Inhalable nano-gelling enzyme for treating pulmonary ECM (extracellular matrix) deposition as well as preparation method and application of inhalable nano-gelling enzyme

Abstract

The invention provides an inhalable nano-gelling enzyme for treating pulmonary ECM (extracellular matrix) deposition as well as a preparation method and application of the inhalable nano-gelling enzyme. According to the preparation method disclosed by the invention, aldehyde groups in the oxidized sodium alginate and amino groups in ECM digestion enzyme molecules are subjected to Schiff base reaction to prepare the nanogelling enzyme, so that intervention of a toxic cross-linking agent is avoided, and the enzyme loading efficiency is greatly improved. The nano gel enzyme has the advantages of enzyme protection, good biocompatibility, high drug loading capacity, targeted delivery and industrial production. The nano-gelling enzyme is applied to preparation of medicines for treating lung diseases, can be administrated through aerosol inhalation, can effectively reduce excessive ECM deposition in the lung diseases, can digest ECM barriers, and can recover pulmonary alveolar structures and relieve dyspnea symptoms at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to an inhalable nano-gel enzyme for treating pulmonary ECM deposition, a preparation method thereof, and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] The abnormal deposition of extracellular matrix (ECM) is the core pathological feature of various lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). The current clinical treatment plans mainly include anti-fibrotic drugs represented by glucocorticoids, pirfenidone, and nintedanib, and mechanical ventilation support, but there are significant defects such as poor drug targeting, low bioavailability, and limited drug efficacy. These defects stem from the highly cross-linked characteristics of the complex physiological structure of the lungs and ECM components (such as collagen and fibronectin), and it is difficult for traditional small molecule drugs to achieve precise clearance.

[0004] The ECM in lung tissue is composed of hundreds of proteins. Directly delivering ECM degrading enzymes (such as collagenase and hyaluronidase) is a breakthrough idea in recent years, but it also faces the following problems: (1) Stability defect: Free enzymes are easily inactivated in the respiratory tract with fluctuating pH and protease-rich environment, and the half-life is greatly shortened; (2) Immunogenicity risk: Heterologous enzymes (such as bacterial-derived collagenase) induce IgE antibody responses, and the allergy reaction rate is high. Currently, in order to protect the activity of the enzyme, methods such as delivering the mRNA of the enzyme are used to degrade the ECM in the lungs, but the cost is relatively high, and the delivery of mRNA also has difficulties.

[0005] Chinese Patent Document CN 118217246 A discloses a multi - level nano - preparation loaded with collagenase and anti - fibrosis drugs and its application. It has a multi - level nano - structure and exhibits cascade stimulus - response characteristics. For the first - level nano - preparation, a thermosensitive liposome composed of thermosensitive phospholipids plays a role first. The carrier excipient undergoes a phase transition depending on the pathological feature of the locally higher temperature in the fibrotic tissue and rapidly releases the second - level nano - preparation and collagenase encapsulated inside. The released collagenase rapidly degrades the extracellular matrix that is overly deposited in the fibrotic lung tissue, thereby promoting the second - level nano - preparation to reach the deep lung tissue to exert a therapeutic effect. For the second - level nano - preparation, after specifically targeting and being effectively internalized into damaged type II alveolar epithelial cells (AECs II) depending on the integrin receptor targeting head, its carrier material P breaks due to the high concentration of ROS inside AECs II and releases the anti - fibrosis drug loaded in the inner shell, further exerting an anti - pulmonary fibrosis therapeutic effect. However, the preparation process of the above nano - preparation is cumbersome, has high requirements for excipients, and due to the formation of a drug delivery barrier by the ECM deposition in the lungs, it is very difficult for the first - level nano - preparation to reach the fibrotic tissue to play a role. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides an inhalable nanogel enzyme for treating pulmonary ECM deposition, its preparation method and application. The present invention uses the aldehyde group in oxidized sodium alginate to react with the amino group in the ECM - degrading enzyme molecule to prepare the nanogel enzyme, avoiding the intervention of toxic cross - linkers and achieving a substantial increase in the enzyme - loading efficiency. The nanogel enzyme of the present invention has the advantages of enzyme protection, good biocompatibility, high drug - loading capacity, targeted delivery and industrial production. The nanogel enzyme of the present invention is applied to the preparation of drugs for treating pulmonary diseases, can be administered by aerosol inhalation, can effectively reduce the excessive deposition of ECM in pulmonary diseases, dissolve the ECM barrier, and at the same time can also restore the alveolar structure and relieve the symptom of dyspnea.

