An mMito-gel mitochondria delivery system, a preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and discloses an mMito-gel mitochondrion delivery system and a preparation method and application thereof. The mMito-gel mitochondrion delivery system comprises: membrane camouflage mitochondria, wherein the membrane camouflage mitochondria comprise mitochondria and a cell membrane component wrapped outside the mitochondria; and an inulin hydrogel matrix, wherein the membrane camouflage mitochondria are dispersed in the inulin hydrogel matrix. The mMito-gel mitochondrion delivery system of the application realizes efficient mitochondrion delivery through multi-level innovative technologies. Compared with a traditional treatment scheme relying on antibiotics, the mMito-gel system can not only control infection, but also fundamentally enhance energy supply of intestinal cells, effectively restore intestinal barrier function related to sepsis, significantly improve repair efficiency, and reduce systemic inflammatory response.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to an mMito-gel mitochondrial delivery system, its preparation method, and its application. Background Technology

[0002] Traditional antibiotic treatment primarily targets bacterial infections, but it fails to effectively repair the intestinal barrier dysfunction commonly seen in sepsis patients. Furthermore, antibiotic use can severely disrupt the balance of the gut microbiota, thereby exacerbating intestinal barrier damage and potentially causing other complications.

[0003] Current antibiotics offer limited effectiveness in repairing the intestinal barrier in sepsis and are prone to drug resistance, affecting long-term treatment outcomes. While some methods aim to repair sepsis-induced intestinal barrier damage, such as nutritional support and anti-inflammatory therapy, these methods largely focus on symptom relief and do not effectively restore energy metabolism in intestinal cells. Existing treatment regimens lack direct interventions targeting the energy supply to intestinal epithelial cells, resulting in a slow repair process and limited effectiveness.

[0004] In recent years, mitochondrial transplantation has gradually attracted attention as an emerging therapeutic strategy. Studies have shown that exogenous mitochondria can be taken up by damaged cells, thereby restoring cellular energy metabolism and function. Mitochondrial transplantation has shown therapeutic potential in various disease models, including myocardial ischemia, nerve injury, and acute kidney injury. However, existing mitochondrial transplantation methods mainly rely on intravenous or local injection, which face many challenges when applied to intestinal barrier repair in sepsis: insufficient stability, low delivery efficiency, and poor targeting.

[0005] Therefore, existing treatments and drug delivery technologies have failed to effectively address key issues in the repair of sepsis-related intestinal barrier damage, particularly how to stably deliver and utilize mitochondria to restore energy metabolism in intestinal cells. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing sepsis treatments, which mainly rely on antibiotics, are difficult to protect / rebuild the intestinal barrier, and are prone to disrupting the intestinal microecology, as well as the technical bottlenecks of oral delivery of active mitochondria, which are difficult to maintain activity in the gastrointestinal tract and are difficult to be effectively taken up by the intestinal epithelium.

[0007] To address the aforementioned objectives, this invention proposes an innovative oral mMito-gel mitochondrial delivery system that directly restores intestinal barrier function by protecting the integrity of mitochondria and ensuring their stable release into intestinal epithelial cells, thus providing a novel therapeutic strategy with significant advancements.

[0008] Specifically, this application adopts the following technical solution: In a first aspect, this application provides an mMito-gel mitochondrial delivery system, comprising: Membrane-masked mitochondria, wherein the membrane-masked mitochondria comprises mitochondria and cell membrane components surrounding the mitochondria; An inulin hydrogel matrix in which the membrane-masked mitochondria are dispersed.

[0009] Furthermore, the membrane-masked mitochondrial surface is modified with triphenylphosphine.

[0010] Furthermore, the inulin hydrogel matrix contains a protease inhibitor.

[0011] Furthermore, the protease inhibitor is aprotinin, and the final concentration of aprotinin in the inulin hydrogel matrix is ​​5 μg / mL.

[0012] Furthermore, the membrane-masked mitochondria are loaded at a rate of 1 × 10⁻⁶ per milliliter of inulin hydrogel matrix. 8 -1×10 9 A membrane disguises the mitochondria.

[0013] Secondly, this application provides a method for preparing the oral mMito-gel mitochondrial delivery system, comprising the following steps: Step 1: Isolate and extract mitochondria; Step 2: Separate and extract cell membrane components; Step 3: Incubate the mitochondria obtained in Step 1 with triphenylphosphine solution to obtain triphenylphosphine-modified mitochondria; Step 4: Mix the cell membrane component obtained in Step 2 with the triphenylphosphine-modified mitochondria obtained in Step 3, and use a membrane extrusion method to coat the outside of the mitochondria with the cell membrane component. After purification, membrane-disguised mitochondria are obtained. Step 5: Dissolve inulin and protease inhibitor in buffer solution, and then heat and cool the mixture sequentially to form a hydrogel from the inulin. Step 6: The membrane-masked mitochondria obtained in Step 4 are loaded into the inulin hydrogel obtained in Step 5, mixed evenly, and allowed to stand to stabilize, thus obtaining the oral mMito-gel mitochondrial delivery system.

