CN110384681B - Nanometer preparation for pulmonary fibrosis and preparation method thereof - Google Patents

Nanometer preparation for pulmonary fibrosis and preparation method thereof Download PDF

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CN110384681B
CN110384681B CN201910688203.7A CN201910688203A CN110384681B CN 110384681 B CN110384681 B CN 110384681B CN 201910688203 A CN201910688203 A CN 201910688203A CN 110384681 B CN110384681 B CN 110384681B
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pulmonary fibrosis
protease
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姜虎林
常鑫
邢磊
林伊君
李玲
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China Pharmaceutical University
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Abstract

The invention discloses a nano preparation for pulmonary fibrosis and a preparation method thereof, namely a protease modified nano preparation with mucin degradation and a carrier thereof. The nanometer preparation is released in a response manner by means of the characteristic that the protease degrades the mucin, so that the therapeutic drug is delivered to the damaged part of the pulmonary fibrosis efficiently, the anti-pulmonary fibrosis drug is delivered in a highly efficient targeted manner, and the aim of highly efficiently treating the pulmonary fibrosis is fulfilled. Aiming at the problem that the deep delivery is difficult in the trachea and inhalation administration dosage forms on the basis of the market at present, namely, mucus is excessively secreted by goblet cells in the airways to cause that a large amount of medicaments are difficult to deliver to the deep part of the alveolus to play a role, the change of the excessive secretion of the mucus in the microenvironment of the pulmonary fibrosis airways is provided, the nano preparation which is released in a response mode and can efficiently pass through the airway barrier is constructed, the therapeutic medicaments are efficiently delivered to the pulmonary fibrosis damage part in the deep part of the alveolus, and the efficiency of the nano preparation reaching the deep part of the alveolu.

Description

Nanometer preparation for pulmonary fibrosis and preparation method thereof
Technical Field
The invention discloses a nano preparation for pulmonary fibrosis and a preparation method thereof, belonging to the technical field of medicines.
Background
Idiopathic Pulmonary Fibrosis (IPF) is one of the severe and common forms of Interstitial Lung Disease (ILD). ILD is a progressive lung disease that causes pulmonary fibrosis as the disease progresses to a terminal stage. During the course of the disease, damaged alveolar epithelial cells secrete a large amount of inflammatory factors, which activate fibroblast hyperproliferation and transformation to myofibroblasts, resulting in excessive deposition of extracellular matrix (ECM), destruction of lung parenchyma, and finally death of patients due to respiratory failure. Although the novel anti-fibrotic drug Pirfenidone (PFD) has been approved for clinical treatment in countries such as europe, certain problems still remain. Since pirfenidone is commonly used as an oral formulation for treating IPF, its bioavailability is low and its administration in large amounts may cause side effects and even toxicity to other organs. Therefore, the proposed strategy of delivering drugs to the lungs by inhalation to achieve efficient delivery of the drugs to the lungs provides new ideas and methods for IPF treatment.
In the form of inhaled administration, the efficiency of drug delivery is generally low due to the limitation of the physiological structure of the airways. The mucus layer in the airways is the first barrier that must be overcome for tracheal or inhalational administration. The mucus layer is mainly composed of dense reticular mucin fibers, and mucin is a negatively charged biomacromolecule containing high-density glycan. In patients with advanced obstructive pulmonary disease and fibrosis, mucus secreted by airway goblet cells serves as part of the clearance system, trapping drugs that reach the airway by steric hindrance or adhesion and subsequently clearing them from the mucus, resulting in a reduction in the amount of drug reaching the deep lung, resulting in a reduction in the therapeutic effect of the drug on the disease. Even though the drug may pass through the mucus layer, the alveoli still present another barrier, the macrophage phagocytic system in the lipid layer, which is a protective defense barrier for type II alveolar epithelial cells (AT II). A large number of drugs are engulfed by macrophages in the lipid layer and are not therapeutically effective.
Therefore, for drugs to be effective deep in the lung, these obstacles must be effectively overcome during delivery to achieve the desired therapeutic effect.
Disclosure of Invention
The purpose is as follows: in order to overcome the defects in the prior art, the invention provides an anti-fibrosis drug nano preparation and a preparation method thereof, and the efficiency of the nano preparation reaching the deep part of alveolus is improved.
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 comprises Y-PEG-MAL and protease X modified with mucin degradation, wherein the protease X modified with mucin degradation is connected with the hydrophilic end of the Y-PEG-MAL to form Y-PEG-MAL-X;
wherein Y is selected from one or more of PLGA, PLA, PGA, PCL, PC and PS; the protease X is selected from one or more of bromelain, papain, acidic or alkaline protease, and chymosin.
