Platelet membrane coated mitochondrial compound, and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a platelet membrane-coated mitochondrial compound and a preparation method and application thereof, and the platelet membrane-coated mitochondrial compound comprises mitochondria extracted from mammalian tissues and a platelet membrane coating the outer layer of the mitochondria. According to the present invention, the high kidney injury targeting enrichment rate is achieved through the PMITO compound constructed by coating the mitochondria with the platelet membrane, the healthy mitochondria is accurately delivered to the focus, and the energy crisis and the cell apoptosis are directly reversed; meanwhile, the biomimetic membrane structure can actively adsorb key inflammatory factors such as TNF-alpha and IL-1beta to synergistically realize dual mechanisms of energy repair and inflammation regulation, so that renal function indexes are remarkably improved in an animal model, and tissue pathological damage is relieved; finally, a new strategy for treating the renal ischemia-reperfusion injury, which is high in targeting property, clear in curative effect and good in biological safety, is formed.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a platelet membrane-coated mitochondrial complex, its preparation method, and its applications. Background Technology

[0002] Renal ischemia-reperfusion injury (RIRI) is the leading cause of acute kidney injury (AKI). Its core pathology lies in the explosive oxidative stress, uncontrolled inflammatory cascade, and renal tubular epithelial cell apoptosis triggered after blood flow restoration. Current clinical treatment strategies, such as diuretics, anti-inflammatory drugs, and antioxidants, mostly target single pathological processes, resulting in systemic bottlenecks such as poor targeting, significant systemic side effects, and an inability to synergistically address multiple injury mechanisms. Consequently, approximately 30%-40% of severely ill patients do not respond to treatment.

[0003] In recent years, mitochondrial transplantation-based cellular energy therapy has provided a new approach to fundamentally repairing renal irritable renal tract infection (RIRI). Healthy mitochondria can directly provide adenosine triphosphate (ATP) to damaged renal tubular epithelial cells, reversing cellular energy metabolism disorders. For example, existing technology (Kubat GB, et al. Toxicol Appl Pharmacol, 2021) discloses a method of directly injecting isolated mitochondria into the renal cortex to treat RIRI in rats. However, this direct transplantation strategy has significant limitations: free mitochondria lack active chemotaxis and have extremely low enrichment efficiency in kidney tissue (usually less than 10%); exogenous mitochondria are easily recognized and cleared by the host immune system, and have a short half-life in vivo (usually less than 30 minutes); mitochondria alone mainly provide energy support and cannot effectively address the complex inflammatory microenvironment in the RIRI lesion area.

[0004] To improve the targeted delivery efficiency of therapeutic agents, existing technologies have explored various biomimetic nanodelivery systems. For example, patent document CN116392610A discloses a "xenon-loaded platelet membrane biomimetic ultrasonic microbubble," which utilizes the targeted delivery of xenon gas with anti-inflammatory and cytoprotective effects via the platelet membrane, while also possessing ultrasound imaging capabilities. However, the core therapeutic component of this technology is gas (xenon), and its mechanism of action is completely different from mitochondrial energy repair. The microbubble structure is complex and requires targeted ultrasonic destruction to release its contents, thus failing to achieve delivery to functional organelles (such as mitochondria) and direct remodeling of intracellular energy metabolism.

[0005] Patent document CN118319876A discloses an "irisin biomimetic nanomedicine" that delivers irisin peptides via a metal-organic framework (MOF) coated on a macrophage membrane to target damaged kidneys and improve mitochondrial function. The core of this technology is the delivery of a protein signaling molecule that regulates mitochondrial function, and its effect is indirect. The carrier itself does not possess core mitochondrial functions such as ATP synthesis, and its therapeutic effect relies on the slow regulation of endogenous mitochondria in the host cell by irisin, failing to reverse the cellular energy crisis as quickly and directly as directly replenishing healthy mitochondria.