[0014] Furthermore, the incubation conditions described in step 3 are: incubation at 4°C in an ice bath for 20 minutes; The membrane extrusion method described in step 4 uses a 1 μm polycarbonate membrane and is repeated 11 times. The heating treatment in step 5 is heating at 70°C for 10 minutes, and the cooling treatment is cooling to room temperature first and then placing at 4°C overnight to complete gelation.

[0015] Thirdly, this application provides the use of the oral mMito-gel mitochondrial delivery system in the preparation of a medicament for repairing intestinal barrier damage.

[0016] Furthermore, the drug is an oral medication.

[0017] In summary, this application has the following beneficial effects: The mMito-gel mitochondrial delivery system of this invention achieves highly efficient mitochondrial delivery through multi-level innovative technologies. First, mitochondria are encapsulated using mouse ileal cell membrane camouflage technology, forming a double-membrane structure that effectively protects them from degradation by gastric acid, bile salts, and digestive enzymes, while significantly enhancing their affinity for intestinal epithelial cells and uptake efficiency. Second, inulin hydrogel is used as a carrier, which not only possesses excellent resistance to gastric acid, degradability, and biocompatibility, but also continuously releases mitochondria in the intestinal environment for up to 12 hours, ensuring sustained repair support during the critical therapeutic window. Furthermore, the introduction of the aprotinin protease inhibitor further protects mitochondrial function, preventing degradation during delivery. More innovatively, this system establishes a dual mechanism: on the one hand, it directly provides exogenous functional mitochondria to restore intestinal cell energy metabolism; on the other hand, it activates the endogenous mitochondrial transport mechanism in macrophages, transferring mitochondria to intestinal epithelial cells via tunnel nanotubes (TNTs) and inhibiting the inflammatory response by regulating the mtDNA-cGAS-STING signaling axis.

[0018] This invention demonstrates significant advantages in clinical applications. Compared to traditional antibiotic-dependent treatments, the mMito-gel system not only controls infection but also fundamentally restores sepsis-related intestinal barrier function by enhancing energy supply to intestinal cells, significantly improving repair efficiency and reducing systemic inflammatory responses. The system uses naturally derived inulin as its base material, ensuring low immunogenicity and excellent biocompatibility, making it suitable for a wide range of patients, including those sensitive to chemically synthesized materials. It is not dependent on specific antibiotics and can be used in combination with different types of antibiotics, exhibiting strong clinical adaptability. Furthermore, the preparation process of this invention is simple and efficient, requiring no complex equipment or conditions. Through precise control of process parameters, it possesses good potential for industrial-scale production, meeting the needs of large-scale manufacturing. In summary, the mMito-gel mitochondrial delivery system, through innovative technological integration, provides a highly efficient, sustainable, and stable treatment option, with broad clinical application prospects in the treatment of sepsis-related intestinal barrier damage. Attached Figure Description

[0019] Figure 1 Preparation and characterization of mMito.

[0020] Among them, (A) Transmission electron microscopy (TEM) images of isolated mitochondria, cell membrane vesicles, and membrane-enclosed mitochondria (mMito). Red arrows indicate membrane-enclosed structures. Scale bar: left and middle images are 100 nm, right image is 200 nm. (B) Mito, isolated cell membrane (CM), and Connexin 43 and Na in mMito. + / K +- Representative Western blot results of ATPase protein. (C) Protein profiles obtained after SDS-PAGE and Coomassie Brilliant Blue staining, showing the protein composition of Mito, TPP-Mito, organelle membrane, cell membrane, and mMito. Experiments shown in (B) and (C) were repeated three times, and similar results were obtained. (D) Particle size and Zeta potential of mitochondria (Mito), TPP-Mito, and mMito (n = 3). (E) Activity of ATP synthase (Complex V) in mitochondria and mMito (n = 3). (F) Scanning electron microscopy (SEM) images of inulin-gel before and after loading mitochondria. (G) In vitro degradation kinetics of inulin-gel under five different conditions: PBS, simulated gastric juice (SGF), simulated intestinal juice (SIF), SIF containing inulinase, and SIF containing inulinase under simulated intestinal peristalsis conditions. The percentage of remaining weight of the hydrogel was determined at 0, 6, 12, 24, and 48 h (n = 3). (H) Schematic diagram of mitochondrial separation after in vitro digestion of inulin hydrogel in SIF for 4 h. The supernatant fraction (S1) was ultrafiltered and used for Western blot analysis of proteins; the precipitate fraction (P1) was the mitochondria released into the SIF. The mitochondria remaining in the hydrogel were released after inulinase treatment and collected as precipitate fraction P2. (I) Representative Western blot results of cytochrome c (Cyt c) in fractions S1, P1, and P2 with or without the addition of the protease inhibitor aprotinin (Apr) during digestion. (J–M) Mitochondria obtained under different treatment conditions were functionally evaluated, including the P2 group released from hydrogels treated with PBS (P2, PBS), the P2 group in hydrogels after 4 h of SIF treatment (P2, 4 h SIF), the P2 group in hydrogels after 4 h of SIF combined with aprotinin treatment (P2, 4 h SIF+Apr), and the P1 group released into the supernatant after SIF or SIF+Apr treatment. The assay parameters included: (J) Complex V activity; (K) ATP production capacity; (L) reactive oxygen species (ROS) levels; and (M) mitochondrial membrane potential (JC-1 assay) (n = 6). Data are expressed as mean ± standard deviation (mean ± SD). The statistical analysis methods are as follows: (E) Two-sided Mann–Whitney U test was used; (G) Two-way ANOVA was used with Tukey post-hoc multiple comparisons; (J–M) One-way ANOVA was used with Tukey multiple comparisons. P<0.0001, P<0.001, P<0.01, P<0.05; ns, P>0.05.

