Application of surface engineered mitochondria for activating mitochondrial autophagy in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of surface-engineered mitochondria for activating mitochondrial autophagy in preparation of a medicine for treating pulmonary fibrosis. According to the present invention, the antibody is covalently coupled to the PLL modified exogenous mitochondrial surface to construct the Ab-mito with characteristics of positive electricity and targeting function, such that the efficient mitochondrial transplantation to the recipient cell is promoted, the problems of poor selectivity and low efficiency confronted by the existing mitochondrial delivery research are overcome, and the new direction is provided for the pulmonary fibrosis treatment. The treatment effect of the traditional Chinese medicine composition on pulmonary fibrosis is equivalent to that of a clinical medicine nintedanib, and the method is simpler and more effective.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and in particular to application of surface-engineered mitochondria for activating mitochondrial autophagy in preparation of a drug for treating pulmonary fibrosis. BACKGROUND

[0002] Pulmonary fibrosis (PF) is a chronic, progressive and irreversible interstitial lung disease characterized by pulmonary interstitial fibrosis, which poses a significant threat to human health. More and more studies have shown that mitochondrial dysfunction in alveolar epithelial cells (AECs) plays a key role in the pathogenesis of PF. Impaired mitochondrial homeostasis is considered to be an important reason for accelerating the progression of PF [1,2] .

[0003] The maintenance of mitochondrial homeostasis depends on the dynamic balance between functional and dysfunctional mitochondria in cells. When the number of functional mitochondria decreases and the accumulation of dysfunctional mitochondria increases, mitochondrial homeostasis is imbalanced, leading to ATP synthesis disorders and excessive generation of reactive oxygen species (ROS) [3,4] . This pathological process eventually damages lung cell function and triggers PF-related clinical manifestations [5-8] . Therefore, restoring mitochondrial homeostasis is a key approach to treating PF.

[0004] Mitochondrial transfer, as an emerging therapeutic technology, delivers healthy mitochondria to recipient cells to increase the number of functional mitochondria and restore mitochondrial homeostasis [9-10] . In recent years, mitochondrial transplantation has made significant progress as an innovative disease intervention strategy

[11] . Studies have shown that human mesenchymal stem cells (hMSCs) can improve mitochondrial function through mitochondrial transfer mechanisms, which is a new strategy for treating PF

[12] . This treatment method works through dual mechanisms: on the one hand, it enhances cellular energy metabolism, and on the other hand, it supplements functional mitochondria to increase the level of healthy mitochondria in lung tissue. Studies have reported that mitochondrial transplantation with Parkin protein plasmid transfection can improve mitochondrial quality by supplementing the number of mitochondria and activating mitochondrial autophagy, thereby slowing the progression of pulmonary fibrosis, but the mechanism of reactivation of mitochondrial autophagy has not been clearly defined.

[0005] Patent application 202180090079X discloses a pharmaceutical composition comprising mitochondria for preventing or treating fibrosis. The fibrosis involves pulmonary fibrosis disease, but the mitochondria still use directly isolated mitochondria, which is limited in terms of targeted therapy and transplantation efficiency, and cannot overcome the problem of limited treatment effect caused by mitochondrial homeostasis imbalance in the treatment approach.

[0006] Although mitochondrial transplantation shows significant efficacy in the treatment of various diseases such as cardiovascular diseases, it is still limited by the technical problem of low transplantation efficiency [13,14] This study aims to enhance the efficiency of mitochondrial transplantation to recipient cells by specific and efficient transplantation of exogenous healthy mitochondria, reactivating mitochondrial autophagy in dysfunctional alveolar cells, achieving mitochondrial quality control and remodeling mitochondrial homeostasis, and playing a therapeutic role in PF.

[0007] [1] Wijsenbeek, M. Progress in the Treatment of Pulmonary Fibrosis. Lancet Respir. Med. 2020, 8, 424−425.

[0008] [2] Mohammadi, A.; Balan, I.; Yadav, S.; Matos, W. F.; Kharawala, A.; Gaddam, M.; Sarabia, N.; Koneru, S. C.; Suddapalli, S. K.; Marzban, S. Post-COVID-19 Pulmonary Fibrosis. Cureus 2022, 14, No. e22770.

[0009] [3] Huang, T.; Zhang, T.; Jiang, X.; Li, A.; Su, Y.; Bian, Q.; Wu, H.; Lin, R.; Li, N.; Cao, H.; Ling, D.; Wang, J.; Tabata, Y.; Gu, Z.; Gao, J. Iron Oxide Nanoparticles Augment the Intercellular Mitochondrial Transfer-Mediated Therapy. Sci. Adv. 2021, 7, No. eabj0534.

[0010] [4] Chung, K. P.; Hsu, C. L.; Fan, L. C; Huang, Z.; Bhatia, D.; Chen, Y. J.; Hisata, S.; Cho, S. J.; Nakahira, K.; Imamura, M.; Choi, M. E.; Yu, C. J.; Cloonan, S. M.; Choi, A. M. K. Mitofusins Regulate Lipid Metabolism to Mediate the Development of Lung Fibrosis. Nat. Commun. 2019, 10, 3390.

[0011] [5] Yu, G.; Tzouvelekis, A.; Wang, R.; Herazo-Maya, J. D.; Ibarra, G. H.; Srivastava, A.; De Castro, J. P. W.; Deliuis, G.; Ahangari, F.; Woolard, T.; Aurelien, N.; Arrojo e Drigo, R.; Gan, Y.; Graham, M.; Liu, X.; Homer, R. J.; Scanlan, T. S.; Mannam, P.; Lee, P. J.; Herzog, E. L.; Bianco, A. C; Kaminski, N. Thyroid Hormone Inhibits Lung Fibrosis in Mice by Improving Epithelial Mitochondrial Function. Nat. Med. 2018, 24, 39−49.

[0012] [6] Katzen, J.; Beers, M. F. Contributions of Alveolar Epithelial Cell Quality Control to Pulmonary Fibrosis. J. Clin. Invest. 2020, 130, 5088−5099.

[0013] [7]Takahashi M, Mizumura K, Gon Y, et al. Iron-Dependent Mitochondrial Dysfunction Contributes to the Pathogenesis of Pulmonary Fibrosis[J]. Frontiers in Pharmacology, 2021, 12: 643980.

[0014] [8]Siekacz K, Piotrowski W J, Iwański M A, et al. The Role of Interaction between Mitochondria and the Extracellular Matrix in the Development of Idiopathic Pulmonary Fibrosis[J]. Oxidative Medicine and Cellular Longevity, 2021, 2021: 9932442.

[0015] [9]Liu Z, Sun Y, Qi Z, et al. Mitochondrial transfer / transplantation: an emerging therapeutic approach for multiple diseases[J]. Cell&Bioscience, 2022, 12(1): 66.

[0016]

[10] Hayashida K, Takegawa R, Shoaib M, et al. Mitochondrial transplantation therapy for ischemia reperfusion injury: a systematic review of animal and humanstudies. J Transl Med, 2021, 19(1): 214.

