment in the amphiphilic block copolymer. Double-drug/single-drug loading, the long circulation of polymer micelles in the body can be realized by PEG, and/or, the nano-preparation of metal ion W coordination drug V and its carrier can be released in response to the mucus layer, so as to realize the Efficient delivery.

Description of drawings

Fig. 1 is the schematic flow sheet of embodiment nano-preparation preparation;

Fig. 2 is the best charging ratio of each composition of the prepared nano-preparation in the embodiment;

Fig. 3 is the nano-preparation particle diameter figure of each preparation of embodiment;

Fig. 4 is the nano-preparation potential diagram of each preparation of embodiment;

Fig. 5 is the transmission electron micrograph of mucin (papain) involved in the embodiment;

Fig. 6 is the transmission electron micrograph that protease can specifically degrade papain in the preparation of the embodiment;

Fig. 7 is the nanoparticle size diagram after the nano-preparation prepared in the embodiment is released in response to different time points under enzyme-sensitive conditions;

The nanoparticle size diagram of the nano-preparation prepared in the embodiment of Fig. 8 after responsive release at different time points under acid-sensitive conditions;

Fig. 9 is the nanoparticle size chart of the nano-preparation prepared in the embodiment after the responsive release at different time points under the condition of simultaneous presence of acid and enzyme;

Fig. 10 is an analysis of the effects of different nano-preparations on reversing pulmonary fibrosis at the in vivo level of the nano-carriers prepared in the example.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1 Synthesis and preparation of nano-preparation components, as shown in Figure 1 and Figure 2:

1. Preparation of double-drug/single-drug PLGA-PEG-MAL nanoparticles

In the present invention, the film dispersion method is preferably used to prepare the PLGA-PEG-MAL nanoparticles loaded with anti-pulmonary fibrosis drug Z. The specific preparation method is as follows:

Weigh 100 mg PLGA-PEG-MAL and 10 mg anti-pulmonary fibrosis drug Z, respectively, and dissolve them in 2 mL ethyl acetate solution, and ultrasonically dissolve. Under stirring conditions, the ethyl acetate solution was spin-dried by a rotary evaporator, then 3 mL of distilled water was added for ultrasonic dissolution for 30 min, and the unencapsulated free drug was removed by centrifugation at 2500 rpm/min for 20 min. Nanoparticles were concentrated to a volume of 500 μL using ultrafiltration tubes with a molecular weight of 10,000.

Antioxidant anti-fibrotic drugs:

1) Antioxidants: Antioxidant active ingredients: metformin, glutathione, α-lipoic acid, carotenoids, ergothioneine;

2) Anti-collagen or inflammatory factor production drugs: imatinib, gefitinib, icotinib, dasatinib, sunitinib, sorafenib, nilotinib, trametinib, Ibuprofen, naproxen, diclofenac, naproxen, ibuprofen, nimesulide, rofecoxib, celecoxib, nintedanib, pirfenidone, cortisone, and prednisone , Cyclosporine.

(1) Preparation of polymer nanoparticles with complex modification mediated by papain and metal ion W

1. Preparation of polymer nanoparticles with complex modification mediated by papain and Fe ³⁺

Mix 30 mg of metal ion Fe ³⁺ with 2 mg of metformin and 30 mg of papain in distilled water overnight, and pass the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Fe ³⁺ mediated complex through thiol and nanoparticles In the MAL reaction with the shell exposed, the enzyme is covalently bound to the coordination compound of Fe ³⁺ at the outer end of the nanoparticle; centrifuge at 10,000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove unreacted sulfhydrylated protease Z.

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Fe ³⁺ mediated complex) and PPZV/(containing Pirfenidone, papain and complexes mediated by Fe ³⁺ ) with a drug loading of 5%-25% and a particle size of 50 nm-500 nm. The particle size distribution of the nano-preparation of the preparation in this example (PPZV/(contains pirfenidone, papain and Fe3 ⁺ -mediated complex at the same time)) is shown in Figure 3 and Figure 4. The particle size distribution of the nano-preparation Uniform, homogeneous shape, obvious core-shell structure.