[0007] Specifically, the present invention relates to the following technical solutions: In the first aspect of the present invention, there is provided an inhalable nanogel enzyme for treating pulmonary ECM deposition, which is prepared by Schiff base reaction of oxidized sodium alginate (OSA) and ECM - degrading enzyme.

[0008] Preferably according to the present invention, oxidized sodium alginate and the ECM - degrading enzyme react to form a three - dimensional network gel structure; the microscopic morphology of the nanogel enzyme is spherical - like and has strong homogeneity. The formation of a three - dimensional network gel structure by oxidized sodium alginate and the ECM - degrading enzyme protects the enzyme structure and greatly extends the active retention time of the enzyme in vivo.

[0009] Preferably according to the present invention, the oxidized sodium alginate is obtained by oxidizing sodium alginate with sodium periodate; the oxidation degree of the oxidized sodium alginate is 30 - 80%, preferably 50%. The meaning of the oxidation degree of the oxidized sodium alginate in the present invention is: the mole percentage of the oxidized uronic acid units in the total uronic acid units of sodium alginate. It has a dialdehyde group structure and can be used instead of crosslinking agents such as formaldehyde and glutaraldehyde that are more toxic to the human body. By regulating the molar ratio of sodium periodate to sodium alginate (SA) monomers, OSA with different oxidation degrees can be obtained. OSA with a higher oxidation degree will have more aldehyde groups, increasing the number of crosslinking points with the ECM-degrading enzymes.

[0010] Preferably according to the present invention, the ECM-degrading enzyme is selected from one or more fusion enzymes of collagenase, matrix metalloproteinases (MMPs), or other enzymes with ECM-degrading functions; the collagenase is selected from one or more fusion enzymes of type I collagenase, type II collagenase, or type III collagenase; the matrix metalloproteinases (MMPs) are selected from one or more fusion enzymes of MMP2, MMP9, or MMP13; other enzymes with ECM-degrading functions are selected from one or more fusion enzymes of hyaluronidase, elastase, or cathepsin. Preferably, the ECM-degrading enzyme is collagenase, which degrades the key collagen component in the ECM.

[0011] Preferably according to the present invention, the mass ratio of oxidized sodium alginate to the ECM-degrading enzyme is 1:1 - 10, preferably 1:7.

[0012] In the second aspect of the present invention, a preparation method of the inhalable nanogel enzyme for treating pulmonary ECM deposition is provided, including the steps of: adding an ECM-degrading enzyme solution to oxidized sodium alginate, stirring and reacting, and then dialyzing to obtain the inhalable nanogel enzyme for treating pulmonary ECM deposition.

[0013] Preferably according to the present invention, the preparation method of oxidized sodium alginate includes the steps of: dissolving sodium alginate (SA) in deionized water; adding sodium periodate and carrying out a stirring reaction under dark and room temperature conditions; adding ethylene glycol to terminate the reaction, adding sodium chloride and stirring to dissolve, then adding absolute ethanol, filtering to obtain a white precipitate product; dissolving the white precipitate product in deionized water, and dialyzing and freeze-drying to obtain oxidized sodium alginate.

[0014] Preferably, the mass ratio of sodium alginate to the volume of deionized water is 1:50 - 1:120 g / mL; the molar ratio of sodium periodate to the uronic acid units of sodium alginate is 0.1 - 1:1, preferably 0.5:1; the stirring reaction time is 3 - 6 h.

[0015] Preferably according to the present invention, the solvent for digesting the ECM enzyme solution is deionized water, and the concentration of the ECM enzyme in the digesting ECM enzyme solution is 0.5 - 5 mg / mL.