Furthermore, the nano preparation carrier also comprises a medicine V coordinated with the metal ion W to form Y-PEG-MAL-X-W-V (PPV), and the Y-PEG-MAL, the protease X modified with mucin degradation and the medicine V coordinated with the metal ion W form the nano preparation carrier;
wherein W is a metal ion selected from Mn2+, Zn2+, Fe2+, Fe3+, Cu2+, Mg2+，Ti4+, Zr4+, Co2+,Al3+，Ca2+One or more of them. More preferably, Fe is used as the metal ion W3+As a coordinating metal ion.
On the other hand, the invention also provides a nano preparation, and the nano preparation carrier is adopted to load the hydrophobic anti-pulmonary fibrosis drug Z. More specifically, the nano preparation comprises:
Z-Y-PEG-MAL-X (PPZ) is formed by Y-PEG-MAL and a medicament Z coated by a modified mucin-degraded protease X carrier;
Z-Y-PEG-MAL-X-W-V (PPZV) is formed by encapsulating a drug Z by Y-PEG-MAL, a medicament V modified with mucin degradation protease X and coordinated with metal ions W.
The hydrophobic anti-pulmonary fibrosis drug Z is one or more of anti-inflammatory factor secretion or anti-fibrosis drugs/drugs for relieving cell oxidative stress, the anti-inflammatory factor is a drug or bioactive molecule for reducing cell secretion inflammatory factors and inhibiting collagen generation, and the drug for relieving cell oxidative stress is a drug or bioactive molecule for reducing cell singlet oxygen, superoxide, peroxide and the like. The hydrophobic anti-pulmonary fibrosis drug Z is selected from one or more of imatinib, gefitinib, erlotinib, dasatinib, sunitinib, sorafenib, nilotinib, trametinib, ibuprofen, naproxen, diclofenac, naproxone, ibuprofen, nimesulide, rofecoxib, celecoxib, nintedanib, pirfenidone, cortisone, prednisone and cyclosporine.
The loaded anti-pulmonary fibrosis drug can simultaneously comprise more than one or one of anti-collagen generation and inflammatory factor secretion reduction, and the anti-oxidation drug in the complex can simultaneously comprise more than one or one antioxidant for reducing singlet oxygen and superoxide secretion. The loaded anti-pulmonary fibrosis drug has certain hydrophobicity, the anti-pulmonary fibrosis drug with certain hydrophobicity is loaded through the hydrophobic section Y in the amphiphilic block copolymer, and long circulation of the polymer micelle in vivo is realized through PEG; the antioxidant medicine in the complex has certain hydrophilicity, and the purpose of reducing mucus secretion is achieved by inhibiting oxidative stress of goblet cells, so that the effective delivery of the nanoparticles is improved. The modified protease X has the function of degrading mucin, and the aim of efficiently delivering the mucin in the lung is fulfilled by avoiding being cleared by mucus. The anti-pulmonary fibrosis drug achieves the purpose of treating pulmonary fibrosis by inhibiting the expression of collagen and/or inhibiting the secretion of inflammatory factors at the damaged part of the lung through the deep lung delivery of the anti-oxidative drug.
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 is loaded with Fe3+The coordination adsorption of metformin is used as a therapeutic system for pulmonary fibrosis. By adopting double drugs to synergistically regulate and control pulmonary fibrosis microenvironment, a new treatment strategy is provided for efficient delivery of anti-pulmonary fibrosis drugs.
Wherein, the loaded hydrophobic anti-pulmonary fibrosis drug can simultaneously comprise a plurality of or one selected from drugs or active molecules for reducing the expression of inflammatory factors or resisting the generation of collagen. Antioxidants are drugs or bioactive molecules that reduce singlet oxygen and superoxide in response to oxidative stress at the cellular level.
1) Antioxidant, including active components of metformin, glutathione, α -lipoic acid, carotenoid and ergothioneine;
2) anti-collagen or inflammatory factor producing drugs: imatinib, gefitinib, erlotinib, dasatinib, sunitinib, sorafenib, nilotinib, trametinib, ibuprofen, naproxen, diclofenac, naproxen, ibuprofen, nimesulide, rofecoxib, celecoxib, nintedanib, pirfenidone, cortisone and prednisone, cyclosporine.
The anti-pulmonary fibrosis drug comprises a drug or a bioactive molecule for relieving oxidative stress at a cellular level and reducing intracellular singlet oxygen or superoxide, and a drug or a bioactive molecule for inhibiting the expression of inflammatory factors or collagen.
