An engineered exosome for repairing intestinal ischemia-reperfusion injury and a construction method and application thereof

Abstract

The application discloses an engineered exosome for repairing intestinal ischemia-reperfusion injury and a construction method thereof, wherein the engineered exosome is internally loaded with Pzp protein and surface-expressed with Adipoq protein. The application successfully realizes efficient and specific targeted delivery of intestinal stem cells by constructing the engineered exosome which is surface-expressed with Adipoq protein and internally loaded with Pzp protein. The engineered exosome exhibits significant treatment advantages in an intestinal ischemia-reperfusion injury model, and provides a new type of nano drug delivery platform which is efficient, precise and has a transformation potential for treating intestinal ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to an engineered exosome for repairing intestinal ischemia-reperfusion injury, its construction method, and its application. Background Technology

[0002] Intestinal ischemia-reperfusion injury is a serious clinicopathological process commonly seen in various clinical situations, including mesenteric artery embolism, shock, severe trauma, cardiac and major vascular surgery, and organ transplantation. The pathological mechanisms of this injury are complex, involving energy metabolism disorders and cell damage in the intestinal tissue during the ischemic phase, as well as a surge of oxygen free radicals, calcium overload, excessive activation and infiltration of inflammatory cells, and a cascade of apoptosis and necrosis after reperfusion. Ultimately, this leads to severe disruption of the intestinal barrier function, bacterial and endotoxin translocation, triggering systemic inflammatory response syndrome and even multiple organ failure.

[0003] Currently, clinical treatment strategies for intestinal ischemia-reperfusion injury mainly focus on supportive care, such as restoring intestinal blood flow as quickly as possible through surgery or interventional procedures, maintaining circulatory stability with vasoactive drugs, using broad-spectrum antibiotics to prevent or control infection, and providing nutritional support. However, these conventional therapies primarily focus on controlling the macroscopic consequences of the injury and maintaining vital signs, lacking direct, efficient, and specific means to promote the regeneration and repair of damaged intestinal tissue, especially the intestinal epithelium. The integrity of the intestinal epithelium depends on the continuous proliferation and differentiation of intestinal stem cells, and intestinal ischemia-reperfusion injury severely disrupts the microenvironment of the intestinal stem cell nests, inhibiting the self-renewal and repair capabilities of stem cells. Therefore, how to effectively target and activate intestinal stem cells to promote their repair function is a key scientific problem and clinical challenge in treating this injury.

[0004] In the development of existing therapeutic drugs and molecules, researchers have explored various strategies to promote intestinal repair. For example, they have used exogenous growth factors, such as epidermal growth factor and hepatocyte growth factor, to stimulate epithelial cell proliferation; used antioxidants such as N-acetylcysteine ​​and superoxide dismutase mimics to scavenge excess oxygen free radicals generated during reperfusion; and used anti-inflammatory cytokines or inhibitors to regulate excessive inflammatory responses. Although these methods have shown some protective effects in animal models, they face significant challenges in practical clinical application and translation. A core common problem is poor targeting. After systemic administration, the distribution of these therapeutic molecules in the body lacks tissue specificity, making it difficult to achieve and maintain effective therapeutic concentrations at damaged intestinal sites, especially in the areas where intestinal stem cells need to be targeted. This non-specific distribution not only reduces efficacy and increases the required dose but may also cause unnecessary side effects by acting on other normal tissues. For example, systemic application of high doses of growth factors may stimulate abnormal proliferation of cells in other sites. Therefore, developing a novel delivery system that can precisely target intestinal injury sites, especially intestinal stem cells, and efficiently deliver therapeutic goods has become a critical technological bottleneck that urgently needs to be overcome in this field.

[0005] Exosomes, as nanoscale vesicles secreted by cells, naturally carry bioactive molecules such as proteins and nucleic acids, playing a crucial role in intercellular communication and becoming a research hotspot in drug delivery in recent years. Compared with artificially synthesized nanocarriers, exosomes have natural advantages such as good biocompatibility, low immunogenicity, and the ability to cross biological barriers (such as the blood-gut barrier). However, natural exosomes also suffer from insufficient targeting; their tropism mainly depends on the characteristics of donor cells, making it difficult to achieve active targeting of specific cell types (such as intestinal stem cells). Therefore, engineering natural exosomes to endow them with active targeting capabilities and specific therapeutic payloads is key to improving their therapeutic efficacy.