[0006] In summary, it is necessary to propose a platelet membrane-coated mitochondrial complex capable of targeted delivery of bioactive mitochondria to the site of kidney injury, while also possessing the ability to regulate the inflammatory microenvironment, along with its preparation method and applications. Summary of the Invention

[0007] The purpose of this invention is to provide a platelet membrane-coated mitochondrial complex, its preparation method, and its application. The PMITO complex constructed by coating mitochondria with platelet membrane achieves a high-efficiency targeted enrichment rate for renal injury, precisely delivering healthy mitochondria to the lesion, increasing the ATP level of renal tubular cells by 2-3 times, and directly reversing energy crisis and apoptosis. At the same time, its biomimetic membrane structure can actively adsorb key inflammatory factors such as TNF-α and IL-1β, synergistically achieving a dual mechanism of energy repair and inflammation regulation. In animal models, it significantly improves renal function indicators (creatinine, blood urea nitrogen) and reduces tissue pathological damage, ultimately forming a new strategy for the treatment of renal ischemia-reperfusion injury with strong targeting, clear efficacy, and good biosafety.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: a platelet membrane-coated mitochondrial complex, comprising mitochondria extracted from mammalian tissue, and a platelet membrane coating the outer layer of the mitochondria.

[0009] The basic principle of this technical solution lies in the synergistic effect of biomimetic camouflage and active targeting: using the natural cell membrane extracted from mammalian platelets as an outer shell to completely encapsulate and protect the functional mitochondria extracted from the tissue.

[0010] Platelet membranes inherit the surface protein characteristics of platelets, which can effectively evade the recognition and clearance of the body's immune system and significantly prolong the circulating half-life of mitochondria in the body.

[0011] When renal ischemia-reperfusion injury occurs, the damaged vascular endothelium is exposed and highly expresses adhesion molecules. Natural adhesion proteins on the platelet membrane surface (such as P-selectin and integrins) can actively recognize and bind to these sites, precisely navigating the complex to and enriching it in the renal lesion area.

[0012] Delivered to healthy mitochondria of damaged cells, it directly provides them with ATP, fundamentally repairing the cells' energy metabolism disorders; at the same time, the platelet membrane itself can also adsorb and neutralize local inflammatory factors (such as TNF-α, IL-6), synergistically achieving the dual therapeutic effects of energy replenishment and inflammation suppression.

[0013] Furthermore, the platelet membrane is derived from platelets of the same or different mammals.

[0014] Furthermore, the platelet membrane is derived from rat or human platelets.

[0015] Furthermore, the mitochondria are derived from mammalian heart tissue.

[0016] A method for preparing a platelet membrane-coated mitochondrial complex includes the following steps: (1) Extracting mitochondria from mammalian heart tissue; (2) Extracting platelet membranes from mammalian blood; (3) The mitochondria and the platelet membrane are mixed and fused to form a coating structure; (4) The fusion product was centrifuged and washed to purify the platelet membrane-coated mitochondrial complex.

[0017] Furthermore, the mitochondria and platelet membranes are mixed in a protein ratio of 1:1.

[0018] Furthermore, the fusion process involves ultrasonic treatment for 2 minutes in a 4°C water bath.

[0019] Furthermore, the centrifugation and washing after fusion were repeated twice, with each instance involving centrifugation at 4°C and 3500g for 10 minutes, followed by resuspending and washing with PBS in the precipitate.

[0020] This preparation method successfully reconstructs two independent biological components—functional mitochondria and platelet membrane with targeting capabilities—into a stable and efficient single therapeutic entity through a standardized and controllable process.

[0021] By leveraging physical force-driven membrane fusion and utilizing platelet membranes as a natural carrier, mitochondria can be biomimeticly camouflaged. A 1:1 protein ratio between mitochondria and platelet membranes is crucial for achieving efficient coating. This ratio ensures that the vast majority of mitochondria are completely covered by the membrane to gain immune camouflage capabilities, while avoiding aggregation or functional masking caused by excessive membrane material. This achieves the optimal solution between maximizing efficacy and controlling costs.

[0022] The subsequent ultrasound treatment in a 4°C water bath is a step to induce fusion. The brief ultrasound energy disturbs the lipid bilayer at the microscopic level, creating kinetic conditions for the fusion of the two membranes. The continuous low temperature environment provides crucial bioactive protection for the fusion process, effectively maintaining the respiratory function of mitochondria and the targeted recognition activity of membrane proteins, and preventing thermal damage that could lead to a decrease in therapeutic efficacy.

[0023] The final centrifugation and washing purification steps are crucial for ensuring the quality and safety of the final product. Repeated operations under specific centrifugal forces aim to achieve precise physical sorting, successfully separating the structurally intact target complex from unfused mitochondria, excess membrane fragments, and free proteins, thereby obtaining a final formulation with uniform composition and qualified purity, laying a solid foundation for its safe and effective application in vivo.