Specifically, the preparation method of Z-Y-PEG-MAL-X-W-V (PPZV) comprises the following steps:
mixing hydrophobic anti-pulmonary fibrosis drug Z and Y-PEG-MAL, and preparing nano-particle Z-Y-PEG-MAL (PPZ) loaded with anti-pulmonary fibrosis drug by a film dispersion method, a direct titration method or a reverse solvent method;
mixing metal ions W, a drug V and a protease X according to a certain proportion to form a metal ion W-mediated complex X-W-V, reacting nanoparticles Z-Y-PEG-MAL (PPZ) loaded with anti-pulmonary fibrosis drugs with the metal ion W-mediated complex X-W-V through sulfydryl on the protease X and MAL exposed from a nanoparticle shell, and covalently modifying a coordination compound containing metal ions W at the outer ends of the nanoparticles to form Z-Y-PEG-MAL-X-W-V; the mucus layer excessively secreted in the air permeability tube can be efficiently penetrated by the nanoparticles; the drug V released in a responsive manner can relieve the oxidative stress of goblet cells, reduce the secretion of mucus and improve the delivery efficiency of nanoparticles; the medicine delivered to the deep layer can play the role of anti-fibrosis so as to achieve the aim of treating fibrosis.
In the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to the protease X is 5: 1-10: 1; more preferably 5: 1.
In the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to the anti-pulmonary fibrosis drug Z is 20: 1-5: 1; more preferably 10: 1.
In the reaction, Fe is added3+The mass ratio of the medicine V is 40: 1-10: 1; more preferably 30: 1.
Specifically, the preparation method of Z-Y-PEG-MAL-X (PPZ) is as follows:
mixing hydrophobic anti-pulmonary fibrosis drug Z and Y-PEG-MAL, and preparing nano-particle Z-Y-PEG-MAL (PPZ) loaded with anti-pulmonary fibrosis drug by a film dispersion method, a direct titration method or a reverse solvent method;
step (2), carrying out grafting reaction on a nanoparticle Z-Y-PEG-MAL (PPZ) loaded with an anti-pulmonary fibrosis drug and a protease X, and covalently modifying the protease X at the outer end of the nanoparticle through the reaction of a sulfydryl on the protease X and the MAL exposed on the nanoparticle shell; the covalent modification of protease X by the nanoparticles is beneficial to the nanoparticles to efficiently penetrate through mucus layers excessively secreted in the trachea; the medicine delivered to the deep layer can play the role of anti-fibrosis so as to achieve the aim of treating fibrosis.
In the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to the protease X is 5: 1-10: 1; more preferably 5: 1.
In the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to the 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:
firstly, preparing nano PP particles from Y-PEG-MAL by a film dispersion method, a direct titration method or a reverse solvent method;
mixing metal ions W, a drug V and a protease X according to a certain proportion to form a metal ion W-mediated complex X-W-V, reacting nanoparticles PP and the metal ion W-mediated complex X-W-V with MAL exposed from a nanoparticle shell through sulfydryl on the protease X, and covalently modifying a coordination compound containing the metal ions W at the outer ends of the nanoparticles to form Y-PEG-MAL-X-W-V; the mucus layer excessively secreted in the air permeability tube can be efficiently penetrated by the nanoparticles; the drug V released in a responsive manner can relieve the oxidative stress of goblet cells, reduce the secretion of mucus and improve the delivery efficiency of nanoparticles.
In the reaction, the mass ratio of the added Y-PEG-MAL polymer nanoparticles to the protease X is 5: 1-10: 1; more preferably 5: 1.
In the reaction, the mass ratio of the added metal ions W to the added medicine V is 40: 1-10: 1; more preferably 30: 1.
As a control, PP/drug was prepared as follows:
firstly, dissolving an anti-pulmonary fibrosis drug (pirfenidone) in DMSO, wherein the ratio of Y-PEG-MAL to the drug is 100: 1-5: 1, the scheme is 10:1 is more preferable, the encapsulation rate is 71.6%, and the drug loading rate is 5.4%.
And (3) mixing the product obtained in the step (2) with Y-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, in the anti-pulmonary fibrosis drug nano preparation, Y-PEG-MAL, 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 anti-pulmonary fibrosis drug nano preparation has 5-25% of drug-loading rate and 20-500 nm of particle size.
The invention requires the application of the nanometer preparation carrier and the anti-pulmonary fibrosis drug nanometer preparation in preparing drugs for treating pulmonary fibrosis diseases.
The invention provides an enzyme-mediated response type high-efficiency delivery therapeutic drug to achieve the purpose of treating pulmonary fibrosis. Is characterized in that the carrier comprises chemical drug loading components and airway response type degradation and effective delivery components. The chemical drug loading component is an amphiphilic polymer with maleimide modification at the tail end, and the response type degraded mucin component is sulfhydrylation polypeptide.
The invention relates to a mucin-degrading protease-modified nano preparation Y-PEG-MAL, wherein Y is selected from PLGA, PLA, PGA, PCL, PC and PS. The hydrophobic segment in the amphiphilic block copolymer is used for realizing the loading of the anti-fibrosis drug with certain hydrophobicity, and the coordination is used for realizing the loading of the anti-oxidation drug. The efficiency of the nanometer preparation reaching the deep part of the pulmonary alveoli is further improved by overcoming the mucus layer barrier in the air passage through the protease degraded by the mucin, and a new treatment strategy is provided for the efficient delivery of the anti-pulmonary fibrosis drug.