[0006] Currently, exosome engineering strategies mainly include: modifying donor cells through genetic engineering to express specific targeting peptides or proteins on the surface of the exosome membrane; or post-modifying exosomes, such as through click chemistry and membrane fusion, to anchor targeting molecules onto the exosome membrane. These technologies offer the possibility of constructing "intelligent" delivery vectors, but how to screen for membrane proteins that can efficiently and specifically target intestinal stem cells, effectively load therapeutic molecules with clear repair functions into exosomes, and form stable, efficient, and safe engineered exosome therapeutic products for the treatment of complex pathological processes such as intestinal ischemia-reperfusion injury, remains a challenging and incompletely solved scientific and engineering problem. Existing technologies lack a mature solution that integrates specific targeting of intestinal stem cells, efficient loading of therapeutic proteins, and a system for verifying damage repair functions. Summary of the Invention

[0007] This invention aims to address the problems of poor drug targeting and difficulty in achieving effective therapeutic concentrations at the site of intestinal ischemia-reperfusion injury (especially intestinal stem cell regions) in existing treatments. Conventional therapies, such as exogenous growth factors, antioxidants, or anti-inflammatory drugs, lack tissue specificity after systemic administration, leading to reduced efficacy, increased required doses, and potential side effects due to their action on normal tissues. This invention aims to provide a novel delivery system that can precisely and actively target the site of intestinal injury, especially intestinal stem cells, and efficiently deliver therapeutic drugs.

[0008] This invention also aims to address the problem of insufficient targeting ability of natural exosomes as drug delivery carriers. The tropism of natural exosomes depends on the characteristics of the donor cells, making it difficult to actively recognize and bind to specific cell types (such as intestinal stem cells), thus limiting their effectiveness in precision medicine. This invention aims to overcome this limitation by engineering natural exosomes to endow them with active targeting capabilities.

[0009] This invention also aims to address the technical challenge of efficiently and stably loading therapeutic molecules with well-defined repair functions into exosomes while ensuring their continued bioactivity after delivery. Existing technologies lack a mature solution that integrates specific targeting, efficient loading, and functional validation systems. This invention aims to construct an engineered exosome system that integrates specific targeting of intestinal stem cells, efficient loading of therapeutic proteins, and validation of damage repair functions.

[0010] In summary, this invention aims to overcome the core problems in the treatment of intestinal ischemia-reperfusion injury in the prior art, such as poor targeting, low delivery efficiency, and difficulty in maintaining the activity of therapeutic molecules. By constructing a novel engineered exosome, it achieves efficient and precise delivery to intestinal stem cells, thereby effectively promoting the repair and regeneration of intestinal tissue.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an engineered exosome for repairing intestinal ischemia-reperfusion injury, wherein the engineered exosome is internally loaded with Pzp protein and expresses Adipoq protein on its surface.

[0012] Preferably, the Adipoq protein targets intestinal stem cells.

[0013] Preferably, the Pzp protein repairs intestinal stem cells.

[0014] Secondly, the present invention provides a method for constructing engineered exosomes for repairing intestinal ischemia-reperfusion injury, comprising the following steps: S1. Prepare a lentiviral expression vector containing the Adipoq protein coding sequence; S2. Infect adipocyte progenitor cells with a lentiviral expression vector containing the Adipoq protein coding sequence, and screen to obtain cell lines that stably express Adipoq. S3. The selected stable cell lines are induced to differentiate into mature adipocytes. After differentiation, the cells are cultured and the cell culture supernatant is collected. S4. Purify exosomes Adipoq-EVs by differential ultracentrifugation; S5. Load the Pzp protein into the internal chamber of the exosome Adipoq-EVs, centrifuge to remove the unloaded free Pzp protein, and resuspend the precipitate with PBS to obtain the engineered exosome.

[0015] In step S1, the lentiviral expression vector is pLVX-Puro.

[0016] In step S3, induction culture medium is used for culturing. The induction culture medium is DMEM high glucose medium containing 10% FBS, and insulin 1 μg / mL, dexamethasone 1 μM and 3-isobutyl-1-methylxanthine 0.5mM are added.