[0017]

[11] Tan YL, Eng SP, Hafez P, Abdul Karim N, Law JX, Ng MH. Mesenchymal stromal cell mitochondrial transfer as a cell rescue strategy in Regenerative Medicine: a review of evidence in preclinical models. Stem Cells Transl Med. 2022. https: / / doi.org / 10.1093 / stcltm / szac044.

[0018]

[12] Huang T, Lin R, Su Y, et al. Efficient intervention for pulmonary fibrosis via mitochondrial transfer promoted by mitochondrial biogenesis [J]. Nature Communications, 2023, 14(1): 5781.

[0019]

[13] Liu Z, Sun Y, Qi Z, Cao L, Ding S. Mitochondrial transfer / transplantation: An emerging therapeutic approach for multiple diseases. Cell Biosci 2022; 12(1):66.

[0020]

[14] Kitani T, Kami D, Kawasaki T, Nakata M, Matoba S, Gojo S. Direct human mitochondrial transfer: A novel concept based on the endosymbiotic theory. Transplant Proc 2014; 46(4): 1233-6. SUMMARY

[0021] The application provides application of surface-engineered mitochondria for activating mitophagy in preparation of a drug for treating pulmonary fibrosis, proposes a strategy of target transplantation of antibody-functionalized exogenous mitochondria Ab-mito, enhances mitochondrial transplantation efficiency, and effectively alleviates pulmonary fibrosis progression by activating mitophagy, and solves the problems in the prior art.

[0022] The technical scheme adopted by the application is: The application provides application of surface-engineered mitochondria for activating mitophagy in preparation of a drug for treating pulmonary fibrosis, and the surface-engineered mitochondria are antibody-functionalized exogenous mitochondria constructed by wrapping mesenchymal stem cell-derived mitochondria with polylysine through electrostatic interaction, and then covalently coupling a myofibroblast marker antibody through amidation reaction.

[0023] Further, the antibody-functionalized exogenous mitochondria effectively eliminate dysfunctional endogenous mitochondria by activating the mechanism of the mitophagy pathway, reshape mitochondrial homeostasis, significantly improve oxidative stress, and effectively alleviate pulmonary fibrosis.

[0024] Further, the myofibroblast marker antibody is an alpha-SMA antibody.

[0025] Further, the antibody-functionalized exogenous mitochondria are prepared as follows: S1, mitochondria are separated by differential centrifugation, resuspended in a mitochondrial storage solution, and reserved; S2, poly-L-lysine solution is added to the mitochondrial storage solution of S1, and incubated at room temperature for 15-25 min; S3, EDC with a final concentration of 100 μg / mL and NHS with a final concentration of 100 μg / mL are added, and incubated at 4°C for 50 min-1.2 h; S4, alpha-smooth muscle actin (alpha-SMA) antibody is added, and incubated at 4°C for 50 min-1.2 h; S5, excess poly-L-lysine, EDC and NHS are removed by centrifugation to obtain antibody-functionalized exogenous mitochondria Ab-mito.

[0026] Further, in S1, 400 μg of mito precipitate is resuspended in 1 mL of a mitochondrial special storage solution; in S2, the concentration of poly-L-lysine solution is 200 μg / mL, and the incubation is performed at room temperature for 20 min; in S3 and S4, the incubation is preferably performed at room temperature for 1 h; and in S5, the centrifugation condition is 11000g, 4°C, and 6 min.

[0027] Specifically, the above preparation process is: first, about 100 μg of mitochondrial protein (PK10016, mitochondrial separation and protein extraction kit, Proteintech, Wuhan, China) is quantitatively detected from 100 million bone marrow mesenchymal stem cells by differential centrifugation. 400 μg of mito precipitate is resuspended in 1 mL of mitochondrial special storage solution. Add 200 μg / mL poly-L-lysine (PLL) solution to the storage solution containing 400 μg of mito, mix, incubate at room temperature for 20 min, allow PLL to adsorb to the mitochondrial membrane surface, then add EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) at a final concentration of 100 μg / mL and NHS (N-hydroxysuccinimide) at a final concentration of 100 μg / mL, 4°C for 1h, finally add 10 μg of α-smooth muscle actin (α-SMA) antibody, 4°C for 1h, EDC and NHS promote the carboxyl-amino crosslinking of the antibody and the mitochondrial surface protein, centrifuge at 11000g, 4°C, 6min to remove excess PLL, EDC and NHS, and finally obtain Ab-mito.

[0028] Further, the medicine comprises an active ingredient and a pharmaceutically acceptable adjuvant, and the active ingredient is the antibody functionalized exogenous mitochondria.

[0029] Further, the dosage form of the medicine comprises one or more of oral liquid, powder, tablet, capsule, granule, decoction, pill, spray, inhalation, atomization and injection.

[0030] Further, the medicine dosage form is an atomization.

[0031] The beneficial effects of the present application are: 1. The present application covalently couples the antibody to the PLL modified exogenous mitochondria surface by EDC / NHS crosslinking, constructs Ab-mito with positive electric nature and targeting function, promotes efficient transplantation of mitochondria to recipient cells, and overcomes the problems of poor selectivity and low efficiency faced by existing mitochondrial delivery research. The bifunctional mitochondria with surface charge change and antibody modification of the present application can be more efficiently transplanted into fibrotic lung epithelial cells by atomization inhalation, which can effectively clear dysfunctional endogenous mitochondria by activating mitochondrial autophagy mechanism, reshape mitochondrial homeostasis, significantly improve oxidative stress, and effectively alleviate the progression of pulmonary fibrosis. This provides a new direction for the treatment of pulmonary fibrosis and an important reference for the research of other mitochondrial related diseases.

[0032] 2、The treatment effect of the application on pulmonary fibrosis is equivalent to that of the clinical drug nintedanib, is equivalent to the effect of the existing literature reported mitochondrial carrier delivery protein transfection plasmid, and the method of the application is simpler and more effective. Specifically, the application uses the method of atomization inhalation to intervene in PF, which is beneficial to the efficient absorption of drugs by alveolar cells and enhances the effect of relieving the progress of PF. The research results show that the Ab-mito constructed by the application shows good ability to relieve oxidative stress of model cells, restore mitochondrial function in vitro research, and the experiment of intervening in the mouse model shows a treatment effect equivalent to that of the clinical drug nintedanib. In addition, the application also describes the mechanism of mitochondrial transplantation to remove damaged mitochondria, regulate mitochondrial homeostasis, and promote the recovery of mitochondrial function to delay the progress of PF through the study of mitochondrial autophagy. Transplanting functional mitochondria with targeting through the method of atomization inhalation can effectively delay the progress of PF by activating mitochondrial autophagy to promote the reconstruction of mitochondrial homeostasis, which has potential clinical application value and provides a reference for the treatment of various mitochondrial-related diseases.