Has the advantages that: the invention provides an enzyme-mediated response type high-efficiency delivery therapeutic drug to achieve the purpose of treating pulmonary fibrosis, and compared with the prior art, the invention has the following advantages: the Y-PEG-MAL in the carrier, wherein Y is a hydrophobic segment, can well realize the entrapment of fat-soluble drugs, the PEG in the carrier can prolong the circulation time of the carrier in blood, metal ions W in the modified carrier in the carrier can responsively release drugs V under the condition that mucus subacid protease X is highly expressed in mucin, the mucus secretion is reduced by regulating and controlling the oxidative stress of goblet cells, so that the delivery efficiency of the nano preparation is improved, and the drugs expressed by anti-fibrosis or anti-inflammatory factors reaching the deep part of the lung can achieve the purpose of treating pulmonary fibrosis by reducing the expression of inflammatory factors or inhibiting the secretion of collagen. The carrier can realize the high-efficiency lung delivery of nano-drugs, the dual responsiveness of enzyme response and acid response realizes the high-efficiency release of anti-oxidative stress drugs, and the nanoparticles reaching the deep part of the alveolus can effectively play a role in resisting the development of pulmonary fibrosis. The invention utilizes microenvironment with high mucin secretion in pulmonary fibrosis trachea to efficiently deliver therapeutic drugs to pulmonary fibrosis injury parts through response type release nano preparations, and simultaneously utilizes anti-pulmonary fibrosis drug Z to inhibit fibrosis progress, thereby providing a new approach and strategy for efficient delivery of anti-pulmonary fibrosis drugs and drug design for effectively treating pulmonary fibrosis. According to the invention, the protease X is used for modifying the nano-carrier Y-PEG-MAL to encapsulate the drug Z, the metal ion W coordinates the nano-preparation of the drug V and the carrier thereof, firstly, during the occurrence process of pulmonary fibrosis, mucus secretions in the trachea are obviously increased, the nano-preparation reaches a mucus layer through trachea administration, and the therapeutic drug is efficiently delivered to the pulmonary fibrosis injury part through the response type release nano-preparation, so that the removal of mucus in the trachea is avoided. The metal ions WV are dissociated from the nano preparation PPZ under the slightly acidic condition of mucus to release the medicine V, mucus secretion is reduced by regulating and controlling oxidative stress of goblet cells to improve the delivery efficiency of the nano preparation, the nano preparation PPZ exposing protease X is released, and the nano preparation PPZ rapidly penetrates through the mucus layer of the airway to reach the deep part of the lung under the condition of high expression of mucin in the mucus layer of the airway to improve the treatment effect of the anti-fibrosis medicine. 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. Meanwhile, the antioxidant is encapsulated in a coordination mode, and the complex has good stability under normal physiological conditions, so that the aim of response type release can be fulfilled under the condition of slightly acid in a mucus layer.
In the invention, protease X modified Y-PEG (X is a hydrophobic segment, and X is PLGA, PLA, PGA, PCL) nanoparticles are used, the loading of double drugs/single drugs with certain hydrophobicity is realized through the hydrophobic segment in an amphiphilic block copolymer, the long circulation of a polymer micelle in a body is realized through PEG, and/or the nano preparation of a metal ion W coordination drug V and a carrier thereof can be released in a response mode in a mucus layer, so that the efficient delivery of the lung is realized.
Drawings
FIG. 1 is a schematic flow diagram of the preparation of an example nano-formulation;
FIG. 2 is the optimum ratio of each component of the nano-formulation prepared in the example;
FIG. 3 is a graph of the particle size of the nanoformulation for each of the formulations of the examples;
FIG. 4 is a potential diagram of the nanoformulation of each of the formulations of the examples;
FIG. 5 is a transmission electron micrograph of mucin (papain) according to the example;
FIG. 6 is a transmission electron micrograph of papain specifically degraded by the protease in the example formulation;
FIG. 7 is a graph of the nanoparticle size of the nanoformulations prepared in the examples after their release in response to various time points under enzyme-sensitive conditions;
FIG. 8 is a graph of the nanoparticle size of the nano-formulations prepared in the example after response-type release at different time points under acid-sensitive conditions;
FIG. 9 is a graph of the nanoparticle size of the nanoformulations prepared in the examples after their response-type release at different time points in the presence of both acid and enzyme;
fig. 10 is an analysis of the effect of different nano-preparations in reversing pulmonary fibrosis, which is examined at the in vivo level by the nano-carriers prepared in the examples.
Detailed Description
The invention will be further described with reference to the following drawings and specific embodiments.