[0017] In step S5, the Pzp protein is a recombinant Pzp protein.

[0018] In step S5, the Pzp protein is loaded into the internal cavity of the exosome Adipoq-EVs using an ultrasonic loading method.

[0019] In step S5, Pzp protein is mixed with purified Adipoq-EVs suspension in an ice bath. The final concentration of Pzp protein is 50-200 μg / mL, and the total protein content of Adipoq-EVs suspension is 100 μg. The mixture is then subjected to sonication.

[0020] On the other hand, the present invention also provides the use of the engineered exosomes in the preparation of drugs for repairing intestinal ischemia-reperfusion injury.

[0021] On the other hand, the present invention also provides a pharmaceutical composition comprising the engineered exosomes described herein.

[0022] This invention successfully achieved efficient and specific targeted delivery of intestinal stem cells by constructing engineered exosomes that express Adipoq protein on the surface and load Pzp protein internally. These engineered exosomes exhibited significant therapeutic advantages in an intestinal ischemia-reperfusion injury model, specifically: compared to free Pzp protein or untargeted empty exosomes, they more effectively accumulated at the injured intestinal site, significantly reducing non-specific distribution throughout the body; they precisely delivered Pzp protein to intestinal stem cells, thereby more effectively promoting the expression of intestinal epithelial tight junction proteins, reducing intestinal permeability, decreasing apoptosis, and significantly improving the survival rate of model animals; simultaneously, these engineered exosomes effectively activated the Wnt / β-catenin repair signaling pathway within intestinal stem cells, directly promoting stem cell proliferation and differentiation, and accelerating the repair and regeneration of the intestinal barrier. The engineered exosome construction method provided by this invention integrates targeting, loading, and functional validation, offering a novel, efficient, precise, and translationally promising nanomedicine delivery platform for the treatment of intestinal ischemia-reperfusion injury. Attached Figure Description

[0023] Figure 1 The purity of Pzp protein is shown. (A) The concentration of Pzp protein was determined by a BCA protein quantification kit; (B) The purity of Pzp protein was identified by SDS-PAGE.

[0024] Figure 2 The engineered exosomes of the present invention are shown. (A) Electron micrograph of engineered Adipoq+Pzp-EVs, the morphology of which conforms to the typical morphology of exosomes; (B) Particle size of engineered Adipoq+Pzp-EVs, which conforms to the size of exosomes; (C) Markers of exosomes from engineered Adipoq+Pzp-EVs, with high purity; (D) Pzp loading determined by BCA method, which significantly increases Pzp loading compared to empty loading; (E) Quantification of surface Adipoq protein by flow cytometry, which significantly increases the expression level of Adipoq on the surface of engineered Adipoq+Pzp-EVs compared to empty loading.

[0025] Figure 3 The results of bioactivity verification of loaded Pzp protein are shown. (A) The efficiency of Pzp delivery by different exosomes, with engineered Adipoq+Pzp-EVs showing the highest delivery efficiency; (B) The effect of different exosomes on stem cell proliferation and differentiation genes, with engineered Adipoq+Pzp-EVs showing the strongest ability to promote intestinal stem cell repair; (C) The effect of different exosomes on stem cell budding, with engineered Adipoq+Pzp-EVs showing the strongest ability to promote intestinal stem cell budding.

[0026] Figure 4The therapeutic effects in an intestinal ischemia-reperfusion model were demonstrated. (A) Compared with natural exosomes, engineered Adipoq+Pzp-EVs showed a significantly enhanced ability to target the intestine; (B) The effects of different exosomes on intestinal injury repair in mice with intestinal ischemia-reperfusion model showed that engineered Adipoq+Pzp-EVs had a significantly better therapeutic effect than free Pzp and empty EVs.