[0016] Preferably according to the present invention, the stirring reaction temperature is 0 - 4°C; the stirring reaction time is 5 - 12 h; the stirring rate is 400 - 1000 rpm.

[0017] Preferably according to the present invention, the dialysis temperature is 0 - 4°C, preferably 0°C; the dialysis time is 3 - 6 h; the cut-off molecular weight of the dialysis bag used for dialysis is 200 - 400 kDa; dialysis is carried out in deionized water. After dialysis, it can be stored as a freeze-dried powder.

[0018] In the third aspect of the present invention, there is provided the use of the above-mentioned inhalable nanogel enzyme for treating pulmonary ECM deposition in the preparation of a medicament for treating pulmonary diseases.

[0019] According to the present invention, the pulmonary disease can be the following diseases or other diseases with ECM deposition as a pathological feature: asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis or emphysema.

[0020] Preferably according to the present invention, the method for preparing a medicament for treating pulmonary diseases includes the steps of: mixing the nanogel enzyme and the drug solution, and stirring and reacting at 0 - 4°C for 0.5 - 5 h to obtain the medicament for treating pulmonary diseases.

[0021] Preferably, the drug can be a protein drug, preferably a polypeptide drug, more preferably KK8, and the amino acid sequence of KK8 is KCSVTCGK. The protein drug has at least any one or more of the following uses: digesting ECM; reducing the content of TGF-β1; reducing the level of pulmonary inflammation; reducing the proliferation and differentiation of pulmonary fibroblasts; reducing the production of extracellular matrix components by pulmonary myofibroblasts.

[0022] Preferably, the solvent for the drug solution is deionized water, and the concentration is 1 - 5 mg / mL; the mass ratio of the ECM-digesting enzyme to the drug in the nanogel enzyme is 1 - 5:1.

[0023] Preferably, the potential of the medicament for treating pulmonary diseases is -24.07 ± 1.44 mV. Negatively charged nanoparticles are less likely to be adhered by the negative charge of mucus in the respiratory tract during inhalation administration, and can increase the accumulation of the drug in the lungs.

[0024] Preferably, the microscopic morphology of the medicament for treating pulmonary diseases is spherical-like, the particle size is 92.18 ± 17.64 nm, and the uniformity is strong.

[0025] Taking the polypeptide KK8 as an example, the present invention has the drug efficacy of reducing the activation of TGF-β1 in the lungs.

[0026] According to the present invention, the medicament of the present invention is used to treat lung diseases by aerosol inhalation, and its pathological feature is the deposition of ECM.

[0027] According to the present invention, the medicament of the present invention can be inhaled into the body by aerosol. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the administration route, the administration method, and so on.

[0028] According to the present invention, the subjects to which the medicament is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc.

[0029] The present invention provides a method for treating lung ECM deposition, the method comprising administering a therapeutically effective dose of the above-mentioned nanogel enzyme or nanogel enzyme medicament to a subject.

[0030] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of an active substance or agent including the nanogel enzyme of the present invention, and this amount can cause a biological or medical response of the tissue system, animal or human pursued by researchers, veterinarians, doctors or other medical personnel, which includes alleviating or partially alleviating the symptoms of the disease, syndrome, disorder or condition being treated. It must be recognized that the optimal dosage and interval of the active ingredient described in the present invention are determined by its nature and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal treatment course, that is, the daily dose of the active substance within a rated time, can be determined by methods well known in the art.

[0031] Technical features and beneficial effects of the present invention: 1. Oxidized sodium alginate (OSA) is derived from natural sodium alginate. The aldehyde group (-CHO) in the OSA structure has high activity and can specifically bind to the primary amino group (-NH2) in the macromolecule of the ECM-degrading enzyme through a Schiff base reaction. And the ECM-degrading enzyme contains multiple amino groups. When the aldehyde group of OSA reacts with the amino group, an imine bond will be formed, thereby crosslinking the sodium alginate chain with the ECM-degrading enzyme to obtain a nanogel enzyme. This crosslinking will form a three-dimensional network gel structure under condition control. While protecting the enzyme structure, this crosslinked structure also has high biosafety and good biocompatibility. The nanogel enzyme of the present invention avoids the intervention of toxic crosslinking agents and realizes a substantial improvement in the enzyme loading efficiency.

