Engineered exosome as well as preparation method and application thereof

Abstract

The invention discloses an engineered exosome and a preparation method and application thereof, the exosome comprises a therapeutically effective amount of CD5L protein, and the surface of the exosome is conjugated with kidney targeting peptide LTH which can specifically target injured renal tubular cells. According to the exosome, the exosome containing CD5L is separated from fibrous reticulate cells engineered to overexpress CD5L protein, and then a kidney targeting peptide LTH is conjugated to the surface of the exosome. The exosome can specifically deliver CD5L to injured renal tubules in a targeting manner, kidney tissue injury caused by S-AKI is effectively relieved, and microcirculation disturbance of the whole body is relieved. The invention provides a novel treatment strategy and candidate medicine with targeting and high efficiency for treating acute kidney injury caused by sepsis.

Description

Technical Field

[0001] The present invention relates to the field of cell biology technology, and in particular to engineered exosomes and a preparation method and application thereof. Background Art

[0002] Sepsis is a syndrome of dysregulated bodily responses to infection that can lead to life-threatening organ dysfunction. Acute kidney injury (AKI) is one of the most common and severe complications of sepsis. Mortality is extremely high in patients with sepsis-induced acute kidney injury (S-AKI), and specific, effective treatments are currently lacking. The pathogenesis of S-AKI is complex, involving multiple mechanisms, including dysregulated inflammatory responses, oxidative stress, microcirculatory disturbances, apoptosis, and metabolic disorders.

[0003] Recent studies have revealed that metabolic reprogramming plays a key role in the pathophysiology of S-AKI. Specifically, fatty acid metabolism in renal tubular epithelial cells (RTECs) is significantly disrupted: on the one hand, the fatty acid synthesis (FAS) pathway is abnormally activated, leading to increased de novo lipid synthesis; on the other hand, fatty acid oxidation (FAO), a key energy metabolism pathway, is impaired, manifesting as abnormal intracellular accumulation of free fatty acids and intermediate metabolites (such as acylcarnitines). This imbalance in lipid metabolism not only leads to the massive accumulation of lipid droplets in renal tubular cells, resulting in lipotoxicity, but also severely impairs cellular energy supply.

[0004] As the "energy factory" of the cell and the main site of FAO, the functional state of mitochondria is closely related to kidney health. In S-AKI, mitochondrial dysfunction is one of the core pathological links. Impaired FAO directly leads to insufficient mitochondrial fuel supply, affecting the normal operation of the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), and reducing ATP production. At the same time, aggravated mitochondrial damage, increased production of reactive oxygen species (ROS), imbalanced mitochondrial dynamics (such as excessive fission), and abnormal regulation of mitochondrial autophagy (Mitophagy) together constitute the destruction of mitochondrial homeostasis, further aggravating renal cell damage and dysfunction. Therefore, targeted intervention of fatty acid metabolism disorders and mitochondrial dysfunction in S-AKI has become an important direction for the development of new treatment strategies.

[0005] Exosomes are a class of nanoscale extracellular vesicles secreted by cells, with a diameter of about 30-150 nm. They naturally carry a variety of biologically active molecules (such as proteins, lipids, mRNA, miRNA) and can mediate intercellular communication. Due to its good biocompatibility, low immunogenicity (especially autologous source or modification), and the ability to cross biological barriers, exosomes are considered as a very potential drug delivery carrier. Through genetic engineering or chemical modification of the source cells or the exosomes themselves, the targeting molecules on the surface of the exosomes and the internal cargo can be customized, i.e. "engineered exosomes".

[0006] Various engineered exosome technologies have been reported in the prior art, aiming to improve targeting or deliver specific therapeutic molecules (such as small nucleic acids, proteins, small molecule drugs). For example: The patent application (CN114292848A) of Duolai Biotechnology (Wuhan) Co., Ltd. discloses an engineered exosome targeting lung cancer, which realizes lung targeting by displaying α6β1 protein on the surface and loading anticancer miRNA (miR-214, miR-770) inside.

[0007] The patent application (CN116970568A) of Tianjin Cancer Hospital describes an engineered exosome targeting breast cancer drug-resistant cells, which can target cells highly expressing ACE2 by expressing CD63-RBD fusion protein in HEK293T cells.