[0024] The use of a platelet membrane-coated mitochondrial complex in the preparation of medicaments for the prevention and / or treatment of renal ischemia-reperfusion injury.

[0025] Furthermore, the renal ischemia-reperfusion injury described herein is acute kidney injury.

[0026] The beneficial effects of the present invention are: (1) By utilizing the natural inflammatory homing characteristics of platelet membrane, it can actively identify and enrich the renal ischemia-reperfusion injury lesion, increase the effective concentration in renal tissue by 8-10 times, and the enrichment rate in renal tissue is as high as 60% or more, fundamentally solving the problem of traditional drugs being distributed throughout the body and delivered inefficiently in local areas.

[0027] (2) The delivered healthy mitochondria can be directly taken up by the damaged renal tubular epithelial cells, increasing their intracellular ATP level by 2-3 times, fundamentally reversing cell apoptosis caused by energy metabolism disorders, and providing direct power for tissue repair.

[0028] (3) The platelet membrane structure can effectively adsorb key inflammatory factors such as TNF-α, IL-6, and IL-1β, significantly reduce local inflammatory infiltration, and synergistically treat kidney injury from another key pathological link.

[0029] (4) It innovatively integrates the two major therapeutic mechanisms of energy repair and inflammation regulation (PMITO), realizing synergistic intervention of multiple pathological pathways in renal ischemia-reperfusion injury, and the therapeutic effect is significantly better than that of single-mechanism therapy.

[0030] (5) Platelet membrane, as a natural biological material of its own origin, endows exogenous mitochondria with "self" identification, which can effectively evade the recognition and clearance of the immune system, significantly prolong the circulating half-life of mitochondria in the body, and reduce the risk of immunogenic reactions, thus having high safety. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the preparation method of platelet membrane-coated mitochondrial complex (PMITO) in Example 1 of the present invention.

[0032] Figure 2 This is a kidney image of an animal model of renal ischemia-reperfusion injury treated with PMITO in Experiment Example 1 of this invention.

[0033] Figure 3 This is a bar chart of the kidney index in an animal model of renal ischemia-reperfusion injury treated with PMITO in Experiment Example 2 of this invention.

[0034] Figure 4 The bar chart shows the changes in serum creatinine (CRE) and blood urea nitrogen (BUN) levels in the animal model of renal ischemia-reperfusion injury treated with PMITO in Experiment Example 3 of this invention (ns P>0.05, *P<0.05, **P<0.01, ***P<0.001).

[0035] Figure 5 The bar chart shows the changes in serum TNF-α, IL-6, and IL-1β levels in an animal model of renal ischemia-reperfusion injury treated with PMITO in Experiment Example 4 of this invention (ns P>0.05, *P<0.05, **P<0.01, ***P<0.001).

[0036] Figure 6 This is a HE staining micrograph of the kidney in an animal model of renal ischemia-reperfusion injury treated with PMITO in Experiment Example 5 of this invention. Detailed Implementation

[0037] The specific implementation method is described below with reference to the accompanying drawings.

[0038] Example 1 This embodiment provides a method for preparing platelet membrane-coated mitochondrial complex (PMITO), the preparation process of which is shown in the attached figure. Figure 1 As shown, the preparation method includes the following steps: (1) Extraction of mitochondria: a) Deeply anesthetize 6-8 week old SD rats with an appropriate amount of anesthetic, immediately remove the heart, and rinse with pre-cooled PBS buffer to remove blood.

[0039] b) After cutting the heart tissue into small pieces, place it in pre-cooled PBS, centrifuge at 4°C and 600 g for 20 seconds, and discard the supernatant.

[0040] c) Add 8 times the volume of pre-cooled trypsin digestion solution to the tissue precipitate, resuspend and mix well, and digest in an ice bath for 5 minutes.

[0041] d) Centrifuge at 4°C and 600g for 20 seconds and discard the supernatant.

[0042] e) Add 8 times the volume of pre-cooled mitochondrial lysis buffer to the precipitate and homogenize 20-30 times under ice bath conditions.

[0043] f) Centrifuge the tissue homogenate at 4°C and 1000 g for 5 minutes and carefully collect the supernatant.

[0044] g) Transfer the supernatant to a new centrifuge tube and centrifuge at 4°C and 3500 g for 10 minutes. Discard the supernatant; the resulting precipitate is the myocardial mitochondria.