[0033] 3、The application specifically separates functional mitochondria from bone marrow mesenchymal stem cells, PLL is a biocompatible positive charge polymer, the surface thereof is modified to regulate the surface potential of the mitochondria, the mitochondria are coupled with an alpha-SMA antibody, the carboxyl and the amino group between the antibody Fc fragment and the PLL amino group are acylated, the mitochondria transplantation efficiency is significantly improved through antibody-mediated specific recognition and positively charged enhanced receptor endocytosis. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The mechanism diagram of the surface engineered mitochondria of the application for treating pulmonary fibrosis; Figure 2 The Ab-mito construction and PF cell model establishment of the application; Figure 3 The cell experiment treatment effect of the Ab-mito of the application for delaying the progress of pulmonary fibrosis; Figure 4 The mechanism research process diagram of the Ab-mito of the application for transferring and relieving PF; Figure 5 The result of the Ab-mito of the application combined with atomization for effectively inhibiting BLM-induced pulmonary fibrosis in mice; Figure 6 The Western blot detection result of the outer membrane protein and matrix protein in the extracted mitochondria of the application; Figure 7 TGF-β1 promotes the growth of C57 / B6-L mouse primary lung fibroblasts; Figure 8The morphology of A549 cells without TGF-β1 treatment and after treatment with 20 ng / mL TGF-β1 for laser confocal microscope observation; Figure 9 The morphology of C57 / B6-L cells without TGF-β1 treatment and after treatment with 20 ng / mL TGF-β1 for inverted fluorescence microscope observation; Figure 10 The ROS fluorescence intensity of C57 / B6-L cells after TGF-β1 induction for DCFH-DA fluorescence probe labeling combined with laser confocal microscope detection; Figure 11 The expression of p53, p21 and Collagen-I in A549 and C57-BL / 6 cell models induced by TGF-β1 for Western Blot detection; Figure 12 The results of JC-1 experiment of C57 / B6-L cells induced by TGF-β1; Figure 13 The results of reactive oxygen species detection of A549 cells and C57 / B6-L cells in each experimental group; Figure 14 The photos of main organs of mice in each experimental treatment group.

[0035] wherein, Figure 2 In a, it is a transmission electron microscope photo of the complete double-layer membrane structure of extracted mitochondria; in b, it is the surface potential data of the mitochondrial solution mixed with different PLL concentrations; in c, it is the dynamic light scattering (DLS) results of pure mitochondria and Ab-mito; in d, it is the mitochondrial fluorescence intensity of each group; in e, it is the toxicity of TGF-β1 on A549 cells detected by cell counting kit; in f, it is the ROS fluorescence intensity of A549 cells after TGF-β1 induction detected by DCFH-DA fluorescence probe labeling combined with laser confocal microscope; Figure 3 In a, it is the results of cell toxicity evaluation of Ab-mito by CCK8 method; in b, it is the intracellular ATP level of A549 cells after 24h of co-incubation with different concentrations of Ab-mito; in c, it is the Western blot graph that Ab-mito can restore the decreased SIRT3 level in TGF-β1 treated damaged cells; in d, it is the results of CLSM analysis after staining of exogenous mitochondria with different modifications and endogenous mitochondria of A549 cells; in e, it is the laser confocal microscopy image of mitochondrial membrane potential detection of A549 cells in different treatment groups; in f, it is the ATP level of A549 cells in different treatment groups; in g, it is the flow cytometry and immunofluorescence analysis of A549 cells in different treatment groups; in h, it is the determination of SOD, GSH content and NADPH content of A549 cells in different treatment groups; in i, it is the SIM mitochondrial morphology of A549 cells in different treatment groups; Figure 4 a is a schematic diagram showing how exogenous mitochondria selectively eliminate damaged mitochondria by activating the mitochondrial autophagy pathway; b-e are Western blot analysis of the expression levels of P62, LC3B and Parkin proteins in A549 cells; f, g are the results of immunofluorescence detection of mitochondrial outer membrane key protein TOM20 and autophagy marker LC3B; Figure 5 a-c are the changes of ALT, AST and BUN in the serum of mice injected with Ab-mito; d is the evaluation results of the targeting and residence ability of Ab-mito on damaged lung tissue; e is the lung CT imaging of one selected model mouse; f is the CT imaging of each group; g is the body weight change curve of mice in each group during treatment; h is the H&E staining results of organs after mouse dissection; j is the Masson staining results of organs after mouse dissection; k is the analysis of collagen deposition area in the Masson staining image. DETAILED DESCRIPTION

[0036] In order to clearly illustrate the technical features of the scheme, the application will be described in detail below with specific embodiments, combined with the drawings.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0038] Pulmonary fibrosis (PF) is a progressive disease characterized by pulmonary interstitial fibrosis, and its pathogenesis is closely related to the number and function of mitochondria in lung cells. Mitochondrial transfer technology improves mitochondrial dysfunction by transferring exogenous functional mitochondria to recipient cells. However, in the pathological process of PF, due to mitochondrial autophagy dysfunction, damaged mitochondria persistently accumulate, exacerbating the progression of PF, and the current mitochondrial transfer technology has low transplantation efficiency. Based on this, the application combines the mechanism of action to propose the application of surface engineered mitochondria that activate mitochondrial autophagy in the preparation of drugs for treating pulmonary fibrosis.

[0039] The term "treatment" above refers to the reduction of symptoms, elimination of symptoms, or prevention or slowing of the appearance of symptoms of the specified disease or condition, on a temporary or permanent basis. This term is intended to encompass therapeutic and prophylactic or preventative measures, including but not limited to alleviating or reducing one or more symptoms, regression, slowing or stopping the progression of the disease or condition, which cause any clinically desirable or beneficial effect.

[0040] As an embodiment, the application provides a use of surface-engineered mitochondria for activating mitochondrial autophagy in the preparation of a drug for treating pulmonary fibrosis, wherein the surface-engineered mitochondria are antibody-functionalized exogenous mitochondria constructed by wrapping polylysine on mitochondria derived from mesenchymal stem cells through electrostatic interaction and then covalently coupling an antibody of a myofibroblast marker through an amidation reaction.

[0041] Specifically, the antibody-functionalized exogenous mitochondria effectively eliminate dysfunctional endogenous mitochondria, reshape mitochondrial homeostasis, significantly improve oxidative stress, and effectively alleviate pulmonary fibrosis through the mechanism of activating the mitochondrial autophagy pathway.

[0042] The application provides a preparation method of the antibody-functionalized exogenous mitochondria: S1, first, 10 million bone marrow mesenchymal stem cells are subjected to differential centrifugation, and about 100 μg of mitochondrial protein (PK10016, mitochondrial separation and protein extraction kit, Proteintech, Wuhan, China) is quantitatively detected. 400 μg of mito precipitate is resuspended in 1 mL of a special mitochondrial storage solution; S2, 200 μg / mL poly-L-lysine (PLL) solution is added to the storage solution containing 400 μg of mito, mixed, and incubated at room temperature for 20 min to allow PLL to adsorb to the mitochondrial membrane surface; S3, 100 μg / mL of EDC and 100 μg / mL of NHS are further added, and incubated at 4°C for 1 h; S4, finally, 10 μg of α-smooth muscle actin (α-SMA) antibody is added, and incubated at 4°C for 1 h. EDC and NHS promote the carboxyl-amino crosslinking of the antibody and the mitochondrial surface protein; S5, centrifugation at 11000 g, 4 °C, 6 min removes excess PLL, EDC and NHS, and finally Ab-mito is obtained.