[0008] The patent (CN115029320B) of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences relates to an exosome targeting tumor cells by surface modification of iRGD peptide for delivering STAT3 siRNA to enhance radiotherapy sensitivity.

[0009] However, despite the progress made in engineered exosome technology, there are still many challenges and limitations: (1) Targeting efficiency and specificity issues: Although targeting molecules are designed, in the complex in vivo environment, whether exosomes can efficiently and specifically reach the target organs / cells (such as damaged kidneys) and avoid being cleared by the immune system or enriched in non-target tissues (off-target effects) is still a great challenge. Many targets (such as ACE2, integrin) are not organ or disease specific. Although Enze Kangtai and other institutions are researching kidney-targeting peptides, efficient, specific, and in vivo large animal model-verified targeted delivery systems still need to be developed.

[0010] (2) Loading efficiency and function maintenance issues: Efficiently loading therapeutic molecules (especially proteins) into the interior of exosomes and ensuring their biological activity during delivery and after reaching target cells are key technical difficulties.

[0011] (3) Insufficient in vivo validation and mechanism research: Many studies are mainly based on in vitro experiments or small animal models. Whether their results can be extrapolated to the clinic (especially in large animal models that better simulate human physiology and pathology) is still uncertain. The specific mechanism by which exosomes play a role in specific diseases (such as S-AKI), especially how they regulate complex metabolic networks (such as lipid metabolism) and organelle functions (such as mitochondria), still needs to be further explored.

[0012] (4) Safety considerations: Exosomes from non-autologous sources or exogenous molecules introduced through engineering modifications may cause immunogenicity.

[0013] Therefore, it is necessary to develop a new type of engineered exosomes and their preparation method and application to solve the above core technical problems in the treatment of S-AKI. Summary of the Invention

[0014] The present invention aims to provide an engineered exosome, its preparation method, and application. The exosome can specifically target and deliver CD5L to damaged renal tubules, effectively alleviating renal tissue damage caused by S-AKI and alleviating systemic microcirculatory disorders.

[0015] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, an engineered exosome is provided, wherein the exosome carries CD5L protein; and the kidney-targeting peptide LTH is conjugated to the surface of the exosome, wherein the amino acid sequence of LTH is shown in SEQ ID NO: 1 (specifically: LTHVVWL).

[0016] In a preferred embodiment, the exosomes are derived from cells genetically engineered to overexpress the CD5L protein. Fibroblastic reticular cells (FRCs) are easy to culture and engineer. By stably overexpressing the CD5L protein in FRCs, the secreted exosomes are enriched with CD5L protein.

[0017] In a preferred embodiment, the kidney-targeting peptide, LTH, is a peptide segment that specifically binds to specific receptors or molecules on the surface of damaged renal tubular epithelial cells. This targeting peptide enables the engineered exosomes to preferentially accumulate in acutely injured kidney sites, particularly the renal tubular regions, following intravenous injection, thereby improving the precision of drug delivery and reducing potential systemic side effects.

[0018] In a preferred embodiment, the CD5L protein is a protein with known biological activity, particularly known to participate in lipid metabolism regulation, such as by directly or indirectly inhibiting the activity of key enzymes in fatty acid synthesis (e.g., FASN). The therapeutically effective amount is a dose of CD5L protein or exosomes that produces the desired therapeutic effect (e.g., improved renal function, reduced lipid accumulation) in a subject (e.g., an animal model or patient with S-AKI). Based on experimental data in the Bama miniature pig model, an exemplary therapeutically effective dose is approximately 3 μg / kg body weight.

[0019] In a preferred embodiment, the kidney-targeting peptide LTH is chemically conjugated to the surface of previously isolated and purified exosomes containing the CD5L protein. This "post-modification" strategy may offer advantages over expressing targeting peptide-membrane protein fusions in source cells in terms of preserving the native structure of exosomes and reducing immunogenicity. Conjugation can be achieved through a variety of established bioconjugation chemistries, such as using amino, sulfhydryl, carboxyl, or azide functional groups.

