Immune-engineered mitochondrial nMITO, preparation method, application and evaluation method

Abstract

The invention discloses an immune-engineered mitochondrial nMITO as well as a preparation method, application and an evaluation method thereof, and relates to the technical field of asthma treatment. According to the application of the immune-engineered mitochondrial nMITO in preparation of the medicine for treating asthma, through the specific immune regulation function of the nMITO, local overactivated immune cells (such as eosinophilic granulocytes and Th2 cells) of an airway are regulated in a targeted mode, abnormal release of proinflammatory factors (such as IL-4, IL-5 and IL-13) is inhibited, meanwhile, activation of an anti-inflammatory signal channel is promoted, and the anti-inflammatory effect is improved. Therefore, the steady state of the airway immune microenvironment is recovered, accurate and efficient intervention on chronic asthma inflammation is achieved, the aims of improving the treatment effect, reducing adverse reactions and reducing disease relapse are finally achieved, and a brand new technical scheme is provided for clinical treatment of asthma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of asthma treatment, in particular to an immune engineered mitochondrion nMITO, a preparation method and application thereof, and an evaluation method. BACKGROUND

[0002] Bronchial asthma (asthma for short) as a chronic airway inflammatory disease, is characterized by chronic airway inflammation, airway hyperresponsiveness and reversible airflow limitation, and its clinical manifestations include recurrent wheezing, shortness of breath, chest tightness or cough, which seriously threatens global public health and brings significant economic burden. The existing treatment drugs are divided into control type, relief type and additional treatment drugs for severe asthma, including glucocorticoids (GCs), bronchodilators and biological target drugs. Compared with traditional small molecule drugs, the way of supplementing healthy mitochondria to damaged cells can repair mitochondrial damage and restore cell function. However, mitochondria themselves lack efficient inflammation regulation ability, and how to endow mitochondria with inflammation regulation ability to improve the effect of mitochondrial transplantation in asthma treatment is a key technical problem that needs to be solved at present.

[0003] The current clinical treatment focuses on controlling airway inflammation and relieving symptoms, mainly including: 1) Glucocorticoids (GCs): play an anti-inflammatory role by inhibiting the NF-κB pathway, and are the basis of asthma control (such as inhaled budesonide and oral prednisone), but long-term systemic use has side effects such as osteoporosis and immunosuppression; 2) Bronchodilators: β2 receptor agonists (such as salbutamol) and anticholinergic drugs (such as ipratropium bromide) rapidly relieve airway spasm and need to be used as needed, and excessive use may cause arrhythmia; 3) Biological target drugs: monoclonal antibodies targeting Th2 inflammatory pathways (such as omalizumab and dupilumab) significantly improve the control of moderate to severe asthma, but are only effective for specific phenotypes, and need to be injected and have high costs (annual treatment cost > $10,000) The administration of asthma treatment drugs focuses on the combination of local targeting and systemic intervention: 1) Inhalation administration is the first choice, including metered dose inhalers (pMDI, portable but dependent on inspiratory coordination), dry powder inhalers (DPI, no propellant and need active inhalation), nebulizer (suitable for severe patients, ultrasonic / compressed air driven) and soft mist inhaler (low-speed aerosol to improve deposition efficiency), which all directly deliver drugs to the airway to achieve local high concentration effect; 2) Oral administration (solid / liquid preparations) depends on gastrointestinal absorption and is suitable for long-term control, with the characteristics of convenient use and high compliance; 3) Injection (intravenous / subcutaneous / intramuscular) for severe emergency or biological agents, intravenous injection has rapid onset (<30 minutes), subcutaneous injection can achieve long-acting delivery of biological agents; 4) Other routes (nasal, transdermal, rectal) as a supplement, among which nasal administration can intervene in the combined inflammation of the upper and lower airways, transdermal / rectal administration is in the invention or standby stage; 5) Emerging technologies (nanometer targeted delivery, intelligent response device, extracellular vesicle carrier) focus on precise delivery and microenvironment regulation, such as targeted peptide modified liposome to improve airway deposition rate, MEMS device to dynamically adjust particle size.