[0045] (2) Preparation of platelet membrane: a) Under appropriate anesthesia, the whiskers of 6-8 week old SD rats were trimmed. 4 mL of blood was collected from the inner canthus of the eye using a capillary tube and placed in a 10 mL centrifuge tube containing 450 μL of anticoagulant. The tube was gently inverted to mix and prevent coagulation.

[0046] b) Centrifuge whole blood at room temperature and 450 g for 10 minutes. Carefully collect the upper plasma layer and discard the lower blood cell precipitate.

[0047] c) Add appropriate PBS to the plasma collected in the previous step, mix well, centrifuge at 400 g for 8-10 minutes, and collect the supernatant.

[0048] d) Centrifuge the supernatant at 2600 g for 10 minutes and discard the supernatant. Add an appropriate amount of red blood cell lysis buffer to the precipitate, lyse on ice for 10 minutes, then centrifuge again at 2600 g for 10 minutes and discard the supernatant.

[0049] e) Add appropriate PBS to the precipitate, centrifuge at 2600 g for 10 minutes, discard the supernatant, and collect the precipitate.

[0050] f) Resuspend the precipitate with an appropriate amount of PBS, then perform three freeze-thaw cycles, sonicate for 10 minutes, centrifuge the suspension at 4000g for 10 minutes, and collect the supernatant.

[0051] g) Centrifuge the supernatant at 100,000 g for 20 minutes. The resulting precipitate is the platelet membrane.

[0052] (3) Preparation of platelet membrane-coated mitochondrial complex (PMITO): a) The platelet membrane and the extracted mitochondria were mixed at a protein ratio of 1:1, and then the mixture was sonicated in a 4°C water bath for 2 minutes.

[0053] b) Centrifuge the ultrasonicated mixture at 4°C and 3500 g for 10 minutes, discard the supernatant, and collect the precipitate.

[0054] c) Resuspend the precipitate in PBS and centrifuge for 10 minutes under the same conditions (4°C, 3500 g); repeat this washing process twice. The final collected precipitate is the purified platelet membrane-modified mitochondria.

[0055] Experimental Example 1 Animal model of renal ischemia-reperfusion injury treated with PMITO prepared in Example 1 The specific experimental steps are as follows: (1) SD rats were fasted for 12 hours before the experiment, but could drink water freely.

[0056] (2) After anesthetizing the SD rats, the skin was prepared and disinfected with 0.5% povidone-iodine.

[0057] (3) The rat made a midline incision with a scalpel, ligated the left kidney and then removed it. The right kidney pedicle (renal artery and vein) was clamped with a vascular clamp to block blood flow for 60 minutes.

[0058] (4) Administer the drug 5 minutes before reperfusion, then release the arterial clamp to allow the right kidney to be reperfused, and suture the incision layer by layer (in the Sham group, only the midline incision was made and no other treatment was done, and the incision was sutured).

[0059] (5) The PRP group was injected with 1 mg / kg of MITO and PMITO via the tail vein 5 minutes before reperfusion. The Sham group and Model group were injected with an equal amount of normal saline via the tail vein as a control. After that, the arterial clamp was released for reperfusion, and the midline abdominal incision was sutured layer by layer.

[0060] (6) The drugs were administered on days 0, 3, 7 and 10 after reperfusion. On day 14, the samples were weighed and collected, including serum, heart, liver, spleen, lung and kidney.

[0061] The experimental results are attached. Figure 2 As shown in the kidney images, the kidneys in the Sham group were plump and rosy in color, exhibiting a normal physiological appearance; the kidneys in the Model group were significantly swollen and dark in color, indicating tissue edema and microcirculatory disturbances. Compared to the Model group, the kidney swelling and color abnormalities in the MITO group were reduced, but the improvement was limited; the kidneys in the PMITO group were close to normal in volume, the swelling had largely subsided, the color had returned to a uniform level, and the appearance was closer to that of the Sham group. Comparing the MITO and PMITO groups, PMITO showed better kidney appearance repair, demonstrating that the enhanced platelet membrane coating provides tissue protection for mitochondria.

[0062] Experiment Example 2 Kidney index detection in an animal model of renal ischemia-reperfusion injury treated with PMITO prepared in Example 1. (1) Animal grouping and model construction: As in Experiment 1, SD rats were randomly divided into Sham group, Model group, MITO group and PMITO group. The right kidney ischemia-reperfusion injury model was constructed in the Model group, MITO group and PMITO group rats according to the surgical method described in Experiment 1. The Sham group only received sham surgery.