[0020] In a second aspect, the present invention provides a method for engineering exosomes, comprising: transfecting FRC cells with a lentiviral vector encoding CD5L, and culturing to obtain cells overexpressing CD5L; isolating and obtaining first exosomes from the supernatant of the CD5L-overexpressing cells, referred to as CD5L-EXO; and conjugating LTH peptide to the surface of the exosomes through a DBCO-azide click chemistry reaction to obtain engineered exosomes, referred to as FRC-EXO.

[0021] Furthermore, the separation method comprises one of differential ultracentrifugation, density gradient centrifugation, size exclusion chromatography, tangential flow filtration, and immunoaffinity capture.

[0022] The nucleotide sequence of CD5L is shown in SEQ ID NO.2.

[0023] In a third aspect of the present invention, provided is a use of the engineered exosomes in the preparation of a drug for treating sepsis-induced acute kidney injury.

[0024] Specifically, the application includes using the engineered exosomes for: Alleviate renal histological damage caused by S-AKI (such as tubular necrosis, loss of brush border, and inflammatory cell infiltration); Improve renal function indicators (such as reducing serum creatinine and urea nitrogen levels); Inhibits kidney inflammation and oxidative stress; Reduce renal cell apoptosis; Regulating the disorder of renal fatty acid metabolism, specifically by inhibiting fatty acid synthesis (FAS), for example by inhibiting FASN activity, and promoting fatty acid oxidation (FAO), for example by up-regulating the expression of key enzymes such as CPT1a, CPT2, etc., thereby reducing the accumulation of lipids in the kidney and the accumulation of lipid toxic metabolites; Restoring mitochondrial function and homeostasis, specifically by increasing mitochondrial respiration rate (OCR), enhancing TCA cycle flux and ETC function, reducing mitochondrial ROS production, regulating mitochondrial dynamics, and possibly activating mitochondrial autophagy.

[0025] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: The engineered exosome provided by the present application and the preparation method and application thereof are obtained by separating the exosome containing CD5L from fibroreticular cells which are engineered to overexpress CD5L protein, and then conjugating the kidney-targeting peptide LTH to the surface of the exosome to obtain the engineered exosome, which has the following advantages: (1) Achieving specific targeting delivery to the S-AKI injured kidney: By conjugating the kidney-targeting peptide LTH to the surface of the exosome containing the therapeutic protein CD5L, the engineered exosome of the present application can be preferentially enriched in the tubular region of the kidney that is acutely injured in vivo after intravenous injection, significantly improving the accuracy of drug delivery. This is directly confirmed by imaging in a large animal model (Bama mini-pig S-AKI model) with high clinical relevance.

[0026] (2) Effectively intervening in the core pathological mechanism of S-AKI, i.e. lipid metabolism disorder and mitochondrial dysfunction: The present application first proves that by delivering the CD5L protein, the engineered exosome can directly act on the disordered fatty acid metabolism pathway in the S-AKI kidney, effectively inhibiting excessive fatty acid synthesis (FAS) and promoting impaired fatty acid oxidation (FAO), thereby significantly reducing pathogenic lipid accumulation and restoring the FAO process as the main energy source for renal cells. Further studies have revealed that this metabolic reprogramming can effectively improve the bioenergetic function of mitochondria and maintain mitochondrial homeostasis.

[0027] (3) Exhibiting significant therapeutic effect and good safety in a clinically relevant large animal model: The engineered exosome of the present application not only significantly improves renal function and histological injury, reduces lipid accumulation, and restores mitochondrial function in a sepsis-induced AKI Bama mini-pig model, but also shows potential to improve systemic microcirculatory disorders and reduce injury to other organs. At the same time, no obvious allergic reaction or systemic toxicity was observed in this large animal model, showing good biological safety.

[0028] (4) In-depth elucidation of the mechanism of action and providing key evidence: This paper uses multi-omics analysis and fatty acid oxidation inhibitor experiments to clearly reveal the mechanism of action of engineered exosomes by delivering CD5L to reprogram lipid metabolism (inhibit FAS and promote FAO) and rely on restoring FAO to improve mitochondrial function and ultimately protect the kidneys from damage. This provides a solid theoretical basis for the clinical translation of this therapy.