[0004] The core bottlenecks of the prior art include: 1) Lack of precision in inflammation regulation: traditional drugs cannot specifically block the complex inflammatory network of asthma (such as the interaction of eosinophils, Th17 cells, and cytokines IL-5, IL-13), resulting in drug resistance in some patients (about 10%-15%); 2) Limitations of the targeting of the drug delivery system: inhalation preparations rely on the coordination of patient inhalation (error rate >40% in children / elderly patients), and the drug deposition rate in the distal airway is low (<20%), which causes side effects due to systemic exposure; 3) Treatment gap for severe asthma: about 5% of severe patients still have frequent acute attacks even after receiving high-dose ICS / LABA treatment, and new intervention means with new mechanisms of action (such as mitochondrial function repair and epigenetic regulation) are urgently needed.

[0005] The existing drug delivery technology has multiple dimensional defects: 1) Inhalation administration: limited by patient inhalation function (error rate >40% in children / elderly), device portability (large size of nebulizer), and particle distribution (>5 μm particles causing oral deposition), the drug concentration in the peripheral airway is insufficient (<30% of the effective concentration); 2) Oral administration: affected by the first-pass effect of the liver (bioavailability generally <70%), multiple dosing is required (missed dose rate >35%), and systemic exposure leads to side effects (such as the risk of glucocorticoid-induced osteoporosis); 3) Injection administration: intravenous injection relies on medical operation, with high risk of vascular stimulation (heart palpitation incidence >15%), subcutaneous injection requires cold chain transportation (cost increase of 20%) and local reaction rate of 20%-30%; 4) Emerging technologies: nanocarrier biocompatibility needs to be verified (hemolysis rate >5%), intelligent device cost is high (> $500 per unit), and extracellular vesicle preparation is difficult (low yield), which have not broken through the clinical translation bottleneck; The common limitations are lack of dynamic dose adjustment (unable to respond to real-time inflammation indicators), insufficient targeting accuracy (central airway deposition >60%), and patient compliance dependent on device operation (error rate >50% for complex steps).

[0006] In summary: Current asthma treatment methods can alleviate symptoms to some extent, but have significant limitations: existing drugs (such as glucocorticoids, bronchodilators, etc.) are mainly single-target or symptomatic treatment, which is difficult to fundamentally correct the immune imbalance of chronic airway inflammation, leading to easy recurrence and poor long-term efficacy; at the same time, long-term use of hormone drugs can easily cause systemic adverse reactions (such as immune suppression, metabolic disorders, etc.), and patients have poor tolerance.

[0007] Therefore, a new solution is needed to solve the above problems. SUMMARY

[0008] The purpose of the present application is to provide an immune engineered mitochondria nMITO and a preparation method, application and evaluation method, to solve the technical problems proposed in the background art.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions: a preparation method of an immune engineered mitochondria nMITO, at least comprising the following steps: S1: extracting mitochondria from the heart of a 5-8 week old male C57 mouse; S2: isolating neutrophils from mouse bone marrow, and extracting cell membranes after activation with LPS; S3: mixing the mitochondria and neutrophil membranes at a protein mass ratio of 1:1, and ultrasonically fusing in an ice water bath for 2 minutes; S4: centrifuging at 3500g for 10 minutes at 4℃, and then performing sedimentation, washing the sediment with PBS twice, to obtain nMITO.

[0010] Further, the S1 at least comprises the following steps: Select a 5-8 week old male C57 mouse, disinfect with 75% alcohol, quickly remove the heart, put it into pre-cooled PBS, wash 5 times, quickly pump the residual blood in the heart, cut it into small pieces, and ice bath for 3 minutes; Centrifuge at 600g for 20 seconds at 4℃, then discard the PBS; Then add 800μL trypsin digestion solution, and ice bath for 5 minutes; Centrifuge at 600g for 20 seconds at 4℃, discard the trypsin digestion solution; Add 1mL mitochondria separation reagent to wash away the residual trypsin; Then perform homogenization treatment, centrifuge at 1000g for 5 minutes at 4℃; Collect the supernatant and centrifuge it at 13500g for 10 minutes at 4°C. The final precipitate is the mitochondria.

[0011] Furthermore, S2 includes at least the following steps: Mice were anesthetized with ether, euthanized by cervical dislocation, and the tibia and femur of the mice were quickly separated to remove excess muscle tissue. The separated tibia and femur were immersed in glass dishes filled with RPIM 1640 medium; Use micro forceps to remove all the tissue around the bone and cut off the joints at both ends of the bone to expose the medullary cavity; Rinse the medullary cavities of the tibia and femur with culture medium; After obtaining the bone marrow suspension, neutrophils were separated by density gradient centrifugation. Neutrophils were activated with 1 μg / mL LPS; Add 5 μL of separation buffer containing PMSF, sonicate for 5 minutes to lyse the cells; Finally, the neutrophil membrane was obtained by centrifugation at 10000g for 10 minutes at 4°C.