[0021] Figure 2 Oral administration of mMito-gel improved the survival rate and intestinal barrier function in septic mice.

[0022] (A) Schematic diagram of the experimental design; 6–8 week old female C57BL / 6J mice underwent cecal ligation and perforation surgery, and were orally administered mMito-gel twice, 4 h and 24 h postoperatively; blood was collected on postoperative day 1, ileal tissue was harvested on day 2, and survival was continuously monitored until day 7. (B) Kaplan-Meier survival curves (n = 15 per group) of mice in the Sham group, CLP group, Inulin-gel (PBS) group, Mito-gel group, and mMito-gel group, were statistically analyzed using the Log-rank (Mantel-Cox) test. (C) Quantitative analysis of peripheral blood leukocyte (WBC), monocyte (Mon), lymphocyte (Lym), and granulocyte (Gran) counts on day 1 postoperatively after CLP (Sham group n = 10, other groups n = 15). Data were presented as box plots (min-maximum), showing all data points; the center line represents the median, and the boxes represent the interquartile range (IQR). (D) Representative HE-stained images of ileal tissue from each group, showing overall tissue morphology (scale bar: 200 μm) and magnified intestinal epithelial structures (scale bar: 50 μm). (E) Transmission electron microscopy (TEM) images of tight junctions in each group. Representative observation areas are marked with red boxes. Scale bar: 2 μm. (F, G) Quantitative analysis of tight junction width and length (3 mice per group, 9–12 tight junctions measured per mouse). (H) Western blot results of tight junction proteins ZO-1, Claudin-1, and Occludin in ileal tissue. (I) Quantitative analysis of ATP content in ileal tissue from each group (n = 6). (J) Oxygen consumption rate (OCR) of mitochondria isolated from ileal tissue in the Sham group, CLP group, and mMito-treated CLP group. OCR was measured under basal conditions, and then detected after sequential addition of oligomycin, FCCP, and rotenone (ROT) combined with antimycin A (AA). (K) Quantitative analysis of maximum respiration and basal respiration in the OCR data. Mitochondrial respiration experiments were performed with n = 3 per group and 3 technical replicates at each time point. Data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test. P<0.0001, P<0.001, P<0.01, P<0.05; ns, no statistically significant difference (P>0.05).

[0023] Figure 3 Effects of mMito on intestinal epithelial cell migration, ROS and mitochondrial membrane potential induced by 2% CLP-MS.

[0024] Among them, (A) Representative images of scratch healing experiments performed on MODE-K cells treated with 2% CLP-MS at 0 h and 24 h after treatment with PBS, Mito, or different doses of mMito (20 or 30 μg). Cells were stained with green viable cell dye. Scale bar: 200 μm. (B) Quantitative analysis of scratch healing rate (%) at 24 h. (C–E) Effect of different doses of mMito (20 μg and 30 μg) on ​​intracellular reactive oxygen species (ROS) levels under 2% CLP-MS treatment. (C) Representative confocal microscopy images of DCFDA staining. Scale bar: 20 μm. (D) Representative flow cytometry histograms of DCFH-DA staining. (E) Quantitative analysis of the proportion of DCFDA-positive cells. (F–H) Detection results of mitochondrial membrane potential in MODE-K cells under the same treatment conditions. (F) Representative confocal microscopy images of JC-1 staining. (G) Representative scatter plot of JC-1 fluorescence transition from aggregates to monomers as detected by flow cytometry. (H) Quantitative results of JC-1 monomer proportion based on flow cytometry analysis. Data are expressed as mean ± standard deviation (mean ± SD) (n = 3). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparisons test. P<0.0001, P<0.001, P<0.01, P<0.05; ns, no statistically significant difference (P>0.05). Detailed Implementation

[0025] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0026] Example 1: Preparation of an oral mMito-gel mitochondrial delivery system I. Isolation and Extraction of Mitochondria Required reagents and materials: Solarbio Mitochondrial Isolation Kit (catalog number: SM0020); PBS buffer (pH 7.4).