[0029] (5) Provides a new S-AKI treatment strategy with potential for clinical transformation: Based on engineered exosome technology, this invention targets the key pathological links of S-AKI and provides a new therapeutic candidate drug with strong targeting, significant efficacy, clear mechanism, and good safety, which has important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 This schematic diagram illustrates the preparation process and characterization results of engineered exosomes according to one embodiment of the present invention. It may include: (A) Schematic diagram of the preparation steps of engineered exosomes (e.g., FRC-EXO); (B) Transmission electron microscopy (TEM) images of exosome morphology; (C) Nanoparticle tracking analysis (NTA) or dynamic light scattering (DLS) results of exosomes, showing particle size distribution and concentration; (D-E) Western blot analysis of exosomal markers (e.g., CD9, CD63, CD81) and cargo (CD5L); (F) Fluorescence microscopy images of engineered exosomes uptake by damaged renal tubular epithelial cells; (G-H) Representative histograms and quantitative analysis of LTH peptide conjugation efficiency for control EXO, FRC-EXO, and CD5L-EXO populations using NanoFCM. (I) Immunofluorescence images of CD31, KIM-1, and LTH in kidney sections from different groups of Bama pigs.

[0032] Figure 2To schematically illustrate the results of the engineered exosomes according to embodiments of the present application improving the systemic indicators and microcirculation of an endotoxemia Bama pig model. May include: (A) the process of injecting LPS and exosomes in pigs of different treatment groups (such as control group, LPS model group, LPS+control exosome group, LPS+FRC-EXO group); (B-F) comparison column charts of serum cardiac injury markers (such as CK-MB, cTnI), liver injury markers (such as ALT, AST, IBIL, DBIL), renin-angiotensin and allergy (leukotrienes, histamine) levels; (G-O) monitoring results of sublingual microcirculation perfusion.

[0033] Figure 3 To schematically illustrate the results of the engineered exosomes according to embodiments of the present application improving the kidney function of an endotoxemia Bama pig. May include: (A) a plot of the serum creatinine (Scr) levels of pigs in different treatment groups over time; (B-C) the contents of HE, TUNEL, beta galactosidase and Ki67 in the kidney tissues of pigs in different treatment groups; (D-E) the contents of kidney creatinine, urea nitrogen, LDH, MDA and inflammatory factors in different groups; (F) the content of LAC in different treatment groups.

[0034] Figure 4 To schematically illustrate the results of the engineered exosomes according to embodiments of the present application reducing the accumulation of lipid droplets in the kidney tissues of an endotoxemia Bama pig and reducing the level of acylcarnitine. Includes: (A) Transmission electron microscopy observation of lipid droplet aggregation around mitochondria in kidney tissues in sepsis acute kidney injury; (B) Oil red O staining section of kidney tissue showing lipid droplet accumulation; Quantitative comparison column chart of lipid droplet area or number; (D-H) Comparison column chart of total free fatty acid content or specific acylcarnitine (such as long-chain acylcarnitine) level in kidney tissue detected by liquid chromatography-mass spectrometry (LC-MS); (I-J) Long-chain fatty acid and acylcarnitine content in metabolic mass spectrometry.

[0035] Figure 5This diagram schematically illustrates the results of restoring mitochondrial bioenergetics (TCA cycle and electron transport chain activity) in kidneys with endotoxemia using engineered exosomes according to an embodiment of the present invention. The results include: (A) detection of carnitine levels in blood, renal tissue, and urine; (B) activity of key TCA cycle enzymes (e.g., citrate synthase); (CG) mRNA levels of key enzymes in lipid metabolism, and the ratios of NADPH / NAD and GSH / GSSG; (H) comparison of electron transport chain complex activity or protein expression; (IJ) detection of fatty acid content, fatty acid metabolic activity, and ATP; (L) visualization of lipid droplet accumulation in renal tubules; (M) detection of creatine kinase (CK) isoenzyme levels in renal tissue, and detection of NADH and NAD+ levels in renal tissue.