[0012] An immunoengineered mitochondrial nMITO is prepared using the method described above.

[0013] An immunoengineered mitochondrial nMITO is used in the preparation of asthma drugs.

[0014] An evaluation method for immunoengineered mitochondrial nMITO, used for the aforementioned immunoengineered mitochondrial nMITO, includes at least the following steps: Construct an OVA-induced mouse model of asthma; Separation of bronchoalveolar lavage fluid from mice.

[0015] Furthermore, the construction of the OVA-induced asthma mouse model includes at least the following steps: Prepare the adjuvant by continuously adding 4 mg of alum to different masses of ovalbumin (OVA) solutions and mixing continuously for 30 minutes. Initially, 50 μg of ovalbumin was injected intraperitoneally into C57 mice. The ovalbumin was adsorbed onto 4 mg of alum, and the total volume for each mouse was 200 μL. On day 10, mice were injected intraperitoneally with 25 μg of ovalbumin adsorbed onto 4 mg of alum. On days 21, 22 and 23, C57 mice were given 25 μg ovalbumin (1 mg / mL ovalbumin) via endotracheal tube. Within 1 hour after challenge, MITO or nMITO 1 mg / kg was administered via tail vein injection. On day 25, samples were collected and the mice were euthanized. The samples collected included orbital venous blood, bronchoalveolar lavage fluid, and tissues from the heart, liver, spleen, lungs, and kidneys. Serum was collected by centrifugation to measure inflammatory factors TNF-α and IL-1β, as well as specific indicators IgE and IL-4 and IL-5. Heart, liver, spleen, lungs and kidneys were collected, weighed and photographed to assess the indices of each organ. Fresh lung tissue was collected to detect inflammatory genes TNF-α, IL-6, IL-1β and IL-4, and lung tissue was fixed with 4% paraformaldehyde for pathological staining.

[0016] Furthermore, the separation of mouse bronchoalveolar lavage fluid includes at least the following steps: After anesthetizing the mice, they were fixed in a supine position on the operating table. After disinfecting the neck skin with 75% alcohol, make a small incision of 0.5-1 cm longitudinally, bluntly peel off the neck skin of the mouse from the midline of the neck to the left and right sides, separate the smooth muscle around the trachea, and expose the trachea. Tie the uppermost end of the trachea with sutures, draw 1 mL of pre-cooled PBS with a 1 mL syringe with a needle, insert it into the trachea at a 30° angle, and then slowly inject the PBS into the mouse's lungs after flattening the needle. At this point, the lungs can be seen bulging; Then slowly draw it back into the syringe; A PBS recovery rate >80% is considered acceptable. Collect the PBS in an EP tube and repeat this process three times.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention provides the application of immunoengineered mitochondrial nMITO in the preparation of drugs for treating asthma. Through the unique immunomodulatory function of nMITO, it targets and regulates overactivated immune cells in the airway (such as eosinophils and Th2 cells), inhibits the abnormal release of pro-inflammatory factors (such as IL-4, IL-5, and IL-13), and promotes the activation of anti-inflammatory signaling pathways, thereby restoring the homeostasis of the airway immune microenvironment. This achieves precise and efficient intervention in chronic inflammation of asthma, ultimately aiming to improve treatment efficacy, reduce adverse reactions, and reduce disease recurrence, providing a novel technical solution for the clinical treatment of asthma.

[0018] 2. The application proposed in this invention has a more significant and lasting therapeutic effect. nMITO improves the immune imbalance of chronic airway inflammation from the root by regulating the function of local immune cells and the balance of cytokines in the airway, rather than simply treating the symptoms. Therefore, it can significantly improve the long-term therapeutic effect and reduce the recurrence of the disease.

[0019] 3. The application proposed in this invention has higher safety and fewer adverse reactions. nMITO is targeted, mainly acting on the local immune microenvironment of the airway, avoiding the widespread impact of systemic administration on normal tissues; at the same time, it works by regulating immune homeostasis, without relying on the strong inhibitory properties of hormones, which can significantly reduce systemic adverse reactions such as immunosuppression and metabolic disorders caused by long-term use, and improve patient tolerance.