[0027] Figure 5 The safety assessment results are shown. (A) Detection of organ toxicity of engineered Adipoq+Pzp-EVs in intestinal ischemia-reperfusion model mice: Engineered Adipoq+Pzp-EVs did not show toxicity to major organs such as liver, lungs, and kidneys; (BD) Effects of engineered Adipoq+Pzp-EVs on systemic inflammatory factors (TNF-α, IL-6, IL-1β) in intestinal ischemia-reperfusion model mice: Engineered Adipoq+Pzp-EVs did not promote the production of inflammatory factors; (E) Liver and kidney function indicators (ALT, AST, BUN, Cr) of engineered Adipoq+Pzp-EVs in intestinal ischemia-reperfusion model mice, indicating that engineered Adipoq+Pzp-EVs are safe and non-toxic. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0031] Example 1: Construction of engineered exosomes loaded with the therapeutic protein Pzp This invention constructs engineered exosomes loaded with the therapeutic protein Pzp. Specifically, a lentiviral vector expressing the Adipoq protein (Gene ID: 9370) was constructed using genetic engineering techniques and transfected into preadipocytes, inducing their differentiation into mature adipocytes, thereby obtaining exosomes expressing the Adipoq protein on their surface. Subsequently, the Pzp protein with repair function was efficiently loaded into the internal chamber of the exosomes using physical or chemical methods, preparing engineered exosomes that simultaneously possess both targeting molecules and therapeutic payloads.

[0032] Specifically, the method for constructing engineered exosomes according to the present invention includes the following steps: (1) Preparation of engineered exosomes expressing Adipoq protein First, we designed and constructed a lentiviral expression vector.

[0033] Specifically, two lentiviral vector plasmids were designed and synthesized: The first type is the pCDH-CMV-Adipoq-EGFP-EF1-puro vector. This vector is based on the pCDH-Puro lentiviral vector. Driven by the strong promoter CMV, it expresses the coding sequence of the Adipoq protein (Chinese and English names: adiponectin; protein number: Q60994; GeneID: 11450; NC_000082.7), and achieves co-expression with the subsequent optional reporter gene (green fluorescent protein EGFP) through the EF1 sequence for subsequent tracking.

[0034] The promoter CMV sequence is as follows: TGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTTTATAAGCAGAGCT.

[0035] The gene sequence encoding the Adipoq protein is as follows: ATGCTACTGTTGCAAGCTCTCCTGTTCCTCTTAATCCTGCCCAGTCATGCCGAAGATGACGTTACTACAACTGAAGAGCTAGCTCCTGCTTTGGTCCCTCCACCCAAGGGAACTTGTGCAGGTTGGATGGCAGGCATCCCAGGACATCCTGGCCACAATGGCACACCAGGCCGTGATGGCAGAGATGGCACTCCTGGAGAGAAGGGAGAGAAAGGAGATGCAGGTCTTCTTGGTCCTAAGGGTGAGACAGGAGATGTTGGAATGACAGGAGCTGAAGGGCCACGGGGCTTCCCCGGAACCCCTGGCAGGAAAGGAGAGCCTGGAGAAGCCGCTTATGTGTATCGCTCAGCGTTCAGTGTGGGGCTGGAGACCCGCGTCACTGTTCCCAATGTACCCATTCGCTTTACTAAGATCTTCTACAACCAACAGAATCATTATGACGGCAGCACTGGCAAGTTCTACTGCAACATTCCGGGACTCTACTACTTCTCTTACCACATCACGGTGTACATGAAAGATGTGAAGGTGAGCCTCTTCAAGAAGGACAAGGCCGTTCTCTTCACCTACGACCAGTATCAGGAAAAGAATGTGGACCAGGCCTCTGGCTCTGTGCTCCTCCATCTGGAGGTGGGAGACCAAGTCTGGCTCCAGGTGTATGGGGATGGGGACCACAATGGACTCTATGCAGATAACGTCAACGACTCTACATTTACTGGCTTTCTTCTCTACCATGATACCAACTGA。

[0036] The amino acid sequence of Adipoq protein is as follows: MLLLQALLFLLILPSHAEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHNGTPGRDGRDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEPGEAAYVYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITVYMKDVKVSLFKKDKAVLFTYDQYQEKNVDQASGSVLLHLEVGDQVWLQVYGDGDHNGLYADNVNDSTFTGFLLYHDTN.

[0037] The sequence of EF1 is as follows: GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG.

[0038] The complete sequence of pCDH-CMV-Adipoq-EGFP-EF1-puro is as follows: >VP073pCDH-CMV-Adipoq-EGFP-EF1-Puro(8830 base pairs)

[0039] Insertion site: The Adipoq gene was cloned into a multiple cloning site (MCS), located downstream of the CMV promoter. Cloning sites include XbaI, BamHI, etc.