[0036] Figure 6 This diagram schematically illustrates the results of a study on the mechanism by which CD5L in engineered exosomes, according to an embodiment of the present invention, reprograms renal lipid metabolism by inhibiting fatty acid synthesis and promoting oxidative stress. The diagram may include: (A-B) Oxygen consumption rate (OCR) measurements of mitochondria isolated from renal tissue (e.g., Seahorse analysis), showing comparisons of parameters such as basal respiration, ATP-coupled respiration, and maximal respiration; (C) Heatmap of differentially abundant proteins involved in the tricarboxylic acid cycle from non-targeted proteomics of porcine kidney tissue; (D) Malate, citrate, and succinate levels in different sepsis groups; (E) Protein profiles showing differentially expressed key proteins involved in fatty acid breakdown; (F) Changes in key proteins involved in electron respiratory chain transport; (G) Changes in mt-Cot mRNA expression in mitochondria; (H) MitoSOX labeling in renal tissue indicating oxidative stress; (I) Changes in mRNA levels of Nd2, Cytb, and Atp6 in mitochondria; and (J) MS-quantified ratios of NADH and NAD+ in renal tissue.

[0037] Figure 7 This diagram schematically illustrates the results of the mechanism validation of engineered exosomes according to an embodiment of the present invention that improve septic kidney injury by regulating fatty acid oxidation to maintain mitochondrial homeostasis. This may include: (AF) observation of mitochondrial morphology in renal tissue mitochondria after administration of engineered exosomes; detection results of mitochondrial homeostasis-related indicators, including mitochondrial number, area, and number of fragmented mitochondria; (G) changes in mRNA levels of mitochondrial fission and fusion, which are related to mitochondrial homeostasis; (H) changes in the levels of mitochondrial fission and fusion proteins; (I) levels of Parkin and PINK1, key mitophagy proteins, in renal tissues of different groups; and (JK) mRNA levels of mitophagy-related genes (PINK1, Parkin, BNIP3, FUNDC1, P62, LC3B).

[0038] Figure 8 This is a map of the LV5 vector. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0040] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, 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 belongs. In the event of any conflict, the present specification shall take precedence.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0042] For the purposes of this invention, "exosomes" refer to vesicles secreted from cells, possessing a lipid bilayer membrane structure. Their diameter typically ranges from 30-150 nm, and they can carry a variety of bioactive molecules from the cells of origin. Exosomes can be isolated and identified using conventional methods in the art, for example, by morphology (saucer or cup-shaped as observed by transmission electron microscopy), size analysis (NTA or DLS peaks between 30-150 nm), and marker protein detection (e.g., Western blot analysis for positive markers such as CD9, CD63, and CD81, and negative markers such as Calnexin).

[0043] "Engineered exosomes" refer to exosomes whose source cells or exosomes themselves are modified through artificial intervention to change their properties (such as targeting) or load specific cargoes (such as therapeutic proteins, nucleic acids).

[0044] "CD5L protein is a known member of the soluble scavenger receptor cysteine-rich (SRCR) superfamily, involved in various biological processes such as immune regulation and lipid metabolism. In this invention, it specifically refers to CD5L protein that can be loaded into exosomes and delivered to target cells to exert functions such as inhibiting fatty acid synthesis. It can be of human, mouse, or other species origin, or recombinantly expressed.

[0045] "LTH" (kidney-targeting peptide) refers to a class of short peptides that can specifically recognize and bind to surface molecules on damaged renal tubular epithelial cells (in pathological conditions such as sepsis). LTH peptides themselves or their derivatives have been reported to exhibit certain kidney-targeting capabilities. The present invention is not limited to a specific LTH sequence; any peptide with similar kidney-targeting capabilities can be used in this invention.

[0046] "Conjugation" refers to the process of stably linking one molecule (such as LTH peptide) to another molecule or structure (such as the surface of exosomes) through chemical bonds.

[0047] A "therapeutically effective amount" refers to the amount of an active ingredient (e.g., CD5L protein) or the dosage of a formulation containing such an active ingredient (e.g., engineered exosomes) that is capable of preventing or treating the target disease (e.g., S-AKI). This amount may vary across species, individuals, and disease severity and can be determined through routine dose-finding studies.

[0048] The following is a detailed description of the SRM-enriched exosomes, their preparation method, and applications in conjunction with the examples and experimental data.

[0049] Example 1: Preparation and Characterization of Kidney-Targeted CD5L Engineered Exosomes (FRC-EXO) 1. Preparation of Engineered Exosomes 1. Source cell construction and culture: (1) Construct a lentiviral vector expressing the human CD5L gene. This vector was purchased from Genephrama, catalog number 230914B7.