[0032] 2. The nano-gel encapsulated enzyme of the present invention has good biocompatibility, high drug loading capacity, targeted delivery, and greatly improves the drug delivery efficiency, and can effectively reduce the excessive deposition of ECM in lung diseases.

[0033] 3. Compared with the prior art, the nano-gel enzyme of the present invention is applied to the preparation of drugs for treating lung diseases. The obtained drugs can be administered only by using a simple atomization instrument, that is, the ECM deposition in lung diseases can be treated by aerosol inhalation to dissolve the ECM barrier, and at the same time, the alveolar structure can be restored, thereby relieving symptoms such as dyspnea. The compliance of patients is high, so it has good practical application value.

[0034] 4. In the present invention, the oxidation degree of oxidized sodium alginate should be controlled at 30%-80%. When the oxidation degree < 30%, the cross-linking sites between oxidized sodium alginate and the enzyme are insufficient, and the particle size of the gel enzyme will be greater than 500 nm, resulting in a decrease in the lung deposition rate of the nano-gel enzyme.

[0035] 5. In the present invention, the types of enzymes are limited to enzymes that can dissolve ECM such as collagenase and MMPs. On the one hand, it can dissolve the drug delivery barrier formed by the deposited ECM in the lungs to clear the way for the delivery of other drugs. On the other hand, it can stop the destruction of the alveoli by ECM and restore the original structure of the alveoli. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0037] Figure 1 It is the transmission electron microscope image of COL@OSA in Example 1 of the present invention; Figure 2 It is the particle size distribution diagram of COL@OSA-KK8 in Example 1 of the present invention; Figure 3 It is the transmission electron microscope image of COL@OSA-KK8 in Example 1 of the present invention; Figure 4 It is the transmission electron microscope image of COL@OSA in Example 2 of the present invention; Figure 5 It is the transmission electron microscope image of COL@OSA in Example 3 of the present invention; Figure 6 It is the aerodynamic distribution diagram of COL@OSA-KK8 in Example 1 of the present invention; Figure 7 It is the in vitro enzyme activity determination diagram of COL@OSA-KK8 in Example 1 of the present invention; Figure 8Transwell penetration of COL@OSA-KK8 of Example 1 of the present invention on the upper chamber of the collagen barrier; Figure 9 Transwell penetration of COL@OSA-KK8 of Example 1 of the present invention on the lower chamber of the collagen barrier; Figure 10 Influence of COL@OSA-KK8 of Example 1 of the present invention and COL+KK8 prepared in Comparative Example 1 on TGF-β1 activation;

[0038] Figure 11 Influence of COL, KK8 and COL@OSA-KK8 of Example 1 of the present invention on the survival period of animals; Detailed implementation manners

[0039] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. If the experimental methods in the following specific implementation manners do not specify specific conditions, they are generally carried out according to the conventional methods and conditions in the biology within the technical field, and such techniques and conditions are fully explained in the literature.

[0041] The present invention will be further explained and illustrated below through examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0042] Example 1 A preparation method of an inhalable nanogel enzyme for treating pulmonary ECM deposition, comprising the steps of: (1) Preparation of oxidized sodium alginate (OSA): Weigh sodium alginate (SA) precisely into a 250 mL round-bottom flask, add deionized water, and stir magnetically for 1 h until SA is completely dissolved. Precisely weigh sodium periodate NaIO4 (so that the molar ratio of sodium periodate to the uronic acid units of sodium alginate is 0.5 to ensure an oxidation degree of 50% for OSA, and the mass ratio of sodium alginate to the volume of deionized water is 1:80 g / mL), add it to the SA aqueous solution, and stir magnetically for reaction for 4 h under dark and room temperature conditions. Add an appropriate amount of ethylene glycol to terminate the reaction, continue stirring for 1 h, then add a small amount of sodium chloride and stir to dissolve. Transfer the solution to a 500 mL beaker, add 200 mL of absolute ethanol, and after a large amount of white flocculent precipitate appears, filter it under reduced pressure to obtain a white precipitate product. Dissolve the product in deionized water, transfer it in aliquots into a 3500 kDa dialysis bag, dialyze it in deionized water for 3 days, change the water every 4 - 6 h, and freeze-dry it for 48 h to obtain the target product OSA with an oxidation degree of 50%.