[0020] 4. The mechanism of action of this invention is more precise and innovative. Unlike the single-target intervention of existing drugs, nMITO achieves overall regulation of the asthma inflammatory network through multi-dimensional regulation (inhibiting the release of pro-inflammatory factors, activating anti-inflammatory pathways, and regulating the activation state of immune cells, etc.), providing a brand-new mechanism of action and technical approach for asthma treatment, with outstanding innovation and application value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram comparing the heart, liver, lung, and kidney indices of four groups of mice in this invention. Figure 2 This is an enlarged schematic diagram of the changes in morphological characteristics of whole lung tissue sections and staining according to the present invention; Figure 3 This is a schematic diagram of the expression level in mouse serum according to the present invention; Figure 4 This is a schematic diagram of the expression level in the mouse lungs of this invention. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Example

[0024] The preparation of an immunoengineered mitochondrial nMITO includes at least the following steps: S1: Mitochondria were extracted from the hearts of 5-8 week old male C57 mice; S2: Neutrophils were isolated from mouse bone marrow, activated with LPS, and the cell membrane was extracted. S3: Mix mitochondria and neutrophil membranes at a protein ratio of 1:1 and sonicate them in an ice-water bath for 2 minutes. S4: Centrifuge at 3500g for 10 minutes at 4℃. After centrifugation, precipitate the precipitate and wash it twice with PBS to obtain nMITO.

[0025] S1 includes at least the following steps: Male C57 mice aged 5-8 weeks were selected. After disinfection with 75% alcohol, the heart was quickly removed, placed in pre-cooled PBS, washed 5 times, and the residual blood in the heart was quickly pumped out. After being cut into pieces, the heart was placed in an ice bath for 3 minutes. Centrifuge at 600g for 20 seconds at 4°C, then discard the PBS; Then add 800 μL of pancreatic enzyme digestion solution and digest in an ice bath for 5 minutes; Centrifuge at 600g for 20 seconds at 4℃ and discard the pancreatic enzyme digestion solution; Add 1 mL of particulate separation reagent to wash away residual trypsin; Then homogenize the mixture and centrifuge it at 1000g for 5 minutes at 4°C. Collect the supernatant and centrifuge it at 13500g for 10 minutes at 4°C. The final precipitate is the mitochondria.

[0026] S2 includes at least the following steps: Mice were anesthetized with ether, euthanized by cervical dislocation, and the tibia and femur of the mice were quickly separated to remove excess muscle tissue. The separated tibia and femur were immersed in glass dishes filled with RPIM 1640 medium; Use micro forceps to remove all the tissue around the bone and cut off the joints at both ends of the bone to expose the medullary cavity; Rinse the medullary cavities of the tibia and femur with culture medium; After obtaining the bone marrow suspension, neutrophils were separated by density gradient centrifugation. Neutrophils were activated with 1 μg / mL LPS; Add 5 μL of separation buffer containing PMSF, sonicate for 5 minutes to lyse the cells; Finally, the neutrophil membrane was obtained by centrifugation at 10000g for 10 minutes at 4°C. Example

[0027] An immunoengineered mitochondrial nMITO was prepared using the method for preparing immunoengineered mitochondrial nMITO proposed in Example 1 above. Example

[0028] This embodiment proposes an immune-engineered mitochondrial nMITO for use in the preparation of asthma drugs, based on the above embodiments. Example

[0029] This embodiment proposes an evaluation method for immune-engineered mitochondrial nMITO based on the above embodiment two, which includes at least the following steps: Construct an OVA-induced mouse model of asthma; Separation of bronchoalveolar lavage fluid from mice.