[0040] The second type is the pCDH-CMV-MCS-EGFP-EF1-Puro empty control vector, which contains only the CMV promoter and the reporter gene EGFP framework, but does not contain the Adipoq coding sequence.

[0041] The pCDH-CMV-MCS-EGFP-EF1-Puro sequence is as follows:

[0042] Subsequently, lentiviruses were packaged and produced in HEK293T cells using a standard three-plasmid system (including packaging plasmids and envelope plasmids), and the viral supernatant was collected and concentrated and titrated.

[0043] The prepared lentiviral particles were used to infect mouse 3T3-L1 adipose precursor cells with a multiplicity of infection (MOI) of 30 (which can be between 10 and 50). Forty-eight hours after infection, the cells were replaced with complete medium containing 3 μg / mL (or 2-5 μg / mL) puromycin (DMEM [PM150210] + 10% FBS [164220] + 1% P / S [PB180120]; Pronos, catalog number CL-0006) for 7-10 days to kill untransfected cells and obtain cell pools that stably express Adipoq or only express the control vector.

[0044] The selected stable cell lines were cultured in induction medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX) (DMEM high-glucose medium containing 10% FBS, with 1 μg / mL insulin, 1 μM dexamethasone, and 0.5 mM IBMX) to induce differentiation into mature adipocytes. This process typically lasts 7-14 days until a large number of lipid droplets appear in the cells. After differentiation, the medium was replaced with serum-free medium (Gibco exosome-free fetal bovine serum, catalog number A2720801) and cultured for another 48-72 hours. The cell culture supernatant was then collected.

[0045] Exosomes were purified using differential ultracentrifugation: First, cell debris was removed by centrifugation at 300×g for 10 minutes at 4°C, followed by centrifugation at 2000×g for 20 minutes to remove apoptotic bodies, and then centrifugation at 10000×g for 30 minutes to remove large vesicles. Finally, the supernatant was ultracentrifuged (using a Beckman ultracentrifuge, Type 70 Ti rotor, 100000×g, 70 minutes, 4°C) to precipitate the exosomes. The precipitate was resuspended in pre-cooled phosphate-buffered saline (PBS) to obtain Adipoq-EVs (experimental group) and Control-EVs (control group, also known as "Mock-EVs").

[0046] (2) Loading of the therapeutic protein Pzp Pzp protein (pregnancy zone protein (PZP); protein number: Q61838; Gene ID: 11287; purchased from Wuhan Yunclone Biotechnology Co., Ltd. (URPG324Mu02)) was obtained. Its purity was verified by SDS-PAGE and high performance liquid chromatography (HPLC), and its concentration was determined by BCA protein quantification kit (Wuhan Yunclone Biotechnology Co., Ltd. (URPG324Mu02)). Figure 1 The purity of the Pzp protein is shown. Figure 1 A in the figure shows the concentration of Pzp protein determined using the BCA protein quantification kit. Figure 1 B in the figure shows that the purity of the Pzp protein was qualified as determined by SDS-PAGE.

[0047] The Pzp protein was introduced into the internal chamber of the prepared Adipoq-EVs using an ultrasonic loading method. Specifically, the Pzp protein was mixed with purified Adipoq-EVs suspension (100 μg total protein in Adipoq-EVs, using 1× PBS (pH 7.4) containing 5 v / v% glycerol as solvent) in an ice bath to achieve a final Pzp protein concentration of 100 μg / mL (range: 50-200 μg / mL). Pulsed sonication was then performed using a Sonics Vibra-Cell ultrasonic homogenizer at a specific power (20% amplitude, 20 kHz frequency) for 5 cycles (10 seconds on / 10 seconds off). This process was carried out in an ice bath to prevent overheating and protein denaturation.

[0048] After loading, unloaded free Pzp protein was removed by ultracentrifugation (100000×g, 70 minutes, 4℃), and the precipitate was resuspended in PBS to obtain the final product "Adipoq+Pzp-EVs (exogenous loading)".