[0043] Further, the drug comprises an active ingredient and a pharmaceutically acceptable excipient, and the active ingredient is the antibody-functionalized exogenous mitochondria.

[0044] The term "pharmaceutically acceptable" means that it is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes that it is acceptable for human pharmaceutical use as well as veterinary use.

[0045] Further, the dosage form of the drug includes one or more of oral liquids, powders, tablets, capsules, granules, decoctions, pills, sprays, inhalants, atomizers, and injections. Preferably, the dosage form of the drug is an atomizer.

[0046] As an embodiment, the above-mentioned drug can be used in combination with other drugs for treating pulmonary fibrosis, or can be used alone.

[0047] The application of the surface-engineered mitochondria activating mitophagy in the preparation of a drug for treating pulmonary fibrosis will be described in detail below through specific examples.

[0048] The following examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods not specified in the following examples are preferably referred to the guidance given in the present application, and can also be carried out according to the experimental manuals or conventional conditions in the art, or by referring to the known experimental methods in the art. Unless otherwise specified, all are conventional methods in the art.

[0049] In the following specific examples, the measurement parameters of the raw material components may, unless otherwise specified, have slight deviations within the weighing accuracy range. The temperature and time parameters allow for acceptable deviations caused by instrument testing accuracy or operation accuracy. The equipment and raw materials used can be purchased from the market or are commonly used in the art.

[0050] Example 1 Preparation and characterization of Ab-mito (antibody-functionalized exogenous mesenchymal stem cell mitochondria) 1.1 Preparation of Ab-mito First, healthy free mitochondria were extracted from 10 million bone marrow mesenchymal stem cells (BMSCs) of rats by differential centrifugation. Quantitative detection showed that about 100 μg of mitochondrial protein (PK10016, mitochondrial separation and protein extraction kit, Proteintech, Wuhan, China) was contained. 400 μg of mito was resuspended in 1 mL of mitochondrial storage solution. 200 μg / mL poly-L-lysine (PLL) solution was added to the storage solution containing 400 μg of mito and mixed, and incubated at room temperature for 20 min to allow PLL to adsorb to the surface of the mitochondrial membrane. Then, 100 μg / mL of EDC and 100 μg / mL of NHS were added and incubated at 4°C for 1 h. Finally, 10 μg of α-smooth muscle actin (α-SMA) antibody was added and incubated at 4°C for 1 h. EDC and NHS promoted the carboxyl-amino crosslinking of the antibody and the surface protein of the mitochondria. The excess PLL, EDC and NHS were removed by centrifugation at 11000 g, 4 °C for 6 min. Finally, Ab-mito was obtained.

[0051] 1.2 Characterization of Ab-mito As Figure 2The transmission electron micrograph in Figure a shows that the mitochondria extracted above have an intact double-layer membrane structure. Western blot analysis confirmed that the mitochondrial marker protein VDAC1 (outer membrane protein) (Voltage-Dependent Anion Channel 1) is a key channel protein located in the mitochondrial outer membrane, maintaining the stability of the outer membrane structure and participating in processes such as energy metabolism, cell apoptosis, and signal transduction. SIRT3 (matrix protein) is mainly involved in various mitochondrial metabolic activities, including energy metabolism, oxidative stress, mitochondrial dynamics, and mitophagy. Both are highly expressed, demonstrating the structural and functional integrity of mitochondria. Figure 6 .

[0052] like Figure 2 The mitochondrial surface potential data in Figure b show a dose-dependent positive correlation between PLL concentration and the zeta potential of the mito solution. Increasing the PLL concentration from 160 to 200 μg / mL resulted in a significant increase in the zeta potential; however, the increase slowed down to 300 μg / mL. Therefore, 200 μg / mL PLL was selected as the optimal working concentration. Compared to pure mitochondria, the surface potential of PLL-modified mitochondria increased from approximately -30 mV to approximately +18 mV, indicating successful PLL modification.

[0053] Dynamic light scattering (DLS) was used to measure the particle size distribution (Z-average) and polydispersity index (PDI) of mito (pure mitochondria) and Ab-mito to evaluate their physical stability and coupling effect. Figure 2 In middle c, DLS data showed that the size of Ab-mito increased by 500 nm-600 nm compared with pure mitochondria with a particle size of 500 nm, which also indicated that the mitochondrial surface modification was successfully achieved.

[0054] Flow cytometry was used to verify whether Ab successfully bound to mito and to remove interference from background signals, nonspecific adsorption of secondary antibodies, and autofluorescence of primary antibodies. Figure 2 As shown in Figure d, the mitochondrial fluorescence intensity of the covalently coupled antibody was significantly higher than that of the other groups. There was no obvious fluorescence in the mito group and the Ab-mito group. Only weak background fluorescence was detected in the mito+secondary antibody group, which ruled out nonspecific binding of the secondary antibody. The strong fluorescence signal in the Ab-mito+secondary antibody group confirmed that the antibody (Ab) was successfully modified to the mitochondrial surface.

[0055] Example 2 Establishment of PF cell model TGF-β1 was used as an inducer, and A549 human alveolar epithelial cells and C57 / B6-L mouse primary lung fibroblasts were used as model cells. A549 cells were purchased from Beyotime (Shanghai, China), and C57 / B6-L cells were purchased from Fenghui Biotechnology (Hunan, China). The specific steps are as follows: A549 cells were seeded in high glucose DMEM medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin and incubated at 37 °C, 5% CO2, constant humidity. The cells were treated with 20 ng / mL TGF-β1 for 24 hours. After induction, the modeling effect was detected by various methods.

[0056] The cytotoxicity of TGF-β1 on A549 and C57 / B6-L cells was detected by Cell Counting Kit-8 (Beyotime, Shanghai, China). The morphological characteristics of A549 cells after TGF-β1 induction were observed by confocal laser scanning microscopy imaging. The morphological characteristics of C57 / B6-L cells were observed under an inverted fluorescence microscope, and the ROS fluorescence changes of A549 and C57 / B6-L cells after the addition of TGF-β1 compared with the PBS group were observed and analyzed. The expression levels of p53, p21 and Collagen-I in cells after TGF-β1 treatment were detected by Western Blot.

[0057] Specifically, the concentration of TGF-β1 was determined by the cell counting kit CCK-8 experiment. The experiment showed that 20 ng / mL TGF-β1 inhibited the proliferation activity of A549 human alveolar epithelial cells after 24 hours of treatment while promoting the growth of C57 / B6-L mouse primary lung fibroblasts, as shown in Figure 2 e、 Figure 7 .

[0058] It was found by laser confocal microscopy that the untreated A549 cells maintained typical epithelial cell morphological characteristics and presented regular cobblestone-like arrangement. After treatment with 20 ng / mL TGF-β1, the cell morphology changed significantly, the cells changed from polygonal to fusiform, the intercellular connection decreased significantly, and presented a typical mesenchymal cell phenotype, as shown in Figure 8 , indicating that TGF-β1 can induce epithelial-mesenchymal transition (EMT) of A549 cells.