2. Preparation of polymer nanoparticles with complex modification mediated by papain and Mn ²⁺

Mix 30 mg of metal ion Mn ²⁺ with 2 mg of metformin and 30 mg of papain in distilled water overnight, and pass the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Mn ²⁺ mediated complex through sulfhydryl and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticle is covalently bound to the Mn2+ coordination compound; centrifugation at 10000 rpm/min is used to resuspend three times, the prepared nanoparticle is collected, and the unreacted sulfhydrylated papain is removed.

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Mn ²⁺ mediated complex) and PPZV/(containing Pirfenidone, papain and complexes mediated by Mn ²⁺ ) have a drug loading of 5%-25%, and a particle size of 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Mn ²⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

3. Preparation of polymer nanoparticles with complex modification mediated by papain and Zn ²⁺

Mix metal ion Zn ²⁺ 30 mg with metformin 2 mg and papain 30 mg in distilled water overnight, and the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Zn ²⁺ mediated complex through thiol and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticles is covalently bound to the Zn ²⁺ coordination compound; centrifuge at 10,000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove the unreacted sulfhydrylated papain .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Zn ²⁺ mediated complex) and PPZV/(containing both Pirfenidone, papain and complexes mediated by Zn ²⁺ ) have a drug loading of 5%-25% and a particle size of 50 nm-500 nm. The particle size distribution of the nano-preparation of the preparation (PPZV/(also containing pirfenidone, papain and Zn ²⁺ mediated complex)) in this example has uniform particle size distribution and uniform shape.

4. Preparation of polymer nanoparticles with complex modification mediated by papain and Fe ³⁺

Mix 30 mg of metal ion Fe ³⁺ with 2 mg of metformin and 30 mg of papain in distilled water overnight, and pass the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Fe ³⁺ mediated complex through thiol and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticles is covalently bound to the coordination compound of Fe ³⁺ ; centrifuged at 10,000 rpm/min to resuspend three times, the prepared nanoparticles are collected, and the unreacted sulfhydrylated papain is removed .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Fe ³⁺ mediated complex) and PPZV/(containing Pirfenidone, papain and complexes mediated by Fe ³⁺ ) with a drug loading of 5%-25% and a particle size of 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Fe ³⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

5. Preparation of polymer nanoparticles with complex modification mediated by papain and Cu ²⁺

Mix metal ion Cu ²⁺ 30 mg with metformin 2 mg and papain 30 mg in distilled water overnight, and the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Cu ²⁺ mediated complex pass through thiol and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticle is covalently bound to the coordination compound of Cu ²⁺ ; centrifuge at 10,000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove the unreacted sulfhydrylated papain .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Cu ²⁺ mediated complex) and PPZV/(containing both Pirfenidone, papain and complexes mediated by Cu ²⁺ ) have a drug loading of 5%-25% and a particle size of 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Cu ²⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

6. Preparation of polymer nanoparticles with complex modification mediated by papain and Mg ²⁺

Mix metal ion Mg ²⁺ 30 mg with metformin 2 mg and papain 30 mg in distilled water overnight, and the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Mg ²⁺ mediated complex through sulfhydryl and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticles is covalently bound to the coordination compound of Mg ²⁺ ; the prepared nanoparticles are collected by centrifugation at 10,000 rpm/min for three times, and the unreacted sulfhydrylated papain is removed .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Mg ²⁺ mediated complex) and PPZV/(containing both The drug loading of pirfenidone, papain and Mg ²⁺ mediated complex) is 5%-25%, and the particle size is 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Mg ²⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

7. Preparation of polymer nanoparticles with complex modification mediated by papain and Si ²⁺

Mix metal ion Si ²⁺ 30 mg with metformin 2 mg and papain 30 mg in distilled water overnight, and the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Si ²⁺ mediate the complex through thiol and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticles is covalently bound to the Si ²⁺ coordination compound; centrifuge at 10,000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove the unreacted thiolated papain .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Si ²⁺ mediated complex) and PPZV/(containing The drug loading of pirfenidone, papain and Si ²⁺ mediated complex) is 5%-25%, and the particle size is 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Si ²⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

8. Preparation of polymer nanoparticles with complex modification mediated by papain and Co ²⁺

Mix 30 mg of metal ion Co ²⁺ with 2 mg of metformin and 30 mg of papain in distilled water overnight, and pass the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Co ²⁺ mediated complex through thiol and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticles is covalently bound to the Co ²⁺ coordination compound; centrifuge at 10,000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove the unreacted thiolated papain .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Co2 ⁺ -mediated complex) and PPZV/(containing The drug loading of pirfenidone, papain and Co ²⁺ mediated complex) is 5%-25%, and the particle size is 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Co ²⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