[0027] Operating steps: 1. Tissue or cell sample preparation: Human umbilical cord mesenchymal stem cells (huMSCs) were used and the procedure was performed on ice.

[0028] 2. Mitochondrial isolation: Strictly follow the Solarbio Mitochondrial Isolation Kit instructions: Homogenize tissue or cell samples mechanically on ice; perform differential centrifugation using the isolation buffer provided in the kit; typically, centrifuge at 800 g for 10 minutes to remove cell nuclei and unbroken cells; collect the supernatant and centrifuge at 12000 g for 10 minutes to precipitate the mitochondria; wash the mitochondrial precipitate 2-3 times with PBS. Figure 1 (A in the middle).

[0029] 3. Mitochondrial concentration adjustment: The isolated mitochondria were resuspended in PBS, and particle counting was performed using a NanoSight NS 300 system (Malvern Instruments). The concentration was adjusted to 1×10⁻⁶. 10 Quantity / ml, store at 4°C for later use.

[0030] 4. Mitochondrial activity verification: A portion of mitochondrial samples were stained with MitoTracker Deep Red FM and the integrity and activity of the mitochondria were verified by confocal microscopy.

[0031] II. Separation and Extraction of Cell Membrane Components Required reagents and materials: Invent Minute TM Plasma membrane / protein and cell component separation kit (Catalog No.: SM-005) Operating steps: 1. Cell membrane separation: using Invent Minute TMThe isolation kit was operated according to the following steps: Fresh mouse ileal epithelial cells were taken and operated on ice; cell lysis and membrane component separation were performed according to the kit instructions; the cytoplasmic membrane / organelle vesicle pellet was separated by differential centrifugation; specific centrifugation conditions were as per the kit instructions (usually 700 g for 5 minutes to remove the nucleus; 16000 g for 15 minutes to collect the membrane component).

[0032] 2. Membrane component purification: Wash the membrane component precipitate 2-3 times with PBS to remove soluble proteins and other impurities.

[0033] 3. Adjustment of membrane component concentration: The purified cell membrane components were resuspended in PBS, and the protein concentration was determined by BCA protein quantification method. The concentration was adjusted to 200 μg / ml, aliquoted, and stored at -80°C for later use.

[0034] III. Preparation of Membrane-Fake Mitochondria (mMito) Required reagents and materials: isolated mitochondrial suspension (1×10⁻⁶) 10 Particles / ml), TPP solution (triphenylphosphine, 1 mg / ml, dissolved in PBS or a suitable solvent); cell membrane component suspension (200 μg / ml); 1 μm polycarbonate membrane (Whatman Inc., USA).

[0035] Detailed operating steps: 1. Mitochondrial pretreatment (TPP modification): Take 200 μl of mitochondrial suspension (1×10⁻⁶) 10 Add particles (1 mg / ml) to a 1.5 ml EP tube; add 200 μl of TPP solution (1 mg / ml); incubate at 4°C on ice for 20 minutes, gently mixing every 5 minutes during this period; Note: Maintain a low temperature of 4°C throughout the process to avoid damage to mitochondrial function.

[0036] 2. Washing of TPP-mitochondria: After incubation, centrifuge at 12000 g, 4°C for 10 minutes; carefully discard the supernatant to avoid disturbing the precipitate; resuspend the precipitate with pre-cooled PBS (500 μl); repeat the centrifugation and washing steps 2-3 times to ensure the removal of excess TPP.

[0037] 3. Fusion of membrane components with mitochondria: Resuspend the washed TPP-mitochondrial precipitate in 100 μl PBS; add 1 ml of cell membrane component suspension (200 μg / ml); incubate at 4°C for 10 minutes, gently mixing every 2-3 minutes; avoid vigorous shaking during incubation to prevent damage to the membrane structure. Figure 1 (A in the middle).

[0038] 4. Membrane extrusion and purification: The fusion mixture was extruded through a 1 μm polycarbonate membrane; the solution was slowly pushed through the filter membrane using a membrane extruder or syringe, and the extrusion was repeated 11 times; the extruded solution was centrifuged at 12000 g for 10 minutes at 4°C; the supernatant was discarded, and the precipitate was washed twice with PBS to remove unbound membrane components.

[0039] 5. mMito resuspending and concentration adjustment: The final mMito precipitate was resuspended in 100 μl PBS; particle counting was performed using a NanoSight NS 300 system, and the concentration was adjusted to 1×10⁻⁶. 9 Quantity / ml; after aliquoting, store at 4°C and use within 24 hours to ensure activity.