Example 1 synthesis and preparation of nano-formulation ingredients, as shown in fig. 1, fig. 2:
preparation of PLGA-PEG-MAL nanoparticles entrapping double drugs/single drug
The invention preferably adopts a film dispersion method to prepare PLGA-PEG-MAL nanoparticles coated with anti-pulmonary fibrosis drug Z. The preparation method comprises the following steps:
100 mg of PLGA-PEG-MAL and 10 mg of anti-pulmonary fibrosis drug Z are weighed and respectively dissolved in 2 mL of ethyl acetate solution and are ultrasonically dissolved. While stirring, the ethyl acetate solution drops were spun dry by a rotary evaporator, then dissolved by adding 3mL of distilled water with ultrasound for 30 min, and centrifuged at 2500 rpm/min for 20 min 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.
Antioxidant anti-fibrotic drugs:
1) antioxidant, including active components of metformin, glutathione, α -lipoic acid, carotenoid and ergothioneine;
2) anti-collagen or inflammatory factor producing drugs: imatinib, gefitinib, erlotinib, dasatinib, sunitinib, sorafenib, nilotinib, trametinib, ibuprofen, naproxen, diclofenac, naproxen, ibuprofen, nimesulide, rofecoxib, celecoxib, nintedanib, pirfenidone, cortisone and prednisone, cyclosporine.
Preparation of polymer nanoparticles modified by papain and metal ion W-mediated complex
1. Has papain and Fe3+Mediated byPreparation of polymer nanoparticles modified by complex
Metal ion Fe3+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Fe3+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to form enzyme and Fe at the outer end of the nanoparticle3+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated protease Z.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Fe) prepared by the method3+Mediated complex) and PPZV/(containing pirfenidone, papain and Fe simultaneously)3+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Fe at the same time)3+Mediated complex), as shown in fig. 3 and 4, the nano preparation has uniform particle size distribution, uniform shape and obvious core-shell structure.
2. Has papain and Mn2+Mediated complex modified polymer nanoparticle preparation
Metal ions Mn2+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Mn2+The mediated complex is covalently combined with a coordination compound of Mn2+ in the covalent bonding of the papain of the nanoparticles through the reaction of sulfydryl and the MAL exposed from the nanoparticle shell; and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Mn) prepared by the method2+Mediated complex) and PPZV/(containing pirfenidone, papain and Mn simultaneously)2+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The formulation of this example(PPZV/(containing pirfenidone, papain and Mn at the same time)2+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
3. Has papain and Zn2+Mediated complex modified polymer nanoparticle preparation
Metal ion Zn2+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Zn2+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to form papain and Zn in the nanoparticle2+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone containing) and PPV/(papain and Zn containing) prepared by the method2+Mediated complex) and PPZV/(containing pirfenidone, papain and Zn simultaneously)2+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Zn at the same time)2+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
4. Has papain and Fe3+Mediated complex modified polymer nanoparticle preparation
Metal ion Fe3+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Fe3+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to obtain papain and Fe in the nanoparticle3+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(containing papain and pirfenidone) prepared by the methodNicotine), PPV/(containing papain and Fe)3+Mediated complex) and PPZV/(containing pirfenidone, papain and Fe simultaneously)3+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Fe at the same time)3+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
5. Has papain and Cu2+Mediated complex modified polymer nanoparticle preparation
Metal ion Cu2+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, mixing the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone with Cu2+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to form papain and Cu in the nanoparticle2+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Cu) prepared by the method2+Mediated complex) and PPZV/(containing pirfenidone, papain and Cu simultaneously)2+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Cu simultaneously)2+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
6. Has papain and Mg2+Mediated complex modified polymer nanoparticle preparation
Metal ion Mg2+Mixing 30Mg, metformin 2 Mg and papain 30Mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Mg2+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to form papain and Mg in the nanoparticle2+Covalently bonding the coordination compound of (a);and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Mg) prepared by the method2+Mediated complex) and PPZV/(containing pirfenidone, papain and Mg simultaneously)2+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Mg at the same time)2+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
7. Has papain and Si2+Mediated complex modified polymer nanoparticle preparation
Metal ion Si2+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Si2+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to form papain and Si in the nanoparticle2+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Si) prepared by the method2+Mediated complex) and PPZV/(containing pirfenidone, papain and Si simultaneously)2+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Si simultaneously)2+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
8. Has papain and Co2+Mediated complex modified polymer nanoparticle preparation
Metal ion Co2+30mg of the mixture, 2 mg of metformin and 30mg of papain are filled in distilled waterMixing the mixture overnight, and mixing the nanoparticles loaded with the anti-pulmonary fibrosis drug pirfenidone with Co2+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to obtain papain and Co in the nanoparticle2+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Co) prepared by the method2+Mediated complex) and PPZV/(containing pirfenidone, papain and Co simultaneously)2+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Co at the same time)2+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
9. Has papain and Al3+Mediated complex modified polymer nanoparticle preparation
Metal ion Al3+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone with Al3+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to obtain papain and Al on the nanoparticle3+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Al) prepared by the method3+Mediated complex) and PPZV/(containing pirfenidone, papain and Al simultaneously)3+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Al simultaneously)3+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
10. Has papain and Ca2+Mediated complex modified polymer nanoparticle preparation
Metal ion Ca2+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Ca2+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to generate papain and Ca in the nanoparticle2+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone containing) and PPV/(papain and Ca containing) prepared by the method2+Mediated complex) and PPZV/(containing pirfenidone, papain and Ca simultaneously)2+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Ca simultaneously)2+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
(II) having proteases X and Fe3+Mediated complex modified polymer nanoparticle preparation
1. Has papain and Fe3+Mediated complex modified polymer nanoparticle preparation
Mixing Fe3+Mixing 30mg, metformin 2 mg and papain 30mg in distilled water overnight, and mixing the nanoparticles loaded with pirfenidone for resisting pulmonary fibrosis with Fe3+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to obtain papain and Fe in the nanoparticle3+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated papain.