[0043] (2) Preparation of nano-gel enzyme (COL@OSA): Add type I collagenase solution (the solvent is deionized water and the concentration is 2 mg / mL) to oxidized sodium alginate, and the feeding mass ratio of collagenase to oxidized sodium alginate is 7:1; stir magnetically (600 rpm) for reaction for 5 h under the reaction condition of ice bath at 0 °C. After the reaction is completed, transfer it to a 300 kDa dialysis bag and dialyze for 4 h, dialyze with ice water at 0 °C to ensure enzyme activity, change the water every 30 min, and store it after freeze-drying after dialysis.

[0044] Preparation of nano-gel enzyme drug (COL@OSA-KK8): KK8 (KK8 amino acid sequence: KCSVTCGK, prepared by the existing solid-phase synthesis method) is a polypeptide drug, which can target and reduce the activation of TGF-β1 in the lungs.

[0045] Add 3 mg / mL KK8 solution (the solvent is deionized water and the mass of KK8 is 0.3 mg) to the nano-gel enzyme (which contains 1.4 mg of type I collagenase), and stir magnetically (600 rpm) for reaction for 1 h under the reaction condition of ice bath at 0 °C.

[0046] The TEM of nano-gel enzyme (COL@OSA) is as Figure 1 shown. Its particle size is larger than that of COL@OSA-KK8. The reason is that after adding the peptide, the network structure of the nano-gel becomes tighter, which instead makes the particle size of COL@OSA-KK8 smaller. The TEM image and particle size distribution diagram of COL@OSA-KK8 are respectively as Figure 2 、 3As shown, COL@OSA-KK8 is spherical, has high uniformity, a particle size of about 92.2 nm, and good dispersibility.

[0047] The zeta potential of COL@OSA-KK8 is -24.07 ± 1.44 mV.

[0048] Example 2 A method for preparing an inhalable nanogel enzyme for treating pulmonary ECM deposition is as described in Example 1, except that: in step (2), the mass ratio of collagenase to oxidized sodium alginate in the feed is 3:1; other steps and conditions are the same as in Example 1.

[0049] As Figure 4 shown, the transmission electron microscopy results show that when the mass ratio of collagenase to oxidized sodium alginate in the feed is 3:1, the resulting nanogel enzyme has a non-uniform particle size.

[0050] Example 3 A method for preparing an inhalable nanogel enzyme for treating pulmonary ECM deposition is as described in Example 1, except that: in step (2), the mass ratio of collagenase to oxidized sodium alginate in the feed is 5:1; other steps and conditions are the same as in Example 1.

[0051] As Figure 5 shown, the transmission electron microscopy results show that when the mass ratio of collagenase to oxidized sodium alginate in the feed is 5:1, the resulting nanogel enzyme has a non-uniform particle size.

[0052] Comparative Example 1 Preparation of nanogel enzyme drug (COL+KK8): Add 3 mg / mL KK8 solution (the solvent is deionized water, and the mass of KK8 is 0.3 mg) to 1.4 mg of type I collagenase, and magnetically stir (600 rpm) for 1 h under the reaction condition of ice bath at 0 °C.