[0040] 6. mMito characterization: Samples were taken for transmission electron microscopy (TEM) observation to confirm the bilayer film structure. Figure 1 (A) Western blot results showed that membrane-encapsulated mitochondria (mMito) express cell membrane marker proteins Connexin 43 and Na+. + / K + -ATPase ( Figure 1 (B in the text); Coomassie Brilliant Blue staining results further showed that mMito not only retained the original protein composition of mitochondria, but also successfully integrated cell membrane proteins, indicating that the membrane fusion process was effectively completed. Figure 1 (C in the text). After membrane encapsulation, the average mitochondrial size increased from 380 nm to 420 nm. Figure 1 (D in the text). Zeta potential analysis showed that TPP treatment effectively reduced the negative charge on the mitochondrial surface, which is beneficial for the binding and encapsulation of cell membrane structures (D). Figure 1 (D in the original text). More importantly, we demonstrated that this rapid modification method can maintain the functional integrity of mitochondria. ATP synthase activity assays showed that the entire modification process (taking less than 40 min) did not affect mitochondrial ATP synthesis capacity. Figure 1 (E in the text).

[0041] IV. Preparation of mMito-Inulin Hydrogel (mMito-gel) Required reagents and materials: Inulin (pharmaceutical grade); PBS buffer (pH 7.4); Aprotinin protease inhibitor (5 μg / ml, MCE, catalog number: HY-P0017); mMito suspension (1×10⁻⁶). 9 Particles / ml); vortex mixer; thermostatic water bath or heating block; 1.5 ml or 2 ml EP tubes.

[0042] Detailed operating steps: 1. Preparation of Inulin-Aprotinin solution: Accurately weigh 2 mg of inulin powder and place it in a 2 ml EP tube; add 1 ml of PBS buffer (pH 7.4); add aprotinin to a final concentration of 5 μg / ml (calculate the volume to be added based on the concentration of the original solution); place the EP tube on a vortex mixer and vortex continuously at room temperature for 2 hours, observing the state of the solution during vortexing until all visible inulin aggregates completely disappear and the solution becomes transparent or translucent.

[0043] 2. Pre-gelation treatment: Transfer the fully dissolved Inulin-Aprotinin solution to a clean 1.5 ml or 2 ml EP tube, and place the EP tube in a 70°C water bath or heating block for 10 minutes. Note: Gently shake the EP tube during heating to promote uniform heating, but avoid splashing the solution.

[0044] 3. Gelation Process: After heating, immediately remove the EP tube from the 70°C environment; allow the EP tube to cool naturally at room temperature (20-25°C); then transfer the EP tube to a 4°C refrigerator overnight (12-16 hours) to promote complete gelation. Key observation point: After gelation, the solution should change from a liquid state to a semi-solid or solid gel, which can be determined by observing the flowability of the EP tube by tilting it.

[0045] 4. mMito encapsulation: Take a gelled Inulin hydrogel EP tube; use a pipette to draw an appropriate amount of mMito suspension (1×10⁻⁶). 9 (particles / ml); inject the mMito suspension directly into the hydrogel matrix; loading capacity: determined according to experimental requirements, typically 1×10⁻⁶ particles per milliliter of hydrogel. 8 -1×10 9 Particle mMito; Immediately place the EP tube on the vortex mixer and vortex at medium intensity for 10 seconds; 5. Stabilization of the final product: After vortexing, the mMito-gel was placed at 4°C and allowed to stand for 30 minutes to allow the gel structure to be restabilized.

[0046] 6. Characterization and functional verification of the final product: Scanning electron microscopy (SEM) observation showed that the inulin-gel has a rich porous microstructure, suitable for loading and embedding mitochondria. Figure 1 The F in the figure. Further hydrogel weight loss experiments showed that Inulin-gel exhibits significant pH-responsive degradation characteristics, with approximately 50% degradation occurring in a simulated intestinal environment after about 12 hours. Figure 1(G in the text). This sustained-release property is particularly suitable for the treatment needs of acute intestinal inflammation. Subsequently, we further evaluated the structural and functional integrity of mitochondria under simulated intestinal release conditions. Figure 1 The results of Western blot analysis of Cytochrome c showed that, compared with the group without aprotinin (Apr), the release of Cytochrome c in the supernatant of the Apr-added group (+Apr) was significantly reduced, indicating that Apr can effectively protect the integrity of the mitochondrial membrane and reduce the leakage of Cytochrome c. Figure 1 (I) To assess the functional stability of mitochondria during the critical transition from hydrogel release to cellular uptake, we evaluated the function of released mitochondria using unprotected mitochondria suspended in PBS as a control. Figure 1 (JM in the text). These results demonstrate that the introduction of Apr into the hydrogel plays a crucial role in reducing mitochondrial functional loss during the release phase. Although mitochondrial activity remained slightly decreased after release, their overall bioenergetic function was well preserved, fully demonstrating the excellent protective efficiency of this delivery platform and providing a reliable basis for further in vivo therapeutic efficacy evaluation.