[0049] In parallel, an "Adipoq+EVs (empty)" control group (i.e., expressing Adipoq protein but not loading Pzp protein) was set up using the same volume of loading buffer (PBS) as Adipoq-EVs to exclude the possible influence of the loading process itself on exosomes.

[0050] Example 2: Physicochemical characterization of engineered exosomes The engineered exosomes constructed in Example 1 were subjected to comprehensive physicochemical characterization and bioactivity verification.

[0051] The physical properties, purity, expression levels of surface target proteins, and loading of internal therapeutic proteins of the prepared Adipoq+Pzp-EVs, Adipoq+EVs (empty) and Control-EVs exosomes were confirmed using a variety of technical means.

[0052] First, the particle size distribution and concentration of exosomes were determined using a nanoparticle tracking analyzer (Malvern NanoSight NS300). An appropriate amount of sample was diluted with PBS to a suitable concentration (typically between 20-100 particles per frame), injected into the sample cell, and analyzed by laser scattering and Brownian motion tracking. The instrument output the average particle size and particle size distribution (polydispersity index, PDI). Figure 2 (B) and the number of particles per milliliter.

[0053] Secondly, the morphology of exosomes was observed using transmission electron microscopy. 10 μL of sample was dropped onto a Formvar / carbon-coated copper grid, allowed to stand for 1-2 minutes, and excess liquid was absorbed with filter paper. Then, the sample was negatively stained with 2% phosphotungstic acid for 1 minute, blotted dry, and allowed to air dry at room temperature. The samples were then observed and photographed at an accelerating voltage of 80 kV to confirm that the exosomes exhibited a typical cup-shaped or spherical morphology with a bilayer membrane structure. Figure 2 (A in the middle).

[0054] Western blotting was used to verify the markers and purity of exosomes. Total exosome protein was extracted, subjected to SDS-PAGE electrophoresis, and transferred to a membrane. Positive exosome markers were detected using antibodies against CD63, TSG101, and CD9, respectively. Anti-Calnexin (an endoplasmic reticulum marker) was used as a negative control to confirm the absence of significant cellular debris contamination in the sample. Figure 2 (C in the middle).

[0055] Flow cytometry was used to quantify surface Adipoq protein. Exosomes were co-incubated with aldehyde / sulfate latex microspheres to induce binding, followed by staining with primary antibody against Adipoq and corresponding fluorescent secondary antibody. Fluorescence signals were detected by flow cytometry and compared with isotype controls. The mean fluorescence intensity (MFI) of Adipoq positive ligands was calculated to quantify the expression level of the target ligand.

[0056] Finally, the loading of Pzp was accurately determined: the total protein concentration of Adipoq+Pzp-EVs was determined using the BCA method, and simultaneously, a specific enzyme-linked immunosorbent assay (ELISA) kit for Pzp (Mouse Pregnancy Band Protein (PZP) ZC-56883 ELISA kit, ZCIBIO Shanghai Zhuocai Biotechnology) was used to quantitatively determine the absolute content of Pzp in the sample according to the standard curve, thereby calculating the amount of Pzp loaded per unit mass of exosomal protein or per unit number of exosomal particles (e.g., ng Pzp / μg total EVs protein or number of molecules / particle). The results are as follows: Figure 2 As shown in D in the figure. Compared with the unloaded state, the engineered Adipoq+Pzp-EVs significantly improve the loading of Pzp. Figure 2The results showed that, using flow cytometry to quantify surface Adipoq protein, the expression level of Adipoq on the surface of engineered Adipoq+Pzp-EVs was significantly increased compared with the empty vector.

[0057] Example 3: Validation of the bioactivity of loaded Pzp protein To confirm that the loading process did not damage the function of the Pzp protein, in vitro cell function experiments were conducted for verification.

[0058] Systematically evaluate the in vivo and in vitro targeting and delivery efficiency of engineered exosomes.

[0059] In animal models, the enrichment of engineered exosomes at the site of intestinal ischemia-reperfusion injury was dynamically observed using in vivo imaging and other techniques, and compared with non-targeted exosomes and free proteins to quantitatively assess their tissue distribution and delivery efficiency.

[0060] At the cellular level, using primary intestinal stem cells and control cells, flow cytometry and other methods were used to verify the specificity of engineered exosomes in binding to and taking up target cells, and to confirm their targeting mechanism dependent on the Adipoq protein.