9. Preparation of polymer nanoparticles with complex modification mediated by papain and Al ³⁺

Mix 30 mg of metal ion Al ³⁺ with 2 mg of metformin and 30 mg of papain in distilled water overnight, and pass the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Al ³⁺ mediated complex through sulfhydryl and nanoparticles In the reaction of MAL exposed shell, the papain in the nanoparticle is covalently combined with the Al ³⁺ coordination compound; centrifuge at 10000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove the unreacted thiolated papain .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Al ³⁺ mediated complex) and PPZV/(containing The drug loading of pirfenidone, papain and Al ³⁺ mediated complex) is 5%-25%, and the particle size is 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Al ³⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

10. Preparation of polymer nanoparticles with complex modification mediated by papain and Ca ²⁺

Mix 30 mg of metal ion Ca ²⁺ with 2 mg of metformin and 30 mg of papain in distilled water overnight, and pass the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and Ca ²⁺ mediated complex through thiol and nanoparticles In the MAL reaction with the shell exposed, the papain in the nanoparticles is covalently bound to the Ca ²⁺ coordination compound; centrifuge at 10,000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove the unreacted thiolated papain .

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Ca2 ⁺ -mediated complex) and PPZV/(containing The drug loading of pirfenidone, papain and Ca ²⁺ mediated complex) is 5%-25%, and the particle size is 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Ca ²⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

(2) Preparation of polymer nanoparticles with complex modification mediated by protease X and Fe ³⁺

1. Preparation of polymer nanoparticles with complex modification mediated by papain and Fe ³⁺

Mix 30 mg of Fe ³⁺ with 2 mg of metformin and 30 mg of papain in distilled water overnight, and expose the nanoparticles loaded with the anti-pulmonary fibrosis drug pirfenidone and the Fe ³⁺ mediated complex through the sulfhydryl group and the shell of the nanoparticle In the MAL reaction, the papain in the nanoparticles was covalently combined with the coordination compound of Fe ³⁺ ; the nanoparticles were collected by centrifugation at 10,000 rpm/min for three times, and the unreacted sulfhydrylated papain was removed.

PP/(pirfenidone), PPZ/(containing papain and pirfenidone), PPV/(containing papain and Fe ³⁺ mediated complex) and PPZV/(containing Pirfenidone, papain and complexes mediated by Fe ³⁺ ) with a drug loading of 5%-25% and a particle size of 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, papain and Fe ³⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

2. Preparation of polymer nanoparticles with acid/alkaline protease and Fe ³⁺ -mediated complex modification

Mix 30 mg of Fe ³⁺ with 2 mg of metformin and 30 mg of acid/alkaline protease in distilled water overnight, and the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone and the Fe ³⁺ mediated complex pass through the In the reaction of MAL exposed shell, the acid/alkaline protease in the nanoparticle is covalently combined with the coordination compound of Fe ³⁺ ; centrifuge at 10,000 rpm/min to resuspend three times, collect the prepared nanoparticles, and remove unreacted thiolated Acid/alkaline protease.

PP/(pirfenidone), PPZ/(containing acid/alkaline protease and pirfenidone), PPV/(containing acid/alkaline protease and Fe ³⁺ mediated complex) and PPZV prepared by this method / (simultaneously containing pirfenidone, acid/alkaline protease and Fe ³⁺ mediated complex) the drug loading is 5%-25%, and the particle size is 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, acid/alkaline protease and Fe ³⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

3. Preparation of polymer nanoparticles with complex modification mediated by bromelain and Fe ³⁺

Mix 30 mg of Fe ³⁺ with 2 mg of metformin and 30 mg of bromelain in distilled water overnight, and expose the nanoparticles loaded with the anti-pulmonary fibrosis drug pirfenidone and the Fe ³⁺ mediated complex through the sulfhydryl group and the shell of the nanoparticle In the MAL reaction, the bromelain in the nanoparticles was covalently combined with the coordination compound of Fe ³⁺ ; the nanoparticles were collected by centrifugation at 10,000 rpm/min for three times, and the unreacted sulfhydryl bromelain was removed.