PP/(pirfenidone), PPZ/(papain and pirfenidone) and PPV/(papain and Fe) prepared by the method3+Mediated complexesAnd PPZV/(containing pirfenidone, papain and Fe at the same time)3+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, papain and Fe at the same time)3+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
2. Having acid/base proteases and Fe3+Mediated complex modified polymer nanoparticle preparation
Mixing Fe3+Mixing 30mg, metformin 2 mg and acid/alkali protease 30mg in distilled water thoroughly overnight, mixing the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone with Fe3+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to obtain acid/alkali protease and Fe in the nanoparticle3+Covalently bonding the coordination compound of (a); and centrifuging and resuspending the mixture for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated acid/alkali protease.
PP/(pirfenidone), PPZ/(acid/alkali protease and pirfenidone), PPV/(acid/alkali protease and Fe prepared by the method3+Mediated complex) and PPZV/(containing pirfenidone, acid/alkali protease and Fe simultaneously)3+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. This example formulation (PPZV/(containing pirfenidone, acid/base protease and Fe at the same time)3+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
3. Has bromelain and Fe3+Mediated complex modified polymer nanoparticle preparation
Mixing Fe3+Mixing 30mg, metformin 2 mg and bromelain 30mg in distilled water overnight, and mixing the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone with Fe3+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to generate bromelain and Fe in the nanoparticle3+Covalently bonding the coordination compound of (a); centrifuging and resuspending for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted sulfydrylA basified bromelain.
PP/(pirfenidone), PPZ/(bromelain and pirfenidone) and PPV/(bromelain and Fe) prepared by the method3+Mediated complex) and PPZV/(containing pirfenidone, bromelain and Fe simultaneously)3+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. This example formulation (PPZV/(containing pirfenidone, bromelain and Fe at the same time)3+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
4. Has rennin and Fe3+Mediated complex modified polymer nanoparticle preparation
Mixing Fe3+Mixing 30mg, metformin 2 mg and chymosin 30mg in distilled water thoroughly overnight, mixing the nanoparticles loaded with anti-pulmonary fibrosis drug pirfenidone with Fe3+The mediated complex reacts with MAL exposed by sulfydryl and the shell of the nanoparticle to obtain chymosin and Fe in the nanoparticle3+Covalently bonding the coordination compound of (a); and centrifuging and resuspending for three times at 10000 rpm/min, collecting the prepared nanoparticles, and removing unreacted thiolated chymosin.
PP/(pirfenidone), PPZ/(containing rennin and pirfenidone), PPV/(containing rennin and Fe) prepared by the method3+Mediated complex) and PPZV/(containing pirfenidone, rennin and Fe simultaneously)3+Mediated complex) with drug loading of 5-25% and particle size of 50-500 nm. The preparation of this example (PPZV/(containing pirfenidone, chymosin and Fe at the same time)3+Mediated complex), the particle size distribution of the nano preparation is uniform, and the shape is uniform.
Example 2 papain can specifically degrade papain
Papain, an analogue of mucin in the mucus layer, was taken as a study object as described in example 1, and added with proteases at different concentrations for 24 h, and morphological changes of papain under a Transmission Electron Microscope (TEM) were photographed. The results of transmission electron microscopy show that the papain and the mucin can be degraded by the mucin after 24 hours of interaction, as shown in fig. 5 and fig. 6.
Example 3 assay of the responsive Release of Nanoparticulates under slightly acid and enzyme sensitive conditions
PP/(pirfenidone), PPZ/(protease X and pirfenidone) and PPV/(protease X and Fe) were prepared as described in example 13+Mediated complex) and PPZV/(containing pirfenidone, protease X and Fe simultaneously)3+Mediated complex). After concentrating the nano preparation to 200 mu L, adding 1mL of PBS with pH 6.8, placing the mixture at 4 ℃ for 24 h, and characterizing the particle size and the potential of the nano particles in different environments by using a particle size meter.