[0061] First, primary mouse stem cells were isolated and cultured. Exosomes from different treatment groups (Adipoq+Pzp-EVs, Adipoq+EVs empty vector, and Control-EVs) were co-cultured with intestinal stem cells at a concentration of 50 μg / mL for 24–48 hours. The expression level of Pzp in the stem cells was detected by Western blot. Figure 3 In the figure, A represents the efficiency of Pzp delivery by different exosomes, showing that engineered Adipoq+Pzp-EVs have the highest delivery efficiency.

[0062] Secondly, a co-culture system for damaged small intestinal organoids was established: crypts were isolated from the mouse small intestine and cultured in Matrigel to form organoids. Hypoxia / reoxygenation treatment was used to simulate ischemia-reperfusion injury. The damaged organoids were co-cultured with different exosomes (exosome concentration 50 μg / mL). After several days of culture, the survival rate, budding number (number of buds per organoid), and diameter changes were observed and quantitatively analyzed under a microscope to assess the ability of Adipoq+Pzp-EVs to promote intestinal epithelial repair, thereby comprehensively confirming that the biological activity of the loaded Pzp protein remained intact.

[0063] Figure 3 B in the figure represents the effect of different exosomes on stem cell proliferation and differentiation genes, showing that engineered Adipoq+Pzp-EVs have the strongest ability to promote intestinal stem cell repair. Figure 3In the figure, C represents the effect of different exosomes on stem cell budding, showing that engineered Adipoq+Pzp-EVs have the strongest ability to promote intestinal stem cell budding.

[0064] I / R: Ischemia-reperfusion modeling; I / R+Mock-EVs: Treatment with negative control exosomes during modeling; I / R+recombinant protein Pzp: Treatment with free recombinant Pzp protein (concentration 50 μg / mL) during modeling; I / R+Natural-Adipoq+EVs: Treatment with natural adipose exosomes expressing only Adipoq during modeling; I / R+Adipoq+Pzp-EVs: Treatment with engineered exosomes Adipoq+Pzp-EVs during modeling.

[0065] Example 4: Evaluation of the therapeutic advantages in an intestinal ischemia-reperfusion model This study comprehensively evaluated the therapeutic efficacy of engineered exosomes in an intestinal ischemia-reperfusion injury model. By setting up multiple control groups, the effects of engineered exosomes on improving histological damage, restoring intestinal barrier function, promoting intestinal stem cell proliferation and differentiation, reducing apoptosis, and improving survival were systematically compared. The therapeutic advantages of engineered exosomes compared to free proteins and empty exosomes were clarified, and the mechanism by which they exert their therapeutic effect by activating downstream repair signaling pathways (such as the Wnt / β-catenin pathway) was explored.

[0066] Mice with intestinal ischemia-reperfusion model were randomly divided into four groups: (1) Mock-EVs treatment group; (2) Free recombinant Pzp protein treatment group; (3) Adipoq+EVs (unloaded) treatment group; (4) Adipoq+Pzp-EVs treatment group.

[0067] Administration method and dosage: administered via intraperitoneal injection; 200 μg / animal; administered 2 hours before ischemia, continued until 1 hour of ischemia, followed by 6 hours of reperfusion to finish material collection, with a treatment time of 9 hours.

[0068] By systematically evaluating histological damage scores, intestinal epithelial tight junction protein (ZO-1) expression, intestinal permeability (FITC-glucan leakage), intestinal stem cell proliferation and differentiation markers (MUC2, Ki-67 cell count), and survival rate, this study aims to determine whether the therapeutic effect of Adipoq+Pzp-EVs is significantly better than that of free Pzp and empty EVs, thus confirming the therapeutic advantages of its targeted delivery system.

[0069] Figure 4The therapeutic effects were demonstrated. In Figure A, the engineered Adipoq+Pzp-EVs showed a significantly enhanced ability to target the intestine compared to natural exosomes. Figure B showed the effects of different exosomes on intestinal injury repair in a mouse model of intestinal ischemia-reperfusion injury; the engineered Adipoq+Pzp-EVs showed significantly better therapeutic effects than free Pzp and empty EVs.