[0050] Conventional methods can also be used to construct: CD5L gene (sequence shown in SEQ ID NO.2) is amplified with the following primer pair and inserted into LV5 vector (vector map shown in Figure 8 The NotI / BamH I restriction enzyme cutting site is shown in FIG.

[0051] Table 1

[0052] (2) The vector was used to infect 293T cells, and 293T cells that stably overexpressed human CD5L protein were obtained through screening.

[0053] (3) 293T cells overexpressing CD5L were seeded into cell culture dishes and cultured at 37°C and 5% CO2 using DMEM complete medium containing exosome-depleted FBS.

[0054] 2. Isolation and purification of CD5L exosomes (CD5L-EXO): When the cell density reached 80-90%, the medium was changed to serum-free medium or medium containing exosome-free serum and cultured for 48-72 hours.

[0055] Collect the cell culture supernatant and remove cell debris and large extracellular vesicles by a series of differential centrifugation steps: for example, 300 g for 10 minutes, 2000 g for 20 minutes, and 10,000 g for 30 minutes.

[0056] The supernatant was filtered through a 0.22 μm filter.

[0057] Pellet exosomes by centrifugation at 100,000 g for 70–90 minutes in an ultracentrifuge (e.g., Beckman Optima L-100 XP).

[0058] The supernatant was discarded, the pellet was resuspended in pre-chilled PBS, and ultracentrifuged again (100,000 g, 70-90 min) to wash the exosomes.

[0059] Finally, resuspend the exosome pellet in an appropriate amount of PBS. This is the first batch of exosomes containing CD5L protein (CD5L-EXO). Alternatively, large-scale purification can be performed using methods based on size exclusion chromatography (SEC) or tangential flow filtration (TFF).

[0060] 3. Conjugation of kidney-targeted peptide LTH: Synthesize or purchase the kidney-targeting peptide LTH. Depending on the chemical conjugation strategy used, the LTH peptide needs to be modified to introduce an azide functional group at one end for attachment.

[0061] Select an appropriate bioconjugation chemistry to conjugate the LTH peptide to CD5L-EXO. Surface click chemistry (Click Chemistry): If azide or alkyne groups have been introduced onto the exosome surface through metabolic labeling, or if the peptide carries a complementary group, the peptide can be linked via a copper-catalyzed (CuAAC) or copper-free (SPAAC) click reaction. After the reaction, unreacted peptide and reagents are removed by ultrafiltration to obtain the final engineered exosome FRC-EXO.

[0062] 2. Characterization of Engineered Exosomes 1. Morphological detection: Take a small amount of FRC-EXO sample and drop it onto a copper grid. After negative staining (such as phosphotungstic acid staining), use a transmission electron microscope (TEM) to observe its morphology.

[0063] The results are as follows Figure 1 As shown in Figure B, typical cup-shaped or spherical vesicle structures with a diameter of approximately 30-150 nm were observed.

[0064] 2. Particle size and concentration The particle size distribution and particle concentration of FRC-EXO were determined using a nanoparticle tracking analyzer (NTA) or a dynamic light scattering (DLS) analyzer.

[0065] The results are as follows Figure 1 Figure C shows the expected peak particle size range of 30-150 nm.

[0066] 3. Marker and load detection Western blot was performed to detect the expression of exosomal markers (CD9, CD63, CD81) (expected to be positive) and the absence of intracellular negative control proteins (e.g., Calnexin). Furthermore, the enrichment of CD5L protein was examined (CD5L content was significantly increased in FRC-EXO compared to exosomes from untransfected cells).

[0067] Whether LTH is successfully conjugated to the exosome surface can be verified by Western Blot or ELISA by introducing a tag (such as His-tag, FLAG-tag) on ​​the LTH peptide or using an antibody against LTH.

[0068] The results are as follows Figure 1 Shown in D: WB: CD9 / CD63 positive, Calnexin negative, CD5L positive, LTH conjugation rate>85%.