[0063] (2) Dosage regimen: Same as in Experiment 1. Five minutes before reperfusion and on days 0, 3, 7 and 10 after reperfusion, the MITO group and PMITO group were injected with the corresponding preparation (1 mg / kg) via tail vein, while the Sham group and Model group were injected with the same amount of saline.

[0064] (3) Sample collection: On the 14th day after the last administration, i.e. after reperfusion, all surviving animals were deeply anesthetized.

[0065] (4) Organ weighing: Open the abdominal cavity, completely separate and remove the right kidney. Gently rinse with pre-cooled saline to remove surface blood, and blot dry with filter paper. Immediately weigh the kidney wet using a precision electronic balance and record the weight.

[0066] (5) Kidney index calculation: Record the final weight of the rats simultaneously. Calculate the kidney index using the following formula: Kidney index (%) = (Kidney wet weight (g) / Rat final body weight (g)) × 100% (6) Statistical analysis: All data are expressed as mean ± standard deviation. One-way ANOVA was performed using appropriate statistical software. The t-test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0067] The experimental results are attached. Figure 3 As shown, compared with the Model group, the renal index in the MITO group (approximately 0.60%) showed no statistically significant difference (ns P>0.05), but the value showed a slight decreasing trend, suggesting that the improvement effect of simple mitochondrial intervention on renal pathological enlargement was not statistically significant. The renal index in the PMITO group (approximately 0.50%) was significantly reduced (*P<0.05), indicating that platelet membrane coating of mitochondria can effectively reduce renal pathological enlargement caused by renal IRI and alleviate tissue pathological burden. Comparing the MITO group and the PMITO group, the renal index in the PMITO group was significantly lower than that in the MITO group (**P<0.01), suggesting that platelet membrane coating enhanced the effect of mitochondrial coating on improving renal pathological burden. This is related to the characteristic of platelet membrane targeting renal injury foci, further demonstrating the tissue protective advantage of PMITO.

[0068] Experimental Example 3 Detection of renal function (serum creatinine, blood urea nitrogen) levels in an animal model of renal ischemia-reperfusion injury treated with PMITO prepared in Example 1 (1) Animal model and drug administration: Animal grouping, model construction and drug administration regimen are the same as in Experiment 2.

[0069] (2) Blood sample collection: On the 14th day after reperfusion, about 3-5 mL of whole blood was collected through the abdominal aorta and injected into a vacuum blood collection tube without anticoagulant.

[0070] (3) Serum separation: The collected blood was left to stand at room temperature for 1-2 hours until it coagulated naturally, and then centrifuged at 4°C and 3000 rpm for 15 minutes. The supernatant was carefully aspirated, which is the serum, and aliquoted into EP tubes.

[0071] (4) Kidney function index testing: Serum creatinine detection: Serum creatinine levels were detected using the picric acid method (or a kit compatible with an automated biochemical analyzer). The procedure was strictly followed according to the kit instructions: an appropriate amount of serum was mixed with the working solution, reacted at a specific temperature, and the absorbance was measured at 510 nm. The creatinine concentration in the sample was calculated using a standard curve.

[0072] Blood urea nitrogen (BUN) detection: Serum BUN levels were measured using the urease-Bohrer colorimetric method (or a kit compatible with an automated biochemical analyzer). The procedure was strictly followed according to the kit instructions: an appropriate amount of serum was mixed with the reagent, and the absorbance was measured at 660 nm after the reaction. The BUN concentration in the sample was calculated using a standard curve.

[0073] (5) Statistical analysis: All data are expressed as mean ± standard deviation. One-way ANOVA was performed using appropriate statistical software. The t-test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0074] The experimental results are attached. Figure 4 As shown, serum creatinine levels were significantly lower in both the MITO and PMITO groups compared to the Model group (**P<0.01), suggesting that both mitochondrial (MITO) and platelet membrane-coated mitochondrial (PMITO) can effectively improve creatinine metabolism and alleviate renal function damage.