[0070] Example 5: Preliminary Safety Assessment Evaluation of the safety of engineered exosomes in an intestinal ischemia-reperfusion injury model.

[0071] After the treatment cycle ended, mouse serum was collected to detect liver and kidney function indicators (ALT, AST, BUN, Cr) and systemic inflammatory factors (TNF-α, IL-6, IL-1β).

[0072] Simultaneously, the liver, lungs, kidneys, and other major organs were subjected to HE staining pathological examination to observe for any abnormal lesions. During multiple drug administration experiments, animals were closely monitored for adverse reactions such as allergic reactions or abnormal weight loss.

[0073] Figure 5 Table A shows the results of organ toxicity testing of engineered Adipoq+Pzp-EVs in a mouse model of intestinal ischemia-reperfusion injury. Engineered Adipoq+Pzp-EVs did not show toxicity to major organs such as the liver, lungs, and kidneys. Tables B to D show the effects of engineered Adipoq+Pzp-EVs on systemic inflammatory factors Tnf-α, IL-6, and IL-1β in a mouse model of intestinal ischemia-reperfusion injury. Engineered Adipoq+Pzp-EVs did not show any promotion of the production of inflammatory factors. Figure 5 The E values ​​in the figure show the liver and kidney function indicators (ALT, AST, BUN, CREA (creatinine)) of engineered Adipoq+Pzp-EVs in mice with intestinal ischemia-reperfusion model, indicating that engineered Adipoq+Pzp-EVs are safe and non-toxic.

[0074] Through the above implementation scheme, this invention details the specific steps, key parameters, and quality control methods for constructing engineered Adipoq+ exosomes loaded with Pzp. This scheme, from the construction of gene engineering vectors, cell engineering modification, exosome production and purification, and efficient loading of therapeutic proteins, to systematic physicochemical characterization and in vitro bioactivity verification, forms a complete and reproducible preparation and quality control process, providing a material basis and quality assurance for subsequent targeted evaluation and therapeutic efficacy studies.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An engineered exosome for repairing intestinal ischemia-reperfusion injury, characterized in that, The engineered exosomes are internally loaded with Pzp protein and express Adipoq protein on their surface.

2. The engineered exosomes according to claim 1, characterized in that, The Adipoq protein targets intestinal stem cells.

3. The engineered exosomes according to claim 2, characterized in that, The Pzp protein repairs intestinal stem cells.

4. A method for constructing engineered exosomes as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Prepare a lentiviral expression vector containing the Adipoq protein coding sequence; S2. Infect adipocyte progenitor cells with a lentiviral expression vector containing the Adipoq protein coding sequence, and screen to obtain cell lines that stably express Adipoq. S3. The selected stable cell lines are induced to differentiate into mature adipocytes. After differentiation, the cells are cultured and the cell culture supernatant is collected. S4. Purify exosomes Adipoq-EVs by differential ultracentrifugation; S5. Load the Pzp protein into the internal chamber of the exosome Adipoq-EVs, centrifuge to remove the unloaded free Pzp protein, and resuspend the precipitate with PBS to obtain the engineered exosome.

5. The method according to claim 4, characterized in that, In step S3, induction culture medium is used for culturing. The induction culture medium is DMEM high glucose medium containing 10% FBS, and insulin 1 μg / mL, dexamethasone 1 μM and 3-isobutyl-1-methylxanthine 0.5 mM are added.

6. The method according to claim 4, characterized in that, In step S5, the Pzp protein is a recombinant Pzp protein.

7. The method according to claim 4, characterized in that, In step S5, the Pzp protein is loaded into the internal cavity of the exosome Adipoq-EVs using an ultrasonic loading method.

8. The method according to claim 4, characterized in that, In step S5, Pzp protein is mixed with purified Adipoq-EVs suspension in an ice bath. The final concentration of Pzp protein is 50-200 μg / mL, and the total protein content of Adipoq-EVs suspension is 100 μg. The mixture is then subjected to sonication.

9. The use of engineered exosomes as described in any one of claims 1 to 3 in the preparation of medicaments for repairing intestinal ischemia-reperfusion injury.

10. A pharmaceutical composition, characterized in that, It comprises engineered exosomes as described in any one of claims 1 to 3.

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