[0059] It was found by inverted fluorescence microscopy that compared with untreated C57 / B6-L cells, after treatment with 20 ng / mL TGF-β1, the cells changed from typical spindle or star-shaped fibroblast morphology to muscle fibroblasts with flat fibrous structure Figure 9 ).

[0060] By DCFH-DA fluorescent probe labeling combined with laser confocal microscope detection, it was found that the ROS fluorescence intensity of the TGF-β1 treatment group (20 ng / mL, 24 h) was significantly enhanced compared with the PBS control group, indicating that TGF-β1 can effectively induce the increase of ROS level in A549 cells and C57 / B6-L cells, such as Figure 2 f and Figure 10 . The activation of p53 / p21 signaling pathway and Collagen-I deposition are key molecular characteristics in the process of pulmonary fibrosis. As Figure 11 , Western Blot confirmed that the expressions of p53, p21 and Collagen-I were significantly up-regulated in the TGF-β1-induced A549 and C57-BL / 6 cell models, confirming the success of the model construction.

[0061] Example 3 Investigation of the effect of Ab-mito on delaying the progression of pulmonary fibrosis based on the PF cell model 1. Cell safety detection of Ab-mito: The cell safety of Ab-mito was evaluated by CCK8 method. The effect of different concentrations of Ab-mito (0, 10, 20, 40, 60 μg / mL) on the activity of A549 cells was detected by CCK-8 method for 24 h. The effect of Ab-mito on the energy metabolism of A549 cells at 0, 10, 20, 40, 60 μg / mL concentration gradient was analyzed, and the ATP level was detected by chemiluminescence method.

[0062] As shown in a of Figure 3 , within the concentration range of 0 ~ 60 μg / mL, the change of cell viability after A549 cells were co-incubated with Ab-mito was negligible, confirming that Ab-mito had good biocompatibility. As shown in b of Figure 3 , after A549 cells were co-incubated with 0, 10, 20, 40, 60 μg / mL Ab-mito for 24 h, the intracellular ATP level was detected. The results showed that the ATP level increased in a concentration-dependent manner with the increase of Ab-mito concentration, and the ATP level in the 40 μg / mL group was significantly increased compared with the blank control group (P<0.001), so 40 μg / mL was selected as the optimal concentration.

[0063] 2. SIRT3 expression level detection: A549 cells were seeded in confocal dishes. A549 cells were treated with different groups: PBS, TGF-β1 (20 ng / mL TGF-β1 for 24 h), TGF-β1 + mito (20 ng / mL TGF-β1 for 24 h, then 40 μg / mL mito was added for 12 h), TGF-β1 + Ab-mito (20 ng / mL TGF-β1 for 24 h, then 40 μg / mL Ab-mito was added for 12 h).

[0064] SIRT3 is a regulator of mitochondrial function, energy metabolism and antioxidant defense, which plays an important role in cell homeostasis. The expression level of SIRT3 protein in A549 cells after different treatments (PBS, TGF-β1, TGF-β1 + mito, TGF-β1 + Ab-mito) was detected by Western blotting method, and the antibody used was anti-SIRT3 antibody (1:1000, 10099-1-AP, proteintech). Western blot analysis showed that Ab-mito could restore the decreased SIRT3 level in damaged cells, as shown in Figure 3 c.

[0065] 3. In vitro delivery efficiency of Ab-mito: To evaluate the transplantation efficiency of Ab-mito, the exogenous mitochondria (PLL-mito, Ab-mito) and the endogenous mitochondria of A549 cells with different modifications were pre-stained with green and red mitochondrial dyes, respectively. Specifically, the in vitro delivery efficiency of Ab-mito was observed by CLSM (Nikon / MM, Japan, Germany). TGF-β1-induced A549 cells were co-incubated with antibody-coupled mitochondria Ab-mito for 6 hours, and MitoTracker Deep Red was used to label cell mitochondria and MitoTracker Green was used to label Ab-mito, and the delivery efficiency of exogenous mitochondria was analyzed by confocal microscopy.

[0066] As Figure 3 d, compared with cells treated with PLL-mito and untreated cells, cells treated with Ab-mito showed the most significant green fluorescence, while maintaining comparable red fluorescence, indicating that surface modification with PLL combined with antibodies effectively enhanced mitochondrial uptake.

[0067] 4. Systematic evaluation of mitochondrial homeostasis of A549 model cells after co-incubation with Ab-mito: A549 cells and C57 / B6-L cells were seeded at 8000 cells per well in a confocal dish (15 mm glass bottom). Cells were treated with different groups: PBS, TGF-β1 (20 ng / mL TGF-β1 for 24 h), TGF-β1 + mito (20 ng / mL TGF-β1 for 24 h, then 40 μg / mL mito for 12 h), TGF-β1 + Ab-mito (20 ng / mL TGF-β1 for 24 h, then 40 μg / mL Ab-mito for 12 h).

[0068] 4.1, Mitochondrial membrane potential detection: Mitochondrial membrane potential was detected using a mitochondrial membrane potential detection kit (JC-1), and the red / green fluorescence ratio was observed and analyzed by laser confocal microscopy.

[0069] Mitochondrial membrane potential was measured to determine the effect of Ab-mito on mitochondrial integrity in cells. As shown in FIG. 4A, the JC-1 experiment showed that in TGF-β1-induced A549 cells, green fluorescence representing monomers increased significantly, and red fluorescence representing J aggregates decreased significantly, indicating that mitochondrial membrane potential was severely damaged. After Ab-mito treatment, the red fluorescence returned to a comparable level to untreated A549 cells (PBS group), while the green fluorescence decreased significantly. The same treatment was performed on C57 / B6-L lung fibroblasts, and consistent results were obtained (FIG. 4B). Figure 3 Figure 12 ). This indicates that Ab-mito can effectively restore mitochondrial membrane potential and reverse TGF-β1-induced mitochondrial depolarization.

[0070] ATP concentration was measured using a chemiluminescence-based ATP detection kit to evaluate the bioenergetics of cells in different treatment groups, as shown in FIG. 4C. Figure 3 Compared with the PBS group, TGF-β1 stimulation led to a significant decrease in intracellular ATP levels. Both mito and Ab-mito treatment increased ATP production, and the Ab-mito treatment group detected more ATP. This is due to improved mitochondrial function and reduced oxidative stress. Ab-mito showed better performance in ATP generation, with better generation capacity. Restoration of ATP homeostasis may promote the alleviation of fibrotic pathological processes during the development of PF.