PP/(pirfenidone), PPZ/(containing bromelain and pirfenidone), PPV/(containing bromelain and Fe ³⁺ mediated complex) and PPZV/(containing Pirfenidone, Bromelain and Fe ³⁺ mediated complexes) have a drug loading of 5%-25% and a particle size of 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, bromelain and Fe ³⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

4. Preparation of polymer nanoparticles with complex modification mediated by chymosin and Fe ³⁺

Mix 30 mg of Fe ³⁺ with 2 mg of metformin and 30 mg of chymosin in distilled water overnight, and the nanoparticles loaded with the anti-pulmonary fibrosis drug pirfenidone and the Fe ³⁺ mediated complex pass through the sulfhydryl group and the shell of the nanoparticle In the exposed MAL reaction, the chymosin in the nanoparticles is covalently combined with the coordination compound of Fe ³⁺ ; the prepared nanoparticles are collected by centrifugation at 10000 rpm/min for three times, and the unreacted thiolated curd is removed enzyme.

PP/(pirfenidone), PPZ/(containing rennet and pirfenidone), PPV/(containing rennet and Fe ³⁺ mediated complex) and PPZV/( At the same time, it contains pirfenidone, chymosin and Fe ³⁺ mediated complex), the drug loading is 5%-25%, and the particle size is 50 nm-500 nm. The particle size distribution of the nano-preparation (PPZV/(also containing pirfenidone, chymosin and Fe ³⁺ mediated complex)) of the preparation in this example has uniform particle size distribution and uniform shape.

Example 2 Papain can specifically degrade papain

According to Example 1, papain, an analog of mucin in the mucus layer, was taken as the research object, and different concentrations of protease were added to act for 24 hours, and the morphological changes of papain under the transmission electron microscope were photographed by transmission electron microscopy (TEM). The results of transmission electron microscopy showed that papain could be degraded by mucin after interacting with mucin for 24 h, as shown in Figure 5 and Figure 6.

Example 3 Determination of Responsive Release of Nano-Preparations under Slightly Acid and Enzyme Sensitive Conditions

Prepare PP/(pirfenidone), PPZ/(containing protease X and pirfenidone), PPV/(containing protease X and Fe ³⁺ mediated complex) and PPZV/(simultaneously) as described in Example 1 Nanoformulation containing pirfenidone, protease X and Fe ³⁺ mediated complex). After the nano-preparation was concentrated to 200 μL, 1 mL of PBS with pH 6.8 was added, and then placed at 4 ºC for 24 h, the particle size and potential of the nanoparticles in different environments were characterized by a particle size analyzer.

(1) After measuring the particle size with the Malvern Panalytical Visible Particle Size Analyzer, the particle size is inspected again after the action of micro-acid and enzyme, and the particle size change is observed;

The particle size data measured in this embodiment are shown in FIG. 7 , FIG. 8 and FIG. 9 , and the particle size change of the nanoparticles was detected before and after stimulation. Before microacid environment and enzyme-responsive stimulation, the particle size distribution of nanoparticles was roughly similar. After microacid and enzyme stimulation, nanoparticles released smaller nanoparticles through microenvironment responsiveness, and Fe ³⁺ mediated The complexes are released responsively into the mucus layer, while smaller nanoparticles can reach deep lungs through osmosis.

The potential changes of the nanoparticles were detected before and after stimulation. Before the slight acidic environment and enzyme-responsive stimulation, the potential of the complex nanoparticles containing Fe ³⁺ should be positive or low. Smaller nanoparticles, while Fe ³⁺ mediated complexes are released into the mucus layer responsively, while smaller nanoparticles are mainly negatively charged. Other studies have also proved that the nanoparticles formed by PLGA-PEG self-assembly are mainly The negative charge further shows that the constructed system can achieve the purpose of responsive release under the dual conditions of slight acidity and high enzyme expression.

Example 4 Analysis of the effects of nano-preparations PP, PPZ, PPV, and PPZV on reversing pulmonary fibrosis after treatment.

According to the preparation method described in Example 1, nano-preparations PP, PPZ, PPV, and PPZV were prepared. Z is pirfenidone, and V is metformin.