[0069] 4. Targeted uptake (in vitro): Fluorescently labeled (e.g., DiI or DiO) FRC-EXOs are co-incubated with cultured renal tubular epithelial cells (e.g., HK-2 cells damaged by LPS treatment). Fluorescence microscopy is used to observe whether the exosomes are effectively taken up by the cells and compare them with CD5L-EXOs not conjugated to LTH or exosomes conjugated to an irrelevant peptide.

[0070] like Figure 1 (E) Compared with CD5L-EXO not conjugated with LTH, enhanced LTH-mediated targeted uptake was demonstrated.

[0071] Example 2: In vivo targeting, efficacy, and safety evaluation of FRC-EXO in the Bama minipig model of sepsis AKI 1. Animal model establishment and grouping: Healthy male Bama miniature pigs (weighing approximately 20 kg) were selected. After adaptive feeding, they were randomly divided into the following groups: Sham group: The mice only received anesthesia and surgical procedures without LPS injection.

[0072] LPS model group (LPS): Lipopolysaccharide (Escherichia coli O111:B4) was injected intraperitoneally or intravenously at a dose sufficient to induce sepsis and AKI.

[0073] LPS + control exosome group (LPS + Ctrl-EXO): After LPS injection, an equal amount of exosomes from non-transfected HEK293T cells and conjugated with irrelevant peptides were administered.

[0074] LPS + FRC-EXO group (LPS + Treatment): After LPS injection, FRC-EXO prepared in Example 1 (dosage of 3 μg / kg, dissolved in normal saline) was injected through the ear vein.

[0075] The number of animals in each group was determined according to statistical requirements (n = 4).

[0076] 2. In vivo targeting observation: Some animals were injected with fluorescently labeled (e.g., DiR) FRC-EXO or control exosomes. Following injection, in vivo imaging was used to observe the distribution of exosomes, specifically their accumulation in the kidneys. The accumulation of FRC-EXO in the injured kidneys increased over time.

[0077] 3. Systemic indicators and microcirculation monitoring During the experiment, the animals' vital signs, including heart rate and mean arterial pressure (MAP), were monitored. Blood samples were collected to measure serum levels of cardiac injury markers (cTnI) and liver injury markers (ALT, AST).

[0078] Lateral flow dark field (SDF) microscopy and other methods are used to monitor the microcirculatory perfusion of the renal cortex or sublingual mucosa and evaluate indicators such as capillary density and blood flow velocity.

[0079] The results are as follows Figure 2 As shown, the LPS group experienced tachycardia, hypotension, elevated cardiac and liver injury markers, and microcirculatory disturbances. FRC-EXO treatment significantly improved these systemic indicators and microcirculatory disturbances.

[0080] 4. Assessment of renal function and tissue damage Blood samples were collected at different time points to measure serum creatinine (Scr) and blood urea nitrogen (BUN) levels and evaluate glomerular filtration function.

[0081] At the end of the experiment (e.g., 48 hours), animals were sacrificed and kidney tissue was obtained. A portion was used for paraffin sectioning and stained with hematoxylin and eosin (HE). Tubular damage (necrosis, brush border loss, cast formation, interstitial edema, inflammatory cell infiltration, etc.) was observed under a microscope and semi-quantitatively scored.

[0082] The results are as follows Figure 3As shown in the results, the Scr and BUN levels in the LPS group were significantly increased, and the kidney tissue showed obvious damage. FRC-EXO treatment was able to significantly reduce the Scr and BUN levels and alleviate the kidney histological damage.

[0083] 5. Assessment of renal lipid metabolism: Kidney tissue was obtained and frozen sections were made. Oil red O staining was performed to observe the accumulation of lipid droplets in renal tubular cells, and image analysis was performed to quantify the area or number of lipid droplets.

[0084] Kidney tissue homogenate was obtained, lipids were extracted, and lipidomics analysis was performed using liquid chromatography-mass spectrometry (LC-MS) technology to detect the total free fatty acid (FFA) content and the levels of various acylcarnitines.

[0085] The results are as follows Figure 4 As shown: The LPS group showed significant lipid droplet accumulation in the kidneys, and elevated levels of FFA and long-chain acylcarnitines. FRC-EXO treatment significantly reduced lipid droplet accumulation and lowered FFA and acylcarnitine levels, indicating that it improved lipid metabolism disorders.