[0071] 4.2, Detection of α-smooth muscle actin (α-SMA) expression level: High expression of α-SMA is a marker of fibrotic cells. The expression level of the key protein α-smooth muscle actin (α-SMA) was studied to evaluate the effect of Ab-mito intervention on PF. The following detection of α-SMA expression in A549 model cells after Ab-mito treatment was performed.​

[0072] 4% paraformaldehyde, 15-20 min at room temperature, PBS wash twice. 0.3% Triton X-100, 20-30 min at room temperature, PBS wash cells 3 times. Add PBS containing 5% BSA to block at room temperature for 30 min, PBS wash 1-2 times. Dilute a-SMA primary antibody (1:200, GB111364, Servicebio) in blocking solution, incubate at room temperature for 2 h in the dark, PBS wash twice. Dilute FITC-labeled secondary antibody (1:50) in blocking solution, incubate at room temperature for 1 h in the dark, PBS wash twice. Dilute DAPI (1:1000) in PBS, incubate at room temperature for 10 min in the dark. PBS wash twice. Observe the fluorescence intensity of the cells under a confocal microscope and take pictures.

[0073] The expression level of a-SMA in the above different groups was detected by flow cytometry (Cuto FLEX, Beckman Coulter, USA). 4% paraformaldehyde, 15 min at room temperature, PBS wash. 0.3% Triton X-100, 10 min at room temperature, PBS wash. Add a-SMA primary antibody, incubate at 4°C for 30 min, PBS wash. Add FITC fluorescent-labeled secondary antibody, incubate at room temperature for 30 min in the dark, PBS wash. Resuspend the cells in PBS, filter out cell clumps using a 40 μm filter screen. Load into the flow cytometer to detect fluorescence signals.

[0074] As Figure 3 The results of flow cytometry and immunofluorescence analysis showed that the fluorescence intensity of Ab-mito-treated A549 model cells was comparable to that of the PBS group. A549 model cells showed stronger fluorescence. The above results consistently showed that Ab-mito intervention significantly reduced the expression of a-SMA. 4.3、Total antioxidant capacity SOD, GSH content, NADPH content, ROS level detection: 4.3.1、Total antioxidant capacity SOD detection: A549 cells were seeded in a 96-well plate and incubated at 37°C in a 5% CO2 cell incubator for 12 h to allow the cells to adhere. The cells were treated with different groups: PBS, TGF-β1 (20 ng / mL TGF-β1 for 24 h), TGF-β1 + mito (20 ng / mL TGF-β1 for 24 h, then add 40 μg / mL mito and incubate for 12 h), TGF-β1 + Ab-mito (20 ng / mL TGF-β1 for 24 h, then add 40 μg / mL Ab-mito and incubate for 12 h). The total SOD activity detection kit (WST-8 method) was used to detect and calculate the content of ATP in the cells.

[0075] 4.3.2, GSH content determination: A549 cells were treated as described in 4.3.2. Reduced glutathione (GSH) assay kit (A006-2-1, Nanjing Jiancheng) was used to detect the absorbance value and calculate the GSH content of each group according to the formula.

[0076] 4.3.3, NADPH content detection: A549 cells were treated as described in 4.3.2. NAD+ / NADH assay kit (S0175, Biyun Tian) was used for detection, and the absorbance of each group was detected by a microplate reader. The concentration of NADPH in the cells was calculated according to the standard curve.

[0077] As shown in Figure 4B, compared with the PBS control group, TGF-β1 treatment significantly reduced intracellular GSH, SOD, and NADPH levels. After Ab-mito treatment, all three indicators returned to near PBS group levels, suggesting that Ab-mito can reverse TGF-β1-induced damage to the antioxidant system. Figure 3 As shown in Figure 4B, compared with the PBS control group, TGF-β1 treatment significantly reduced intracellular GSH, SOD, and NADPH levels. After Ab-mito treatment, all three indicators returned to near PBS group levels, suggesting that Ab-mito can reverse TGF-β1-induced damage to the antioxidant system.

[0078] 4.3.4, A549 mitochondrial morphology characterization: A549 cells were seeded in a confocal dish and incubated at 37°C, 5% CO2 in a cell incubator for 12 h. Different groups were treated: PBS, TGF-β1 (20 ng / mL TGF-β1 for 24 h), TGF-β1 + mito (20 ng / mL TGF-β1 for 24 h, then add 40 μg / mL mito for 12 h), TGF-β1 + Ab-mito (20 ng / mL TGF-β1 for 24 h, then add 40 μg / mL Ab-mito for 12 h). Mito-Tracker Deep Red FM (C1032, Biyun Tian) was diluted in cell culture medium to a working concentration of 200 nM and added to the cells at 37°C for 30 min. Wash with PBS for 2-3 times. Use Nikon N-SIM super-resolution microscope to observe, select 100x oil lens. Set the excitation wavelength to 644 nm and the emission wavelength to 665 nm. Use the microscope software to reconstruct the image and obtain the super-resolution image.

[0079] Structural illumination microscopy (SIM) was used to observe mitochondrial morphology. After MitoTracker Deep Red staining, SIM imaging was used to capture mitochondria in cells. As shown in Figure 4C, compared with the PBS control group, TGF-β1 treatment significantly reduced the number of mitochondria and increased mitochondrial fragmentation. After Ab-mito treatment, the number of mitochondria returned to near PBS group levels, suggesting that Ab-mito can reverse TGF-β1-induced mitochondrial damage. Figure 3Mitochondria in TGF-β1 treated cells appeared fragmented, swollen and reticular network was destroyed, in sharp contrast to the typical tubular reticular structure observed in PBS control group, which showed severe mitochondrial damage, possibly due to oxidative stress and bioenergy dysfunction in the pathogenesis of pulmonary fibrosis. After treatment with mito or Ab-mito, respectively, the mitochondrial morphology returned to the typical tubular reticular structure. This finding suggests that Ab-mito can rescue mitochondrial function by rebuilding the mitochondrial network structure.

[0080] 4.3.5, Detection of reactive oxygen species level: A549 cells and C57 / B6-L cells were seeded at 8000 per well in confocal dishes (15 mm glass bottom). After seeding, the confocal dishes were incubated in a cell incubator at 37°C, 5% CO2 for 12 h to allow cell adhesion. According to the experimental requirements, the cells were treated as follows: PBS, TGF-β1 (20 ng / mL TGF-β1 for 24 h), TGF-β1 + mito (20 ng / mL TGF-β1 for 24 h, then 40 μg / mL mito for 12 h), TGF-β1 + Ab-mito (20 ng / mL TGF-β1 for 24 h, then 40 μg / mL Ab-mito for 12 h). The reactive oxygen species (ROS) level in cells was determined using a reactive oxygen species detection kit (DCFH-DA method), and images were collected and observed by laser confocal microscopy (AI+ CellManipulatorPlus, Nikon, Japan). As shown in FIG. 9, PBS control group A549 cells and C57 / B6-L cells showed weak and uniform green fluorescence, and after TGF-β1 stimulation, the fluorescence intensity of intracellular ROS was significantly enhanced, and after Ab-mito treatment, the fluorescence intensity was reduced. Figure 13

[0081] 4.3.6, Detection of autophagy-related proteins and exploration of Ab-mito transfer mechanism to alleviate PF: ​Western blotting was used to detect the expression levels of p62, Parkin, and LC3B proteins in A549 cells treated with different methods. Primary antibodies used were SQSTM1 / p62 Rabbit pAb (1:1000, A11483, ABclonal), Parkin Antibody (1:1000, AF0235, Affinity), LC3B Rabbit Polyclonal Antibody (1:1000, AF5225, Beyotime), and β-Actin Rabbit mAb (1:50000, AC026, ABclonal). Secondary antibodies were HRP-goat anti-rabbit (1:5000, 111-032-003, Jackson) and HRP-goat anti-mouse (1:5000, SA00001-1, Proteintech).