[0053] Test Example 1 In vitro aerodynamic particle size distribution Referring to the method for determining the aerodynamic characteristics of fine particles in inhalation preparations in the Pharmacopoeia of the People's Republic of China, evaluate the aerodynamic particle size distribution (APSD) of the inhaled droplets. Pre-cool the NGI impactor device for at least 90 min, adjust the vacuum pump to control the gas flow rate at about 15 L / min, atomize COL@OSA-KK8 prepared in Example 1, and the atomization time is 90 s. Transfer the atomized active substance in the L-type connecting tube, each layer and the additional filter paper, and use the BCA protein quantification kit to measure the protein concentration in each layer. As Figure 6As shown, approximately 90% of the atomized droplets are less than 4 μm, and most of the droplet sizes are in the range of 1-4 μm, demonstrating that the atomized COL@OSA-KK8 droplets can be delivered to the lungs via the respiratory system with a high lung deposition rate.

[0054] Test Example 2 In Vitro Enzyme Activity Characterization To determine the protective effect of the nanogel enzyme on enzyme activity in the present invention, an enzyme activity assay kit was used. According to the instructions, reaction buffer, DQ collagen solution, diluted collagenase solution (Collagenase) to be tested, and the COL@OSA-KK8 solution prepared by the method of Example 1 were sequentially added to a black 96-well plate, incubated in the dark at room temperature, and the fluorescence after incubation for different times was measured at 515 nm using a multimode microplate detection system. As Figure 7 shown, it indicates that after the collagenase is crosslinked with oxidized sodium alginate to form a nanogel, the activity time of the collagenase can be effectively extended.

[0055] Test Example 3 In Vitro Ability to Penetrate the Collagen Barrier Characterization Mouse tail collagen was laid on a Transwell chamber to simulate the excessive deposition of ECM in the environment of idiopathic pulmonary fibrosis. Cells were seeded in the upper chamber of the Transwell and cultured with PBS at pH 7.4 or the COL@OSA-KK8 prepared by the method of Example 1. The ability of COL@OSA-KK8 to penetrate the collagen barrier in vitro was verified by flow cytometry of the cells in the upper and lower chambers of the chamber. As Figure 8 (upper chamber cell count), 9 (lower chamber cell count) shown, for the crosslinked nanogel preparation, almost all the cells in the upper chamber passed through the collagen layer and entered the lower chamber of the chamber. In the control group without collagenase, a large number of cells remained in the upper chamber of the cells and could not pass through the collagen barrier.

[0056] Test Example 4 TGF-β1 Activation Influence Characterization An ELISA detection kit for TGF-β1 was used to measure the content of TGF-β1 in the lung tissues of mice with pulmonary fibrosis and mice after aerosol inhalation treatment with PBS at pH 7.4 or the COL@OSA-KK8 prepared by the method of Example 1 or the COL+KK8 prepared in Comparative Example 1. The results are as Figure 10 shown, and there is a significant downward trend in the TGF-β1 content in the COL@OSA-KK8 treatment group.

[0057] Test Example 5 Animal Survival Period Influence Characterization Using mice as an experimental animal model, a mouse pulmonary fibrosis model was established by intratracheal instillation of bleomycin. The main pathological feature of mouse pulmonary fibrosis disease is the excessive and abnormal deposition of ECM in the lungs. The pulmonary fibrosis mice were raised under the same conditions and randomly grouped, and were respectively atomized with PBS at pH 7.4 or COL@OSA-KK8 prepared by the method of Example 1 or collagenase (COL) alone or KK8 polypeptide, and the survival period of the mice was recorded. The results are as Figure 11 shown. The survival period of the mice in the COL@OSA-KK8 treatment group was significantly prolonged, demonstrating that the inhalable COL@OSA-KK8 prepared by the method of Example 1 can treat the ECM deposition in the lungs, thereby restoring the lung structure of the pulmonary fibrosis mice and prolonging the survival period of the mice.

[0058] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An inhalable nanogel enzyme for treating pulmonary ECM deposition, characterized in that, The nano-gel enzyme is prepared by Schiff base reaction of oxidized sodium alginate (OSA) and ECM-degrading enzyme.