[0075] Blood urea nitrogen (BUN): Compared with the Model group, serum BUN levels in the MITO group showed no significant change (ns P>0.05), while serum BUN levels in the PMITO group were significantly lower (**P<0.01), suggesting that PMITO has a significant effect in reducing blood urea nitrogen, while MITO did not show a statistically significant improvement in this indicator. Comparing the MITO and PMITO groups, serum BUN levels in the PMITO group were significantly lower than those in the MITO group (*P<0.05), indicating that PMITO is superior to MITO in regulating blood urea nitrogen.

[0076] PMITO showed better results in improving renal function indicators (especially BUN), while MITO could effectively reduce serum creatinine levels; both were equally effective in regulating serum creatinine, but PMITO was more advantageous in regulating blood urea nitrogen.

[0077] Experiment Example 4 Detection of inflammatory factors (TNF-α, IL-6, IL-1β) levels in an animal model of renal ischemia-reperfusion injury treated with PMITO prepared in Example 1 (1) Animal model and drug administration: Animal grouping, model construction and drug administration regimen are the same as in Experiment 2.

[0078] (2) Blood sample collection and serum separation: The method is the same as in Experiment 3.

[0079] (3) Use ELISA to detect the concentration of inflammatory factors.

[0080] (4) Statistical analysis: All data are expressed as mean ± standard deviation. One-way ANOVA was performed using appropriate statistical software. The t-test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0081] The experimental results are attached. Figure 5 As shown, TNF-α: Compared with the Model group, there was no statistically significant difference in serum TNF-α levels in the MITO group (approximately 11 pg / ml) (ns P>0.05), while the serum TNF-α levels in the PMITO group (approximately 10 pg / ml) were significantly reduced (*P<0.05), suggesting that PMITO can effectively inhibit TNF-α-mediated early inflammatory responses.

[0082] IL-6: Compared with the Model group, there was no statistically significant difference in serum IL-6 levels between the MITO group and the PMITO group (both ns P>0.05), but the values ​​showed a decreasing trend, suggesting that MITO and PMITO may have some potential to improve the regulation of IL-6-mediated inflammatory response.

[0083] IL-1β: Compared with the Model group, serum IL-1β levels were significantly lower in both the MITO group (*P<0.05) and the PMITO group (**P<0.01), suggesting that both MITO and PMITO can effectively inhibit IL-1β-mediated inflammatory responses and alleviate renal tissue inflammatory damage. Comparing the MITO and PMITO groups, serum IL-1β levels in the PMITO group were significantly lower than those in the MITO group (*P<0.05), indicating that PMITO has a superior anti-inflammatory effect compared to MITO in regulating IL-1β levels.

[0084] PMITO showed a more significant effect in inhibiting TNF-α and IL-1β-mediated inflammatory responses, especially in regulating IL-1β; while MITO only had a significant inhibitory effect on IL-1β, and its regulatory effect on TNF-α and IL-6 was limited.

[0085] Experimental Example 5 Safety test of kidney HE staining in an animal model of renal ischemia-reperfusion injury treated with PMITO prepared in Example 1 (1) Animal model and drug administration: Animal grouping, model construction and drug administration regimen are the same as in Experiment 2.

[0086] (2) Kidney tissue collection: On the 14th day after reperfusion, after weighing, a portion of the right kidney tissue (usually near the hilum, including the cortex and medulla) was taken and immediately fixed in 4% paraformaldehyde phosphate buffer for paraffin embedding.

[0087] (3) Preparation of paraffin sections: Fixation and dehydration: After the tissue blocks were fixed in 4% paraformaldehyde for 24-48 hours, they were dehydrated by a series of ethanol solutions (70%, 80%, 90%, 95%, 100%).

[0088] Clearing and embedding: The dehydrated tissue was cleared with xylene and then embedded in molten paraffin.

[0089] Sectioning: The tissue block was continuously sectioned using a paraffin microtome to a thickness of 4 μm.

[0090] (4) Hematoxylin-eosin staining: Dewaxing and hydration: The sections were dewaxed in xylene and then hydrated in a gradient of ethanol (100%, 95%, 85%, 70%) to distilled water.

[0091] Hematoxylin staining: Immerse in Harris hematoxylin staining solution for 5-10 minutes, then rinse with tap water.

[0092] Differentiation and blueing: Differentiate with 1% hydrochloric acid ethanol for a few seconds, rinse with tap water, and then blue with 0.6% ammonia.

[0093] Eosin staining: After dehydration with graded ethanol, immerse in eosin staining solution for 1-3 minutes.