[0082] like Figure 4 The schematic diagram in Figure a illustrates how exogenous mitochondria selectively eliminate damaged mitochondria by activating the mitophagy pathway. Figure 4 Middle b, Visualized bands and quantitative gray values ​​obtained by Western blot analysis ( Figure 4 Figures (c, d, and e) show that compared with the PBS group, the TGF-β1 group showed a significant decrease in Parkin protein expression (P < 0.001), while P62 and LC3-II expressions were significantly upregulated (P < 0.001). Downregulation of Parkin suggests that mitophagy may be inhibited because the lack of the key E3 ubiquitin ligase Parkin impairs the clearance of damaged mitochondria. Accumulation of P62 typically reflects a blockage in autophagic flux. As an adaptor protein for autophagic substrates, P62 is typically degraded during autophagy. Increased LC3-II and upregulation of P62 indicate enhanced autophagosome formation, but subsequent autophagic progression is hindered by lysosomal dysfunction or impaired autophagosome maturation. However, after Ab-mito treatment, Parkin expression was significantly increased (P < 0.001), indicating that mitochondrial transfer successfully reactivated mitophagy. P62 protein levels were significantly reduced, and LC3-II protein levels returned to normal, indicating restoration of autophagic function. This finding indicates that the intervention of the exogenous mitochondria prepared in the present application effectively restored the autophagic flux, enabling the autophagosome to successfully fuse with the lysosome for subsequent degradation.

[0083] Immunofluorescence detection of TOM20, a key protein in the mitochondrial outer membrane, and LC3B, a widely used autophagy marker, was performed. Figure 4Fig. 4a shows that compared with the Ab-mito group, LC3II and TOM20 labeled mitochondria both showed strong green and red fluorescence, but the co-localization of the TGF-β1 group was less. In the Ab-mito treatment group, due to the co-localization of LC3II and mitochondria, the green fluorescence of LC3II and the red fluorescence of TOM20 labeled mitochondria combined to appear yellow areas, indicating that mitochondrial autophagy was activated. The co-localization of lysosomes and mitochondria was observed with structured illumination microscopy. As shown in Fig. 4b, compared with the TGF-β1 group, the co-localization of mitochondria red fluorescence and lysosome green fluorescence in the Ab-mito group was significantly enhanced, indicating that lysosome-mitochondria fusion to form autolysosomes was promoted by mitochondrial intervention. In summary, Ab-mito transfer may activate mitochondrial autophagy through the PINK1 / Parkin pathway, clear damaged mitochondria, and restore mitochondrial homeostasis in pulmonary fibrosis. Figure 4 Fig. 4a shows that compared with the Ab-mito group, LC3II and TOM20 labeled mitochondria both showed strong green and red fluorescence, but the co-localization of the TGF-β1 group was less. In the Ab-mito treatment group, due to the co-localization of LC3II and mitochondria, the green fluorescence of LC3II and the red fluorescence of TOM20 labeled mitochondria combined to appear yellow areas, indicating that mitochondrial autophagy was activated. The co-localization of lysosomes and mitochondria was observed with structured illumination microscopy. As shown in Fig. 4b, compared with the TGF-β1 group, the co-localization of mitochondria red fluorescence and lysosome green fluorescence in the Ab-mito group was significantly enhanced, indicating that lysosome-mitochondria fusion to form autolysosomes was promoted by mitochondrial intervention. In summary, Ab-mito transfer may activate mitochondrial autophagy through the PINK1 / Parkin pathway, clear damaged mitochondria, and restore mitochondrial homeostasis in pulmonary fibrosis.

[0084] Example 4 In vivo treatment of animal modeling and PF mouse model 1.1, Animal modeling C57BL / 6J male mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. C57BL / 6J male mice were induced by intraperitoneal instillation of 50 μL of bleomycin (BLM) at a concentration of 2 U / kg. From the BLM modeling group, C57BL / 6J male mice were randomly selected, and high-resolution micro-CT imaging technology was used to perform multi-planar imaging analysis of the mouse lung to systematically evaluate the morphological characteristics and spatial distribution of pulmonary fibrosis and confirm the establishment of the mouse PF model.

[0085] 1.2, In vivo treatment of PF mouse model After 28 days of modeling, PF male mice were treated by aerosolization, and various preparations were inhaled through nasal aerosolization: PBS, Nintedanib (0.3 mg per mouse per time, given in multiple times, twice a day), Ab-mito (10 μg per mouse per time, given in multiple times, twice a day). During the experiment, the behavior of the mice was observed and recorded daily, including activity, fur, diet, breathing, and mental state. From the 0th day of the experiment, the body weight of the mice was measured every 4 days until the end of the experiment, and the change in body weight of the mice over time was recorded. After 24 days of treatment, the lung status of the mice in the PBS group, the BLM group, the Nintedanib group, and the Ab-mito group was evaluated by mouse CT imaging technology. The right lung tissue of the mice in each group was fixed, and the largest section of the right lung tissue was stained with HE and Masson. The Masson staining images were quantitatively analyzed by ImageJ software.

[0086] During the modeling process, the biosafety of Ab-mito was first evaluated by detecting the changes in serum ALT, AST, and BUN in mice after Ab-mito injection. Figure 5 As shown in a, b, and c, serum biochemical indicators were all within the normal range, indicating that Ab-mito treatment had no adverse effects on liver and kidney function. Subsequently, a PF mouse model was established by intratracheal instillation of bleomycin (BLM). Mice in the PBS group were intratracheally instilled with normal saline as a control. 21 male C57 BL / 6 J mice were instilled with 50 μL 2 U / kg of BLM through the trachea. 28 days after induction, one model mouse was randomly selected and lung CT imaging was performed using high-resolution micro-CT imaging technology. Figure 5 Middle e, Two-dimensional (2D) coronal imaging shows areas of pulmonary fibrosis as hyperattenuating shadows located within the peripheral lung parenchyma. Reticular and honeycombing patterns are shown, suggesting alveolar wall thickening and interstitial fibrosis. Two-dimensional cross-sectional images further demonstrate that areas of fibrosis appear as heterogeneous hyperattenuating shadows, accompanied by bronchiectasis and vascular distortion, with marked regional differences in the distribution of lesions. Furthermore, two-dimensional sagittal imaging demonstrates hyperattenuating shadows aligned along the long axis of the lung lobes, which is associated with disruption of alveolar architecture. Three-dimensional (3D) reconstruction provides a visual representation of the diffuse distribution and spatial structural characteristics of fibrosis, highlighting the reduction in lung volume and distorted parenchymal architecture.