(1) After the particle size is measured by a Malverapaceae visual particle size analyzer, the particle size is inspected again after the action of the micro-acid and the enzyme, and the change condition of the particle size is observed;
the particle size data obtained in this example are shown in fig. 7, 8 and 9, and the change in particle size of the nanoparticles before and after the stimulation was examined. Before the micro-acid environment and the enzyme response type stimulation do not exist, the particle size distribution of the nanoparticles is approximately similar, after the micro-acid environment and the enzyme response type stimulation, the nanoparticles release smaller nanoparticles through micro-environment responsiveness, and meanwhile, Fe3+The mediated complex is released responsively to the mucus layer, while smaller nanoparticles can reach the deep lung by osmosis.
And detecting the potential change condition of the nanoparticles before and after stimulation. Fe before no slightly acidic environment and enzyme response type stimulation3+The potential of the mediated complex nano-particle is positive or low, after the micro-acidity and enzyme stimulation, the nano-particle releases smaller nano-particles through micro-environment responsiveness, and simultaneously Fe3+The mediated complex is released to a mucus layer in a responsive manner, while smaller nanoparticles are mainly electronegative, and other researches prove that the nanoparticles formed by PLGA-PEG self-assembly are mainly electronegative, further showing that the constructed system can achieve the purpose of releasing in a responsive manner under the dual conditions of high expression of micro-acid and enzyme.
Example 4 the effect of the nanopreparative formulations PP, PPZ, PPV, PPZV on the treatment of over-reversed pulmonary fibrosis was analyzed.
The nanoformulations PP, PPZ, PPV, PPZV were prepared as described in example 1. Z is pirfenidone and V is metformin.
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 2 USP/Kg, and after three weeks, the effect analysis of the nanometer preparations PP, PPZ, PPV and PPZV in the treatment of over-reversed pulmonary fibrosis is continuously carried out. Mice molded for three weeks were randomly assigned to 5 groups of 5 mice each, and the nanopreparations PP, PPZ, PPV, PPZV and physiological saline were injected through the tail vein, respectively, with the injected drug Z being 2 μ g/g mouse body weight as a standard. Reversal of pulmonary fibrosis effect for different nanopreparations after 4 weeks of treatment the effect of different dosing groups against pulmonary fibrosis was analyzed by H & E staining. From the H & E staining results, it can be seen that the final preparation PPZV group has a significantly reduced alveolar space, a uniform alveolar wall thickness, and a significantly reduced degree of pulmonary fibrosis, as compared to the BLM group, and can achieve the purpose of treating pulmonary fibrosis, indicating that efficient lung targeting and dual drug combination treatment can be achieved by mediating the circulating fiber cell nano-formulation delivery system to effectively reverse pulmonary fibrosis, and the anti-fibrosis effects of the preparation groups are compared with PPZV > PPZ > PPV > PP, with the results shown in fig. 10.
PLGA (polylactic-polyglycolic acid), PLA (polylactic acid), PGA (polyglycolide), PCL (polycaprolactone) are common hydrophobic blocks with good biocompatibility, are often used as hydrophobic cores in amphiphilic block copolymers, and have good affinity for most drugs with certain hydrophobicity. In the above embodiments, PLA-PEG-MAL, PGA-PEG-MAL, PCL-PEG-MAL block copolymers are used instead of PLGA-PEG-MAL block copolymers, and drugs with certain hydrophobicity can be loaded to form polymer micelles, which will be 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 (10)

1. A nanometer preparation carrier is characterized by comprising Y-PEG-MAL and protease X modified with mucin degradation, wherein the protease X modified with mucin degradation is connected with a hydrophilic end of the Y-PEG-MAL to form Y-PEG-MAL-X;
wherein Y is selected from one or more of PLGA, PLA, PGA, PCL, PC and PS; the protease X is selected from one or more of bromelain, papain, and acidic or alkaline protease.
2. The Nanoprotein nanoparticle formulation vehicle of claim 1, further comprising a drug V coordinated to the metal ion W to form a Y-PEG-MAL-X-W-V, wherein the drug V coordinated to the metal ion W comprises Y-PEG-MAL, a mucin-degradation-modified protease X, and the metal ion W;
wherein W is a metal ion selected from Mn2+, Zn2+, Fe2+, Fe3+, Cu2+, Mg2+，Ti4+, Zr4+, Co2+, Al3+，Ca2+One or more of the medicines V is selected from metformin, glutathione, α -lipoic acid, carotenoid and ergothioneine.
3. A nano-formulation, characterized in that the nano-formulation carrier of claim 1 or 2 is used for loading a hydrophobic anti-pulmonary fibrosis drug Z.
4. The nano-formulation according to claim 3, comprising:
Z-Y-PEG-MAL-X, formed by Y-PEG-MAL and a medicament Z encapsulated by a carrier modified by mucin degradation protease X;
the Z-Y-PEG-MAL-X-W-V is formed by Y-PEG-MAL, a medicament V modified with mucin degradation protease X and coordinated with metal ions W and carrying a medicament Z.