[0047] Example 2: mMito-gel restores intestinal barrier integrity and improves survival rate in CLP mice A cecal ligation-perforation (CLP) sepsis model was established using 6-8 week old female C57BL / 6J mice to systematically evaluate the therapeutic potential of mitochondrial supplementation therapy in sepsis. Following systemic inflammation induction, mMito-gel treatment was administered at 4 h and 24 h post-surgery. Figure 2 (A) The survival of mice was continuously monitored for 7 days, and blood samples were collected on the first day (24 h) after surgery, and ileum tissue was collected on the second day (48 h) for mechanistic studies. Figure 2 (A) CLP induction led to severe mortality, with only 13.3% of mice surviving to day 7, compared to 100% survival in the sham-operated group. Inulin-gel (PBS) treatment showed no therapeutic effect, Mito-gel provided only a mild and statistically insignificant improvement, while mMito-gel treatment significantly improved mouse survival, increasing the 7-day survival rate to 53.3% (P=0.03). Figure 2 (B in the text). Notably, peripheral blood leukocyte analysis showed that CLP led to a significant leukopenia, manifested as a significant decrease in total white blood cell (WBC), monocyte, and lymphocyte counts compared to the Sham group. Figure 2 C in the text). mMito-gel can partially restore the total white blood cell and lymphocyte counts (C). Figure 2(C) further confirms that mitochondrial replacement therapy helps alleviate immune exhaustion caused by sepsis. These results indicate that mMito treatment not only significantly improves the survival rate of septic mice but also restores immune cell homeostasis.

[0048] To comprehensively evaluate intestinal barrier function, we performed a combined histological, ultrastructural, and molecular analysis of the intestinal mucosal barrier on day 2 post-CLP. HE staining results showed that CLP caused severe inflammatory damage to the intestinal mucosa, manifested as significant villous atrophy, epithelial shedding, and crypt structural disorder. Figure 2 (D in the text). Transmission electron microscopy (TEM) further revealed that the tight junction structures were significantly damaged, the intercellular spaces were widened, and the continuity of the junctional complex was disrupted. Figure 2 The expression of key tight junction proteins ZO-1, Claudin-1, and Occludin was significantly downregulated, suggesting that intestinal barrier function is severely impaired at the molecular level. Figure 2 Inulin-gel failed to improve CLP-induced intestinal damage. In contrast, Mito-gel could protect the intestinal barrier to some extent, while mMito-gel showed superior and synergistic protective effects at multiple levels, including tissue structure, ultrastructure, and molecular markers. Figure 2 These results indicate that efficient targeted delivery achieved by mMito-gel is a key factor in restoring the integrity of the intestinal barrier in sepsis. To further clarify whether mitochondrial therapy can improve ileal energy metabolism, we examined ATP content and mitochondrial respiratory function in the ileum. The results showed that ATP levels in the ileum and basal, maximal, and reserve respiratory capacities of mitochondria were significantly decreased in both the CLP and PBS groups. Figure 2 (IK in the middle).

[0049] Experimental steps: 1. Animal experiments and establishment of a CLP sepsis model Female C57BL / 6J mice aged 6–8 weeks were selected for in vivo experiments. Mice were randomly divided into 5 groups: Sham group, CLP group, Inulin-gel (PBS) group, Mito-gel group, and mMito-gel group. For survival experiments, each group contained 15 mice; for peripheral blood cell analysis, the Sham group contained 10 mice, and the other groups contained 15 mice; for tissue ATP assays, each group contained 6 mice; and for mitochondrial respiratory function assays, each group contained 3 mice, with 3 technical replicates.

[0050] Before establishing the CLP model, mice were anesthetized with 3% isoflurane under 60% oxygen. After adequate anesthesia, the abdominal hair was shaved, and the skin was disinfected with povidone-iodine. The skin and abdominal wall were then incised along the midline of the abdomen to expose the cecum. Approximately 50% of the cecum tissue was ligated with 4-0 silk sutures, and the cecum was punctured twice with a 21G needle, gently squeezing out approximately 10 μL of fecal contents to induce abdominal infection and sepsis. The cecum was then repositioned, and the abdominal wall and skin were sutured in two layers to complete the surgical procedure. Mice in the Sham group underwent only laparotomy, without cecal ligation, puncture, or squeezing out of fecal contents. Immediately after surgery, mice were given 1 mL of pre-warmed Ringer's solution for fluid resuscitation and subcutaneously injected with ibuprofen / buprenorphine (0.05 mg / kg). All mice were kept in a warm environment for recovery and were routinely observed postoperatively.

[0051] 2. Animal grouping and administration: Each treatment group received two oral administrations at 4 h and 24 h post-CLP surgery. The administration details were as follows... Figure 2 The experimental design remained consistent, evaluating the effects of different mitochondrial delivery methods on survival, inflammatory response, intestinal barrier function, and mitochondrial energy metabolism in septic mice. Each mouse was orally administered 100 μL of gel containing 1 × 10⁻⁶ mitochondrial gel via gavage. 8 Each mitochondrial particle.