[0087] After the PF mouse model was established on day 28, the targeting and retention ability of Ab-mito on damaged lung tissue was evaluated. Three groups (n=3) were set up, namely the healthy group, the mito treatment group and the Ab-mito treatment group. MitoTracker Deep Red was used to pre-stain mito and Ab-mito. Mice in each group were euthanized after inhaling different aerosol particles, and major organs such as heart, liver, spleen, lung, and kidney were dissected. Figure 14 , use the in-vivo imaging system to take pictures of major organs. Figure 5 As shown in Figure d, compared with the healthy control group and the mito-treated group, obvious red fluorescence was observed in the lungs of the Ab-mito-treated group, and the fluorescence in the liver and kidneys was slightly weakened. This finding indicates that Ab-mito exhibits effective targeting ability and a good residence time in the damaged lung tissue in vivo.

[0088] After 24 days of treatment, mice in each group were randomly selected and CT imaging was used to evaluate the progression of PF disease in mice. Figure 5The results shown in FIG. 6F show that compared with the healthy group, the BLM group of mice showed obvious grid-like shadows in the lungs, bronchial dilatation, honeycomb changes, which are typical PF imaging manifestations. In contrast, the Nintedanib group and the Ab-mito group both showed a significant reduction in PF lesions, and the CT imaging of the Ab-mito group of mice was closer to the healthy group. This suggests that the efficacy of Ab-mito is better than the clinical drug Nintedanib.

[0089] In the present study of pulmonary fibrosis treatment, the remaining 15 model mice were randomly divided into Nintedanib group, Ab-mito group, BLM group (n=5). During the treatment, the Nintedanib group and the Ab-mito group were respectively aerosolized and inhaled Nintedanib (10 mg / kg / day, twice a day), Ab-mito (10 μg / each / time, twice a day). The healthy control group and the BLM group were both aerosolized with PBS. The body weight of all mice was recorded throughout the experiment, and the body weight change curve was drawn, as shown in FIG. 6G. Figure 5 The results show that the mice in the healthy control group steadily increased in weight over time, with no obvious fluctuations. In contrast, the body weight of the mice in the BLM group decreased significantly and remained at a low level in the later stage of the experiment. The mice in the Nintedanib group also showed weight loss in the early stage of drug administration, but gradually recovered. In the Ab-mito treatment group, the body weight of the mice decreased to a lesser extent and recovered faster. The above results show that Ab-mito treatment has good tolerability for PF intervention.

[0090] In order to systematically analyze the pathological progression of PF and its multi-organ effects after treatment, the mice were dissected, and the main organs, including the heart, liver, spleen, lung, and kidney, were collected. Masson staining (FIG. 6I) and H&E staining (FIG. 6H) were used to assess collagen deposition in lung tissue in more detail. Image J software was used to quantitatively analyze the collagen deposition area in the Masson staining images. As shown in FIG. 6I, the results show that the collagen deposition area in the lungs of the BLM group of mice was significantly larger than that in the healthy control group. In contrast, the Nintedanib group and the Ab-mito group both showed a significant reduction in collagen deposition in the lungs. The above results show that Ab-mito treatment has good tolerability for PF intervention. Figure 5 Figure 5 In order to systematically analyze the pathological progression of PF and its multi-organ effects after treatment, the mice were dissected, and the main organs, including the heart, liver, spleen, lung, and kidney, were collected. Masson staining (FIG. 6I) and H&E staining (FIG. 6H) were used to assess collagen deposition in lung tissue in more detail. Image J software was used to quantitatively analyze the collagen deposition area in the Masson staining images. As shown in FIG. 6I, the results show that the collagen deposition area in the lungs of the BLM group of mice was significantly larger than that in the healthy control group. In contrast, the Nintedanib group and the Ab-mito group both showed a significant reduction in collagen deposition in the lungs. The above results show that Ab-mito treatment has good tolerability for PF intervention. Figure 5 ​As shown in FIG. 12, the blue area (collagen deposition) of the Masson section of the BLM group mice was significantly larger than that of the healthy group. In contrast, the lung collagen deposition of the Nintedanib group mice was less than that of the BLM group, and the alveolar structure was also improved. Notably, the Ab-mito group had the highest degree of improvement in lung histopathology (***p < 0.001 vs BLM; *p < 0.05 compared with Nintedanib). HE staining histological analysis showed that the lung tissue of the BLM group mice had obvious destruction of the alveolar structure, and vacuoles were visible in the alveolar cavity, suggesting damage to the alveolar wall or inflammatory response. Other organs such as the heart, liver, spleen, and kidney showed no obvious pathological changes, and all maintained a complete structure, normal cell morphology, and no abnormalities such as inflammation and necrosis. Nintedanib and Ab-mito both showed significant efficacy in improving fibrosis by reducing collagen deposition and inflammatory infiltration, and even Ab-mito showed better improvement. These findings indicate that Ab-mito shows superior efficacy in alleviating PF progression compared to the clinical drug Nintedanib.

[0091] The above describes in detail the use of the surface-engineered mitochondria for activating mitochondrial autophagy in the preparation of a drug for treating pulmonary fibrosis. The above specific embodiments are not considered limiting to the scope of protection of the present application, and any alternative improvements or changes made by those skilled in the art to the embodiments of the present application are within the scope of protection of the present application.

[0092] The details not described in the present application are well known to those skilled in the art.

Claims

1. Use of surface engineered mitochondria for activating mitophagy in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The surface engineered mitochondria are antibody-functionalized exogenous mitochondria constructed by wrapping polylysine with mitochondria derived from mesenchymal stem cells through electrostatic action and then covalently coupling with a myofibroblast marker antibody through an amidation reaction.

2. The use according to claim 1, characterized in that The myofibroblast marker antibody is an α-smooth muscle actin antibody.

3. The use according to claim 1, characterized in that The antibody-functionalized exogenous mitochondria were prepared as follows: S1. Isolate mitochondria by differential centrifugation and resuspend in mitochondrial storage solution for later use; S2. Add poly-L-lysine solution to the S1 mitochondrial stock solution and incubate at room temperature for 15-25 minutes. S3. Add EDC and NHS to a final concentration of 100 μg / mL and incubate at 4°C for 50 min-1.2 h. S4, add α-smooth muscle actin antibody and incubate at 4°C for 50 min-1.2 h; S5. Remove excess poly-L-lysine, EDC, and NHS by centrifugation to obtain antibody-functionalized exogenous mitochondria Ab-mito.

4. The use according to claim 1, characterized in that In S1, 400 μg of mito pellet was resuspended in 1 mL of mitochondrial storage solution. In S2, the concentration of poly-L-lysine solution was 200 μg / mL and incubated at room temperature for 20 min. In S3 and S4, the incubation time at room temperature was preferably 1 h. In S5, the centrifugation conditions were 11,000 g, 4°C, and 6 min.

5. The use according to claim 1, characterized in that The medicine comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is the antibody-functionalized exogenous mitochondria.

6. The use according to claim 1, characterized in that The pharmaceutical dosage form is an aerosol.

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