5. The nano-preparation according to claim 3, wherein the hydrophobic anti-pulmonary fibrosis drug Z is one or more of anti-inflammatory factor secretion, anti-fibrosis or drugs for relieving cell oxidative stress, the anti-inflammatory factor is a drug or bioactive molecule for reducing cell secretion inflammatory factors and inhibiting collagen production, and the drug for relieving cell oxidative stress is a drug or bioactive molecule for reducing cell singlet oxygen, superoxide and superoxide production;
the hydrophobic anti-pulmonary fibrosis drug Z is selected from one or more of imatinib, gefitinib, erlotinib, dasatinib, sunitinib, sorafenib, nilotinib, trametinib, ibuprofen, naproxen, diclofenac, naproxone, nimesulide, rofecoxib, celecoxib, nintedanib, pirfenidone, cortisone, prednisone and cyclosporine.
6. The anti-pulmonary fibrosis drug nano-preparation according to claim 4, wherein the preparation method of Z-Y-PEG-MAL-X-W-V comprises the following steps:
firstly, mixing hydrophobic anti-pulmonary fibrosis drug Z and Y-PEG-MAL, and preparing nano-particle Z-Y-PEG-MAL loaded with anti-pulmonary fibrosis drug by a film dispersion method, a direct titration method or a reverse solvent method;
mixing metal ions W, a drug V and a protease X according to a certain proportion to form a metal ion W-mediated complex X-W-V, reacting nanoparticles Z-Y-PEG-MAL loaded with anti-pulmonary fibrosis drugs with the metal ion W-mediated complex X-W-V through sulfydryl on the protease X and MAL exposed on a nanoparticle shell, and covalently modifying a coordination compound containing the metal ions W at the outer ends of the nanoparticles to form Z-Y-PEG-MAL-X-W-V; the mucus layer excessively secreted in the air permeability tube can be efficiently penetrated by the nanoparticles; the drug V released in a responsive manner can relieve the oxidative stress of goblet cells, reduce the secretion of mucus and improve the delivery efficiency of nanoparticles; the medicine delivered to the deep layer can play the role of anti-fibrosis so as to achieve the aim of treating fibrosis.
7. The anti-pulmonary fibrosis drug nano-preparation according to claim 4, wherein the Z-Y-PEG-MAL-X is prepared by the following method:
firstly, mixing hydrophobic anti-pulmonary fibrosis drug Z and Y-PEG-MAL, and preparing nano-particle Z-Y-PEG-MAL loaded with anti-pulmonary fibrosis drug by a film dispersion method, a direct titration method or a reverse solvent method;
step (2), carrying out grafting reaction on the nanoparticle Z-Y-PEG-MAL loaded with the anti-pulmonary fibrosis drug and protease X, and covalently modifying the protease X at the outer end of the nanoparticle through the reaction of sulfydryl on the protease X and the MAL exposed on the nanoparticle shell; the covalent modification of protease X by the nanoparticles is beneficial to the nanoparticles to efficiently penetrate through mucus layers excessively secreted in the trachea; the medicine delivered to the deep layer can play the role of anti-fibrosis so as to achieve the aim of treating fibrosis.
8. The anti-pulmonary fibrosis drug nano-preparation according to claim 2, wherein the Y-PEG-MAL-X-W-V is prepared by the following method:
firstly, preparing Y-PEG-MAL nanoparticles by a film dispersion method, a direct titration method or a reverse solvent method;
mixing metal ions W, a drug V and a protease X according to a certain proportion to form a metal ion W-mediated complex X-W-V, reacting nanoparticles Y-PEG-MAL and the metal ion W-mediated complex X-W-V with MAL exposed from a nanoparticle shell through sulfydryl on the protease X, and covalently modifying a coordination compound containing the metal ions W at the outer ends of the nanoparticles to form Y-PEG-MAL-X-W-V; the mucus layer excessively secreted in the air permeability tube can be efficiently penetrated by the nanoparticles; the drug V released in a responsive manner can relieve the oxidative stress of goblet cells, reduce the secretion of mucus and improve the delivery efficiency of nanoparticles.
9. The anti-pulmonary fibrosis drug nano-preparation as claimed in any one of claims 3-8, wherein in Y-PEG-MAL, the molecular weight range of Y is 1000-50000, and the molecular weight range of PEG is 200-10000;
and/or the drug-loading rate of the anti-pulmonary fibrosis drug nano preparation is 5-25%, and the particle size is 20 nm-500 nm.
10. Use of the nano-formulation carrier of any one of claims 1 to 2, the anti-pulmonary fibrosis drug nano-formulation of any one of claims 3 to 8 in the preparation of a drug for treating pulmonary fibrosis diseases.
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