First, 6-8 week-old male C57BL/6 mice were used for the modeling experiment of the pulmonary fibrosis model, and the lungs of the mice were directly modeled by tracheal intubation. Bleomycin hydrochloride was used as an inducer of pulmonary fibrosis in mice during modeling, and the concentration was 2 USP/Kg. Three weeks later, when the pulmonary fibrosis of mice was formed, the nano-preparation PP, PPZ, PPV, and PPZV were administered after treatment. Analysis of the effect of reversing pulmonary fibrosis. The mice that had been modeled for three weeks were randomly assigned to 5 groups, 5 in each group, and the nano-preparation PP, PPZ, PPV, PPZV and normal saline were injected respectively through the tail vein, and the injected drug Z was 2 μg/g mouse body weight as standard. After 4 weeks of treatment, the effect of different nano-preparations on reversing pulmonary fibrosis was analyzed by H&E staining to analyze the anti-pulmonary fibrosis effect of different administration groups. From the results of H&E staining, it can be seen that compared with the BLM group, the final preparation PPZV group has significantly narrowed alveolar spacing, and the alveolar wall is uniform in thickness, and the degree of pulmonary fibrosis is significantly weakened, which can achieve the purpose of treating pulmonary fibrosis, indicating that by mediating circulation The nano-preparation delivery system of fibroblasts can achieve efficient lung targeting and double-drug combination therapy can effectively reverse pulmonary fibrosis. The anti-fibrosis effects of each preparation group were compared PPZV>PPZ>PPV>PP, and the results are shown in Figure 10.

PLGA (polylactic acid-polyglycolic acid), PLA (polylactic acid), PGA (polyglycolide), PCL (polycaprolactone) are common hydrophobic blocks with good biocompatibility, often in amphiphilic block As a hydrophobic core in the block copolymer, it has a good affinity for most drugs with certain hydrophobicity. In the above-mentioned examples, by using PLA-PEG-MAL, PGA-PEG-MAL, and PCL-PEG-MAL block copolymers instead of PLGA-PEG-MAL block copolymers, drugs with certain hydrophobicity can also be loaded to form The purpose of polymer micelles is clear to those skilled in the art.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also possible. It should be regarded as the protection scope of the present invention.

Claims (10)

1. a kind of nano-preparations carrier, which is characterized in that the nano-preparations carrier includes Y-PEG-MAL and is modified with mucin The protease X of degradation, the water-wet side that the protease X for being modified with mucin degradation is connected to Y-PEG-MAL form Y-PEG- MAL-X；

Wherein, Y is selected from one or more of PLGA, PLA, PGA, PCL, PC, PS；Protease X is selected from bromelain, wood One or more of melon protease, acidity or alkali protease, renin.

2. nano-preparations carrier according to claim 1, which is characterized in that further include the drug with metal ion W coordination V, formed Y-PEG-MAL-X-W-V, by Y-PEG-MAL, be modified with mucin degradation protease X and metal ion W coordination Drug V composition；

Wherein W is metal ion, is selected from Mn²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, Mg²⁺, Ti⁴⁺, Zr⁴⁺, Co²⁺, Al³⁺, Ca²⁺One or more of；The drug V is selected from melbine, glutathione, alpha-lipoic acid, carotenoid, ergot sulphur Cause.

3. a kind of nanometer formulation, which is characterized in that anti-using nano-preparations carrier of any of claims 1 or 2 load hydrophobicity Pulmonary fibrosis medicine Z.

4. nanometer formulation according to claim 3 characterized by comprising

Z-Y-PEG-MAL-X, by Y-PEG-MAL, be modified with mucin degradation protease X carrier contain drug it is Z-shaped at；

Z-Y-PEG-MAL-X-W-V, by Y-PEG-MAL, be modified with mucin degradation protease X and metal ion W coordination Drug V contain drug it is Z-shaped at.