[0086] 6. Assessment of renal mitochondrial bioenergetics function: Blood, urine and kidney tissue samples were collected to test the carnitine content in the blood, kidney tissue and urine; the mRNA expression levels of key enzymes of fat metabolism, NADPH / NAD content and GSH / GSSG content, and creatine kinase CK isoenzyme content in the kidney were tested; and the NADH and NAD+ contents in the kidney tissue were tested.

[0087] Detection of electron transport chain complex activity or protein expression levels, fatty acid content, fatty acid metabolic activity, and ATP content. Transmission electron microscopy shows lipid droplet aggregation in renal tubules. The results are as follows Figure 5 As shown: In the LPS group, the carnitine content in the kidney increased, the lipase increased, the FAO decreased, the ATP decreased, and the fatty acid metabolism decreased. FRC-EXO treatment was able to restore the carnitine content, repair the electron transport chain function, restore energy metabolism, improve fatty acid metabolism disorders, and maintain renal mitochondrial energy metabolism homeostasis.

[0088] 7. Assessment of mitochondrial function and homeostasis Mitochondria were isolated from fresh kidney tissue. Mitochondrial oxygen consumption rate (OCR) was measured using a Seahorse XF analyzer to assess basal respiration, ATP synthesis-coupled respiration, maximal respiratory capacity, and spare respiratory capacity.

[0089] Detect the activity or protein expression level of key enzymes in the mitochondrial TCA cycle (e.g., citrate synthase) or related metabolites. Detect the activity or protein expression level of electron transport chain complex (IV).

[0090] Detection of mitochondrial homeostasis-related indicators: Use probes such as MitoSOX Red to detect mitochondrial ROS levels.

[0091] The results are as follows Figure 6 As shown, the LPS group showed a significant decrease in mitochondrial OCR, impaired TCA cycle and ETC function, increased ROS production, decreased membrane potential, kinetic imbalance, and abnormal autophagy. FRC-EXO treatment significantly restored mitochondrial OCR, improved TCA / ETC function, reduced ROS, stabilized membrane potential, and regulated kinetic balance, potentially promoting effective mitophagy.

[0092] 8. Verification of the mechanism of action By measuring key enzymes of fatty acid metabolism, the expression or activity of FASN, a key enzyme in fatty acid synthesis, in renal tissue was detected. The mRNA or protein expression levels of key enzymes of fatty acid oxidation were also detected.

[0093] The results are as follows Figure 7 As shown: FRC-EXO treatment inhibited fatty acid synthesis and upregulated the expression of key FAO enzymes such as CPT1a / CPT2. Etomoxir significantly weakened or reversed the renal protective effect of FRC-EXO, demonstrating that the therapeutic efficacy of these exosomes is at least partially dependent on their ability to promote fatty acid oxidation.

[0094] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0095] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0096] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An engineered exosome, characterized in that: The exosomes are loaded with CD5L protein; the surface of the exosomes is conjugated with the kidney-targeting peptide LTH, and the amino acid sequence of the LTH is shown in SEQ ID NO:

1.

2. The exosome according to claim 1, wherein The exosomes are derived from fibrous reticular cells that overexpress CD5L protein.

3. The exosome according to claim 1, wherein The therapeutically effective amount of the CD5L protein is 3 μg / kg body weight.

4. The exosome according to claim 1, wherein The LTH peptide was conjugated to exosomal membrane proteins via click chemistry.

5. A method for preparing the engineered exosomes according to any one of claims 1 to 4, characterized in that: The method comprises: obtaining CD5L-overexpressing fibroblasts through genetic engineering, isolating exosomes from the supernatant of the CD5L-overexpressing cells, and conjugating LTH peptide to the surface of the exosomes through a DBCO-azide click chemistry reaction to obtain engineered exosomes.

6. The method according to claim 5, characterized in that The genetic engineering modification includes: A lentiviral vector encoding CD5L was constructed and used to transfect FRC cells.

7. The method according to claim 5, characterized in that The exosomes are separated by ultracentrifugation or size exclusion chromatography.

8. Use of the engineered exosomes according to any one of claims 1 to 4 in the preparation of a drug for treating sepsis-induced acute kidney injury.

9. The use according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

10. The use according to claim 8, characterized in that The dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections and dispersions.

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