[0030] Constructing an OVA-induced asthma mouse model includes at least the following steps: Prepare the adjuvant by continuously adding 4 mg of alum to different masses of ovalbumin (OVA) solutions and mixing continuously for 30 minutes. Initially, 50 μg of ovalbumin was injected intraperitoneally into C57 mice. The ovalbumin was adsorbed onto 4 mg of alum, and the total volume for each mouse was 200 μL. On day 10, mice were injected intraperitoneally with 25 μg of ovalbumin adsorbed onto 4 mg of alum. On days 21, 22 and 23, C57 mice were given 25 μg ovalbumin (1 mg / mL ovalbumin) via endotracheal tube. Within 1 hour after challenge, MITO or nMITO 1 mg / kg was administered via tail vein injection. On day 25, samples were collected. Mice were euthanized, and samples were collected including orbital venous blood, bronchoalveolar lavage fluid, and tissues from the heart, liver, spleen, lungs, and kidneys. Serum was collected by centrifugation to measure inflammatory factors TNF-α and IL-1β, as well as specific indicators IgE and IL-4 and IL-5. Heart, liver, spleen, lungs and kidneys were collected, weighed and photographed to assess the indices of each organ. Fresh lung tissue was collected to detect inflammatory genes TNF-α, IL-6, IL-1β and IL-4, and lung tissue was fixed with 4% paraformaldehyde for pathological staining.

[0031] The separation of mouse bronchoalveolar lavage fluid includes at least the following steps: After anesthetizing the mice, they were fixed in a supine position on the operating table. After disinfecting the neck skin with 75% alcohol, make a small incision of 0.5-1 cm longitudinally, bluntly peel off the neck skin of the mouse from the midline of the neck to the left and right sides, separate the smooth muscle around the trachea, and expose the trachea. Tie the uppermost end of the trachea with sutures, draw 1 mL of pre-cooled PBS with a 1 mL syringe with a needle, insert it into the trachea at a 30° angle, and then slowly inject the PBS into the mouse's lungs after flattening the needle. At this point, the lungs can be seen bulging; Then slowly draw it back into the syringe; A PBS recovery rate >80% is considered acceptable. Collect the PBS in an EP tube and repeat this process three times.

[0032] Based on the above, the following experimental demonstration is further proposed: Effects of nMITO on organ indices in OVA-induced asthmatic mice This invention assesses the safety of nMITO and its regulatory effect on an asthma model by detecting organ indices in four groups of mice (① healthy control group (Control), ② model group (Model), ③ ordinary mitochondrial therapy group (MITO), and ④ immune-engineered mitochondrial therapy group (nMITO)). Figure 1 The results showed that there were no statistically significant differences in the heart, liver, lung, and kidney indices among the four groups of mice (P > 0.05), indicating that nMITO did not produce significant toxic effects on the major organs of mice, providing a basis for its safety and having no macroscopic toxic effects on the major organs.

[0033] nMITO can alleviate OVA-induced pathological damage in mouse lung tissue. Hematoxylin and eosin (H&E) staining of paraffin sections of lung tissue can assess the extent of lung damage. H&E staining reflects the infiltration of inflammatory cells. Figure 2 As can be seen, H&E staining of lung tissue revealed alveolar structure disruption and significantly increased inflammatory cell infiltration in the OVA-induced model group, directly reflecting the degree of lung tissue damage. The nMITO treatment group showed significant improvement in these symptoms and exhibited a relieving effect on asthma. (See also...) Figure 2 .

[0034] nMITO can inhibit the production of immunoglobulin E (IgE), Th2 cytokines, and inflammatory factors. To evaluate the effects of nMITO on changes in inflammatory factors and Th2 cytokines in asthmatic mice, we divided the experiment into four groups: ① healthy control group (Control), ② model group (Model), ③ ordinary mitochondrial therapy group (MITO), and ④ immune-engineered mitochondrial therapy group (nMITO).

[0035] In pulmonary immunology, abnormal Th2 cell function in the lungs is a core characteristic of asthma pathogenesis, and the resulting immune response dysregulation can be manifested through abnormal expression of IgE and cytokines such as IL-4 and IL-5. Serum inflammatory factor levels, as key indicators reflecting systemic inflammatory status, have important reference value in evaluating the efficacy of asthma treatment.

[0036] This invention uses ELISA to detect the expression levels of TNF-α, IL-1β, IL-6 (pro-inflammatory factors) and IgE, IL-4, IL-5 (Th2 immune-related factors) in mouse serum. Figure 3The results showed that the levels of all the above-mentioned factors in the OVA-induced model group mice were significantly higher than those in the normal control group, confirming that OVA successfully constructed an asthma inflammation model. RT-qPCR can detect the mRNA levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and IL-4 in lung tissue. Figure 4 The results showed that gene expression in the model group was significantly upregulated (P<0.05), which, together with serum detection, formed a "circulatory-tissue" dual-dimensional verification, confirming the successful construction of the asthma model.