5. nanometer formulation according to claim 3, which is characterized in that the hydrophobicity anti-fibrosis drug Z is anti-inflammatory The drug Z/ of inflammation factor secretion or anti-fibrosis alleviates one or more of cellular oxidation stress medicine, and anti -inflammatory cytokine is drop Low cell secretion inflammatory factor and inhibit collagenogenic drug or bioactive molecule, alleviate cellular oxidation stress drug be Reduce the drug or bioactive molecule of the generations such as cell singlet oxygen, superoxides and peroxide；

The hydrophobicity anti-fibrosis drug Z is replaced selected from Imatinib, Gefitinib, Conmana, Dasatinib, Buddhist nun of relaxing Buddhist nun, Sorafenib, nilotinib, Trimetinib, brufen, naproxen, Diclofenac, Nabumetone, brufen, aulin, One or more of rofecoxib, celecoxib, Nintedanib, pirfenidone, cortisone and prednisone, cyclosporin.

6. anti-fibrosis drug nanometer formulation according to claim 4, which is characterized in that Z-Y-PEG-MAL-X-W-V Preparation method:

Step (1) first mixes hydrophobicity anti-fibrosis drug Z with Y-PEG-MAL, passes through film dispersion method, direct titration The nanoparticle Z-Y-PEG-MAL of method or reversed solvent method preparation load anti-fibrosis drug；

Metal ion W and drug V and protease X are mixed in a certain ratio to form metal ion W mediation complex X- by step (2) The nanoparticle Z-Y-PEG-MAL for loading anti-fibrosis drug and metal ion W is mediated complex X-W-V to pass through albumen by W-V The MAL reaction of sulfydryl and the exposure of nanoparticle shell on enzyme X, contains matching for metal ion W in the outer end covalent modification of nanoparticle Position compound, forms Z-Y-PEG-MAL-X-W-V；Facilitate nanoparticle and efficiently penetrates rete malpighii in tracheae by excessive secretion； The drug V of responsiveness release can alleviate the oxidative stress of goblet cell, reduce the secretion of mucus, improve the delivering effect of nanoparticle Rate；The drug for being delivered to deep layer can play the effect of anti-fibrosis to achieve the purpose that treatment of fibrosis.

7. anti-fibrosis drug nanometer formulation according to claim 4, which is characterized in that the system of Z-Y-PEG-MAL-X Preparation Method is as follows:

Step (1) first mixes hydrophobicity anti-fibrosis drug Z with Y-PEG-MAL, passes through film dispersion method, direct titration The nanoparticle Z-Y-PEG-MAL of method or reversed solvent method preparation load anti-fibrosis drug；

The nanoparticle Z-Y-PEG-MAL and protease X that load anti-fibrosis drug are carried out graft reaction by step (2), are passed through The MAL reaction of sulfydryl and the exposure of nanoparticle shell on protease X, in the outer end covalent modification protease X of nanoparticle；Nanoparticle By the protease X of covalent modification, facilitates nanoparticle and efficiently penetrate rete malpighii in tracheae by excessive secretion；It is delivered to deep layer Drug can play the effect of anti-fibrosis to achieving the purpose that treatment of fibrosis.

8. anti-fibrosis drug nanometer formulation according to claim 2, which is characterized in that Y-PEG-MAL-X-W-V's The preparation method is as follows:

Y-PEG-MAL is prepared nanoparticle PP by film dispersion method, direct titrimetric method or reversed solvent method first by step (1)；

Metal ion W and drug V and protease X are mixed in a certain ratio to form metal ion W mediation complex X- by step (2) Nanoparticle PP and metal ion W are mediated complex X-W-V to pass through sulfydryl on protease X and the exposure of nanoparticle shell by W-V MAL reaction contains the complex of metal ion W in the outer end covalent modification of nanoparticle, forms Y-PEG-MAL-X-W-V； Facilitate nanoparticle and efficiently penetrates rete malpighii in tracheae by excessive secretion；It is thin that the drug V of responsiveness release can alleviate cup-shaped The oxidative stress of born of the same parents reduces the secretion of mucus, improves the delivery efficiency of nanoparticle.

9. according to the described in any item anti-fibrosis drug nanometer formulations of claim 3-8, which is characterized in that Y-PEG-MAL In, wherein the molecular weight ranges of Y are 1000-50000, and the molecular weight ranges of PEG are 200-10000；

And/or the drugloading rate of the anti-fibrosis drug nanometer formulation, in 5-25%, particle size is in 20 nm-500 nm.

10. the described in any item nano-preparations carriers of claim 1-2, the described in any item pulmonary fibrosis resistants of claim 3-8 Application of the medicament nano-preparation in preparation treatment pulmonary fibrosis disease drug.