[0094] Dehydration, clearing and mounting: The slides are dehydrated again with a gradient of ethanol, cleared with xylene, and finally mounted with neutral resin.

[0095] (5) Pathological observation and image acquisition: Observe the pathological changes in kidney tissue under an optical microscope.

[0096] Key areas to observe and assess: glomeruli (whether they are congested and whether their structure is intact), renal tubules (whether epithelial cells are edematous, vacuolar degeneration, necrosis and shedding, and whether the lumen is dilated), and renal interstitium (whether there is edema and the degree of inflammatory cell infiltration).

[0097] Images from multiple fields of view are randomly captured using the microscope's digital camera system for recording and analysis.

[0098] The staining results are attached. Figure 6 As shown, in the Sham group: the glomerular structure was intact, the renal tubular epithelial cells were tightly arranged and regularly shaped, and there was no edema or inflammatory cell infiltration in the renal interstitium, which is consistent with the histological characteristics of normal renal tissue.

[0099] Model group: glomerular congestion, mild dilation of the mesangial area, extensive edema and vacuolar degeneration of renal tubular epithelial cells, necrosis and shedding of some epithelial cells, tubular dilation, significant edema of renal interstitium, accompanied by a large number of inflammatory cell infiltration, indicating that the model was successfully constructed and the renal tissue structure and function were severely damaged.

[0100] MITO group: Compared with the Model group, the degree of renal tissue damage in the MITO group was significantly reduced, as evidenced by the relief of glomerular congestion and mesangial expansion, a reduction in the extent of edema and necrosis of renal tubular epithelial cells, and a significant decrease in renal interstitial edema and inflammatory cell infiltration. This indicates that mitochondria can alleviate kidney damage by improving the morphological structure of renal tissue.

[0101] PMITO group: The morphological improvement of renal tissue was more significant in the PMITO group, with near-normal glomerular structure, neatly arranged renal tubular epithelial cells, further reduction in edema and necrosis, and almost complete disappearance of renal interstitial edema and inflammatory cell infiltration. Compared with the MITO group, the morphological recovery of renal tubular epithelial cells in the PMITO group was more thorough, suggesting that the enhanced platelet membrane coating enhances the histological protective effect of mitochondria on damaged kidneys.

[0102] Histomorphological analysis stained with hematoxylin and eosin (HE) showed that PMITO could significantly alleviate histological damage to renal intraepithelial neoplasia (IRI) by improving pathological changes such as glomerular congestion, renal tubular epithelial cell necrosis, and interstitial inflammatory infiltration, and its protective effect was superior to that of MITO.

Claims

1. A platelet membrane-coated mitochondrial complex, characterized in that, This includes mitochondria extracted from mammalian tissues, as well as the platelet membrane that covers the outer layer of the mitochondria.

2. The platelet membrane-coated mitochondrial complex according to claim 1, characterized in that, The platelet membrane is derived from platelets of the same or different mammals.

3. The platelet membrane-coated mitochondrial complex according to claim 2, characterized in that, The platelet membrane is derived from rat or human platelets.

4. The platelet membrane-coated mitochondrial complex according to claim 1, characterized in that, The mitochondria are derived from the heart tissue of mammals.

5. A method for preparing a platelet membrane-coated mitochondrial complex as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Extracting mitochondria from mammalian heart tissue; (2) Extracting platelet membranes from mammalian blood; (3) The mitochondria and the platelet membrane are mixed and fused to form a coating structure; (4) The fusion product was centrifuged and washed to purify the platelet membrane-coated mitochondrial complex.

6. The method for preparing platelet membrane-coated mitochondrial complex according to claim 5, characterized in that, The mitochondria and platelet membranes were mixed at a protein ratio of 1:

1.

7. The method for preparing platelet membrane-coated mitochondrial complex according to claim 5, characterized in that, The fusion process involves ultrasonic treatment for 2 minutes in a 4°C water bath.

8. The method for preparing platelet membrane-coated mitochondrial complex according to claim 5, characterized in that, The centrifugation and washing after fusion were repeated twice, with each cycle involving centrifugation at 4°C and 3500g for 10 minutes, followed by resuspending and washing the precipitate with PBS.

9. The use of the platelet membrane-coated mitochondrial complex as described in any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of renal ischemia-reperfusion injury.

10. The application according to claim 9, characterized in that, The renal ischemia-reperfusion injury is acute kidney injury.

Images