[0052] 3. Blood Collection and Hematological Analysis: Peripheral blood was collected under anesthesia via cardiac puncture on the first day after CLP, i.e., 24 hours post-procedure. A portion of the blood sample was used for hematological analysis, including white blood cell, monocyte, lymphocyte, and granulocyte counts. Blood samples were tested using an automated blood analyzer (Element Ht5, Heska) within 6 hours of collection.

[0053] 4. Ileal Tissue Collection and Histological Analysis: Mice were sacrificed and ileal tissue was collected on the second day after CLP surgery (48 hours post-surgery). Tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4-5 μm. Hematoxylin and eosin (H&E) staining was then performed to observe ileal mucosal structure, villus morphology, epithelial integrity, and the degree of inflammatory damage. Tissue sections were scored blinded by gastrointestinal pathologists unaware of their grouping information according to a pre-defined scoring standard.

[0054] 5. Transmission electron microscopy: To assess the ultrastructure of tight junctions in the intestinal epithelium, fresh ileal tissue was fixed in 2% glutaraldehyde. After dehydration and epoxy resin embedding, ultrathin sections of approximately 50 nm were prepared. The tight junction structure between intestinal epithelial cells was observed using a Philips CM100 transmission electron microscope, and the width and length of tight junctions were measured. Three mice were used in each group, and 9-12 tight junction regions were measured in each mouse.

[0055] ATP detection and mitochondrial respiratory function analysis of 6-ileum tissue To evaluate the effects of mitochondrial intervention on ileal energy metabolism, ileal tissues were collected from each group for ATP content detection and mitochondrial respiratory function analysis. ATP levels were measured according to the kit instructions and normalized to tissue protein content. Fresh mitochondria were isolated from the ileal tissue, and oxygen consumption rate (OCR) was measured using a Seahorse XFe96 analyzer. During the detection process, oligomycin (1 μg / mL), FCCP (0.5 μM), and a rotenone / antimycin A mixture (1 μM) were added sequentially to assess mitochondrial basal respiration, ATP-related respiration, maximal respiration, and reserve respiratory capacity. The data were normalized to protein content and then statistically analyzed using GraphPadPrism software.

[0056] 7. Detection of cellular mitochondrial respiratory function MODE-K cells at 1 × 10 5 Cells / wells were seeded in gelatin-coated Seahorse XFe96 cell culture plates and cultured overnight. Before assay, the medium was replaced with XF assay medium, i.e., DMEM medium containing 11 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate. Subsequently, the cellular oxygen consumption rate was measured using a Seahorse XFe96 analyzer, with oligomycin (1.5 μM), FCCP (50 nM), and a mixture of rotenone / antimycin A (1 / 5 μM) added sequentially to analyze basal respiration, ATP-related respiration, and maximal respiratory capacity.

[0057] Example 3: mMito alleviates oxidative stress in intestinal epithelial MODE-K cells mMito treatment can restore MODE-K cell function and alleviate oxidative stress. First, we evaluated the effects of mMito on basic cellular functions. Scratch healing assays showed that, under 2% CLP-MS stimulation, mMito treatment significantly promoted scratch closure in MODE-K cells, suggesting its ability to restore the barrier repair capacity of intestinal epithelial cells. Figure 3 (A and B in the original text). Further analysis of intracellular reactive oxygen species (ROS) levels revealed that mMito significantly reduced intracellular oxidative stress. Specifically, this was manifested in a significant decrease in the fluorescence intensity of DCFH-DA under confocal microscopy (…). Figure 3 C in the middle); ROS positive cells (DCFH-DA) + The proportion decreased significantly. Figure 3(D and E in the original text). Given the central role of ROS in mitochondrial dysfunction and cell damage, we further evaluated the effect of mMito on mitochondrial bioenergetics. JC-1 staining results showed that under 2% CLP-MS stimulation, mMito treatment could partially restore the mitochondrial membrane potential (Δψm), as evidenced by a significant increase in the red / green fluorescence ratio (D and E in the original text). Figure 3 (F–H in the text). It is worth noting that mMito showed similar therapeutic effects at both 20 μg and 30 μg doses, suggesting that its efficacy had reached a plateau, and further increasing the dose within the tested range did not bring additional benefit.

[0058] Experimental steps: The mouse intestinal epithelial cell line MODE-K was purchased from Shanghai Crown Biotech Co., Ltd. (Shanghai, China) and cultured in Eagle's minimum essential medium (EMEM) containing 10% FBS, 2 mM L-glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin, in a 37°C, 5% CO2 incubator. An in vitro sepsis model was established using mouse serum after CLP surgery (CLPmouse serum, CLP-MS). Peripheral blood was collected from mice 24 h after CLP or Sham surgery. After coagulation at room temperature, serum was separated, inactivated by a 56°C water bath for 30 min, filtered through a 0.22 μm filter for sterilization, aliquoted, and placed in... Store at 80°C for later use. Before the experiment, MODE-K cells were treated with 1%, 2%, and 3% CLP-MS for 24 h, respectively, with 5% Sham-MS as the normal control. Cell viability was detected by CCK-8 kit (MCE, HY-K0301). Finally, 2% CLP-MS, which could induce significant inflammatory damage and maintain a cell viability of about 75%, was selected as the experimental condition for subsequent experiments.