[0037] Further comparison of treatment effects revealed that the levels of inflammatory factors in both the conventional mitochondrial therapy group and the immunoengineered mitochondrial nMITO therapy group were significantly downregulated compared to the model group (P < 0.05); and the downregulation in the nMITO therapy group was significantly greater than that in the conventional mitochondrial therapy group (P < 0.05).

[0038] Experimental data show that, compared with ordinary mitochondria, nMITO can more significantly reduce the levels of pro-inflammatory factors and Th2 immune-related factors in the serum of asthma model mice, directly proving that nMITO has a superior effect in inhibiting systemic inflammatory response in asthma and regulating Th2 immune imbalance. Its anti-inflammatory enhancement properties are strongly supported, highlighting the technical advantages of nMITO in the treatment of asthma.

[0039] In summary, the present invention possesses the following indispensable technical advantages: 1) Unique Fusion Preparation Technology: This invention utilizes a specific process to fuse mitochondria with neutrophil membranes to prepare immunoengineered mitochondria (nMITO). This preparation process involves the precise extraction and purification of mitochondria and neutrophil membranes, as well as fusion operations under specific conditions. This ensures that nMITO inherits the cell repair function of mitochondria and the damage-targeting and anti-inflammatory properties of neutrophil membranes, which are fundamental technical steps for achieving subsequent therapeutic effects. For example, when obtaining mitochondria, appropriate tissue sources and separation techniques must be used to ensure the high activity and integrity of the mitochondria; for the extraction of neutrophil membranes, it is necessary to ensure that key components such as surface proteins are not damaged to maintain targeting and anti-inflammatory functions. The conditional parameters of the entire fusion process, such as temperature, fusion time, and mixing ratio, are all key technical points.

[0040] 2) Targeting airway inflammation sites: nMITO possesses the ability to precisely target airway inflammation sites in asthma patients, relying on specific proteins and receptors derived from the neutrophil membrane on its surface. These components can recognize and bind to corresponding ligands on the surface of abnormally activated immune cells and damaged airway epithelial cells in the airway, thereby achieving high enrichment of nMITO at the inflammation site. This is a key innovation, distinguishing it from traditional treatments and conventional mitochondrial therapy.

[0041] 3) Multidimensional immune regulation mechanism: nMITO regulates the inflammatory microenvironment and damaged cell function in asthma through multiple pathways. It can inhibit over-activated immune cells in the airway, such as eosinophils and Th2 cells, and reduce the release of pro-inflammatory factors (such as IL-4 and IL-5). This multidimensional mechanism of action is the core technical advantage of this invention, fundamentally improving the pathological state of asthma.

[0042] 4) Application Forms of Asthma Treatment Drugs: This invention covers techniques for preparing nMITO into drug formulations suitable for treating asthma, including but not limited to selecting suitable carriers and excipients, determining optimal drug formulations and preparation processes to ensure the stability, activity, and bioavailability of nMITO in pharmaceutical preparations. For example, how to formulate nMITO into an aerosol to efficiently and accurately deliver it to the site of airway lesions; or developing suitable sustained-release formulations to prolong the duration of action of nMITO in vivo and maintain a lasting therapeutic effect. These application forms of drugs for asthma treatment and related preparation techniques are key areas for protection.

[0043] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for preparing immunoengineered mitochondrial nMITO, characterized in that: At least the following steps are included: S1: Mitochondria were extracted from the hearts of 5-8 week old male C57 mice; S2: Neutrophils were isolated from mouse bone marrow, activated with LPS, and the cell membrane was extracted. S3: Mix mitochondria and neutrophil membranes at a protein ratio of 1:1 and sonicate them in an ice-water bath for 2 minutes. S4: Centrifuge at 3500g for 10 minutes at 4℃. After centrifugation, precipitate the precipitate and wash it twice with PBS to obtain nMITO.