2. The inhalable nanogel enzyme for treating pulmonary ECM deposition according to claim 1, wherein It includes one or more of the following conditions: i. Oxidized sodium alginate reacts with ECM-degrading enzyme to form a three-dimensional network gel structure; the microscopic morphology of the nano-gel enzyme is spherical-like. ii. The oxidized sodium alginate is obtained by oxidizing sodium alginate with sodium periodate; the oxidation degree of oxidized sodium alginate is 30-80%, preferably 50%. iii. The ECM-degrading enzyme is selected from one or a fusion enzyme of two or more of collagenase, matrix metalloproteinases (MMPs), or other enzymes with the function of degrading ECM; collagenase is selected from one or a fusion enzyme of two or more of type I collagenase, type II collagenase, or type III collagenase; matrix metalloproteinases (MMPs) are selected from one or a fusion enzyme of two or more of MMP2, MMP9, or MMP13; other enzymes with the function of degrading ECM are selected from one or a fusion enzyme of two or more of hyaluronidase, elastase, or cathepsin; preferably, the ECM-degrading enzyme is collagenase. iv. The mass ratio of oxidized sodium alginate to ECM-degrading enzyme is 1:1-10, preferably 1:

7.

3. The preparation method of the inhalable nano-gel enzyme for treating pulmonary ECM deposition as described in claim 1 or 2 includes the steps: adding an ECM-degrading enzyme solution to oxidized sodium alginate, stirring and reacting, and dialyzing to obtain the inhalable nano-gel enzyme for treating pulmonary ECM deposition.

4. The preparation method of the inhalable nanogel enzyme for treating pulmonary ECM deposition according to claim 3, characterized in that, The preparation method of oxidized sodium alginate includes the steps: dissolving sodium alginate (SA) in deionized water; adding sodium periodate, and carrying out stirring reaction under dark and room temperature conditions; adding ethylene glycol to terminate the reaction, adding sodium chloride and stirring to dissolve, then adding absolute ethanol, filtering to obtain a white precipitate product; dissolving the white precipitate product in deionized water, and carrying out dialysis and freeze-drying to obtain oxidized sodium alginate. Preferably, the mass ratio of sodium alginate to the volume of deionized water is 1:50-1:120 g / mL; the molar ratio of sodium periodate to the uronic acid unit of sodium alginate is 0.1-1:1, preferably 0.5:1; the stirring reaction time is 3-6 h.

5. The preparation method of the inhalable nanogel enzyme for treating pulmonary ECM deposition according to claim 3, characterized in that, It includes one or more of the following conditions: i. The solvent used for the ECM-degrading enzyme solution is deionized water, and the concentration of the ECM-degrading enzyme in the ECM-degrading enzyme solution is 0.5-5 mg / mL. ii. The stirring reaction temperature is 0-4°C; the stirring reaction time is 5-12 h; the stirring rate is 400-1000 rpm. iii. The dialysis temperature is 0-4°C, preferably 0°C; the dialysis time is 3-6 h; the cut-off molecular weight of the dialysis bag used for dialysis is 200-400 kDa; dialysis is carried out in deionized water.

6. The application of the inhalable nano-gel enzyme for treating pulmonary ECM deposition as described in claim 1 or 2 in the preparation of drugs for treating pulmonary diseases.

7. The application according to claim 6, wherein The pulmonary disease can be the following diseases or other diseases with ECM deposition as a pathological feature: asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, or emphysema.

8. The application according to claim 6, characterized in that, The method for preparing a drug for treating lung diseases comprises the steps of: mixing a nano-gel enzyme and a drug solution, and stirring and reacting at 0-4 °C for 0.5-5 h to obtain the drug for treating lung diseases.

9. The application according to claim 8, characterized in that, The drug can be a protein drug, preferably a polypeptide drug, and further preferably KK8, and the amino acid sequence of KK8 is KCSVTCGK.

10. The application according to claim 9, characterized in that It includes one or more of the following conditions: i. The solvent used for the drug solution is deionized water, and the concentration is 1-5 mg / mL; the mass ratio of the ECM-degrading enzyme to the drug in the nano-gel enzyme is 1-5:1; ii. The potential of the drug for treating lung diseases is -24.07 ± 1.44 mV; iii. The microscopic morphology of the drug for treating lung diseases is spherical-like, and the particle size is 92.18 ± 17.64 nm.

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