[0059] MODE-K cells were seeded in 6-well plates. Once cell confluence reached 90-100%, a vertical scratch was made along the center of each well using a 10 μL sterile pipette tip. The cells were then gently rinsed twice with PBS to remove detached cells. The medium was then replaced with 2% CLP-MS (2% Sham-MS was added to the Sham group), and PBS, unmembrane mitochondria (Mito), or membrane-bound mitochondria (mMito) were added for treatment. Two protein doses were set for mMito: 20 μg and 30 μg, with the corresponding protein amount added to each well. After 24 h of treatment, Calcein-AM live cell staining was performed, and phase-contrast and fluorescence images were acquired at 0 h and 24 h. The scratch area was measured using ImageJ software, and the scratch closure rate was calculated as: Scratch closure (%) = (initial scratch area - 24 h scratch area) / initial scratch area × 100%. All image analyses were performed blinded.

[0060] Intracellular reactive oxygen species (ROS) were detected using the H2DCFDA fluorescent probe (Invitrogen, D-399). After treatment of MODE-K cells in each group, a final concentration of 5 μM H2DCFDA was added, and the cells were incubated at 37°C in the dark for 30 min. After washing three times with PBS, fluorescence images were acquired using a confocal laser scanning microscope, and the proportion of DCFH-DA positive cells and mean fluorescence intensity (MFI) were detected by flow cytometry to comprehensively evaluate intracellular ROS levels.

[0061] Mitochondrial membrane potential (Δψm) was detected using the JC-1 fluorescent probe (BD Pharmingen, 551302). After treatment, cells in each group were added to 5 μM JC-1 working solution and incubated at 37°C in the dark for 30 min. After washing with PBS and JC-1 buffer, the fluorescence signals of JC-1 aggregates (red) and monomers (green) were observed using a confocal laser scanning microscope, and the changes in JC-1 red / green fluorescence and the monomer ratio were detected by flow cytometry. The recovery of mitochondrial membrane potential was quantitatively analyzed using the red / green fluorescence ratio and the JC-1 monomer ratio.

[0062] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An mMito-gel mitochondrial delivery system, characterized in that, include: Membrane-masked mitochondria, wherein the membrane-masked mitochondria comprises mitochondria and cell membrane components surrounding the mitochondria; An inulin hydrogel matrix in which the membrane-masked mitochondria are dispersed.

2. The mMito-gel mitochondrial delivery system according to claim 1, characterized in that, The membrane-masked mitochondrial surface is modified with triphenylphosphine.

3. The mMito-gel mitochondrial delivery system according to claim 1, characterized in that, The inulin hydrogel matrix contains a protease inhibitor.

4. The mMito-gel mitochondrial delivery system according to claim 3, characterized in that, The protease inhibitor is aprotinin, and the final concentration of aprotinin in the inulin hydrogel matrix is ​​5 μg / mL.

5. The mMito-gel mitochondrial delivery system according to claim 1, characterized in that, The membrane-masked mitochondria were encapsulated at a concentration of 1 × 10⁻⁶ per milliliter of the inulin hydrogel matrix. 8 -1×10 9 A membrane disguises the mitochondria.

6. A method for preparing the mMito-gel mitochondrial delivery system according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Isolate and extract mitochondria; Step 2: Separate and extract cell membrane components; Step 3: Incubate the mitochondria obtained in Step 1 with triphenylphosphine solution to obtain triphenylphosphine-modified mitochondria; Step 4: Mix the cell membrane component obtained in Step 2 with the triphenylphosphine-modified mitochondria obtained in Step 3, and use a membrane extrusion method to coat the outside of the mitochondria with the cell membrane component. After purification, membrane-disguised mitochondria are obtained. Step 5: Dissolve inulin and protease inhibitor in buffer solution, and then heat and cool the mixture sequentially to form a hydrogel from the inulin. Step 6: Load the membrane-masked mitochondria obtained in Step 4 into the inulin hydrogel obtained in Step 5, mix evenly, and let stand to stabilize to obtain the mMito-gel mitochondrial delivery system.

7. The preparation method according to claim 6, characterized in that, The incubation conditions described in step 3 are: incubation at 4°C in an ice bath for 20 minutes; The membrane extrusion method described in step 4 uses a 1 μm polycarbonate membrane and is repeated 11 times. The heating treatment in step 5 is heating at 70°C for 10 minutes, and the cooling treatment is cooling to room temperature first and then placing at 4°C overnight to complete gelation.

8. The use of the mMito-gel mitochondrial delivery system according to any one of claims 1-5 in the preparation of a medicament for repairing intestinal barrier damage.

9. The application according to claim 8, characterized in that, The drug is an oral medication.

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