2. The method for preparing immunoengineered mitochondrial nMITO according to claim 1, characterized in that: S1 includes at least the following steps: Male C57 mice aged 5-8 weeks were selected. After disinfection with 75% alcohol, the heart was quickly removed, placed in pre-cooled PBS, washed 5 times, and the residual blood in the heart was quickly pumped out. After being cut into pieces, the heart was placed in an ice bath for 3 minutes. Centrifuge at 600g for 20 seconds at 4°C, then discard the PBS; Then add 800 μL of pancreatic enzyme digestion solution and digest in an ice bath for 5 minutes; Centrifuge at 600g for 20 seconds at 4℃ and discard the pancreatic enzyme digestion solution; Add 1 mL of mitochondrial separation reagent and wash away any residual trypsin; Then homogenize the mixture and centrifuge it at 1000g for 5 minutes at 4°C. Collect the supernatant and centrifuge it at 13500g for 10 minutes at 4°C. The final precipitate is the mitochondria.

3. The method for preparing immunoengineered mitochondrial nMITO according to claim 1, characterized in that: S2 includes at least the following steps: Mice were anesthetized with ether, euthanized by cervical dislocation, and the tibia and femur of the mice were quickly separated to remove excess muscle tissue. The separated tibia and femur were immersed in glass dishes filled with RPIM 1640 medium; Use micro forceps to remove all the tissue around the bone and cut off the joints at both ends of the bone to expose the medullary cavity; Rinse the medullary cavities of the tibia and femur with culture medium; After obtaining the bone marrow suspension, neutrophils were separated by density gradient centrifugation. Neutrophils were activated with 1 μg / mL LPS; Add 5 μL of separation buffer containing PMSF, sonicate for 5 minutes to lyse the cells; Finally, the neutrophil membrane was obtained by centrifugation at 10000g for 10 minutes at 4°C.

4. An immunoengineered mitochondrial nMITO, characterized in that: nMITO is prepared using the method described in any one of claims 1-3 for preparing immunoengineered mitochondrial nMITO.

5. An application of a method for preparing immunoengineered mitochondrial nMITO, characterized in that: The immunoengineered mitochondrial nMITO described in claim 4 is used in the preparation of asthma drugs.

6. An evaluation method for immunoengineered mitochondrial nMITO, used for the immunoengineered mitochondrial nMITO described in claim 4, characterized in that: At least the following steps are included: Construct an OVA-induced mouse model of asthma; Separation of bronchoalveolar lavage fluid from mice.

7. The method for evaluating immunoengineered mitochondrial nMITO according to claim 6, characterized in that: The construction of the OVA-induced asthma mouse model includes at least the following steps: Prepare the adjuvant by continuously adding 4 mg of alum to ovalbumin solutions of different masses and mixing continuously for 30 minutes. Initially, 50 μg of ovalbumin was injected into the peritoneum of C57 mice. The ovalbumin was adsorbed onto 4 mg of alum, and the total volume for each mouse was 200 μL. On day 10, mice were injected intraperitoneally with 25 μg of ovalbumin adsorbed onto 4 mg of alum. On days 21, 22 and 23, C57 mice were given 25 μg ovalbumin via endotracheal tube. Within 1 hour after challenge, MITO or nMITO 1 mg / kg was administered via tail vein injection. On day 25, samples were collected and the mice were euthanized. The samples collected included orbital venous blood, bronchoalveolar lavage fluid, and tissues from the heart, liver, spleen, lungs, and kidneys. Serum was collected by centrifugation to measure inflammatory factors TNF-α and IL-1β, as well as specific indicators IgE and IL-4 and IL-5. Heart, liver, spleen, lungs and kidneys were collected, weighed and photographed to assess the indices of each organ. Fresh lung tissue was collected to detect inflammatory factors TNF-α, IL-6, IL-1β and IL-4, and lung tissue was fixed with 4% paraformaldehyde for pathological staining.

8. The method for evaluating immunoengineered mitochondrial nMITO according to claim 6, characterized in that: The separation of mouse bronchoalveolar lavage fluid includes at least the following steps: After anesthetizing the mice, they were fixed in a supine position on the operating table. After disinfecting the neck skin with 75% alcohol, make a small incision of 0.5-1 cm longitudinally, bluntly peel off the neck skin of the mouse from the midline of the neck to the left and right sides, separate the smooth muscle around the trachea, and expose the trachea. Tie the uppermost end of the trachea with sutures, draw 1 mL of pre-cooled PBS with a 1 mL syringe with a needle, insert it into the trachea at a 30° angle, and then slowly inject the PBS into the mouse's lungs after flattening the needle. At this point, the lungs can be seen bulging; Then slowly draw it back into the syringe; A PBS recovery rate >80% is considered acceptable. Collect the PBS in an EP tube and repeat this process three times.

Images