Engineered exosome derived from iPSC-MSC and preparation method and application thereof

Abstract

The invention relates to an iPSC-MSC-sourced engineered exosome as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention provides a double-module engineering strategy which comprises the following steps: firstly, overexpressing hypoxia regulatory factor FIH-1 protein by induced pluripotent stem cell-derived mesenchymal stem cells through genetic engineering, and extracting exosomes loaded with the protein from the hypoxia regulatory factor FIH-1 protein; secondly, coupling a kidney targeting peptide to the surface of the exosome through chemical modification, so as to construct an engineered exosome (EXos-FIH1-KT) with an active targeting capability; the exosome can be efficiently enriched in renal tubular epithelial cells, and can accurately inhibit HIF-alpha and NF-kB / NLRP3 inflammation pathways and autophagy pathways at the downstream of the HIF-alpha by delivering FIH-1 protein, so that the renal fibrosis process is remarkably reversed in an animal model, and good biological safety is shown. The invention provides a brand new delivery platform and strategy for targeted therapy of organ fibrosis diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to engineered exosomes derived from iPSC-MSCs, their preparation methods, and applications. Background Technology

[0002] Uric acid (UA) is a product of purine metabolism and is considered a mediator of pathological processes such as oxidative stress, inflammation, and endothelial dysfunction. Hyperuricemia (HUA) is characterized primarily by elevated serum uric acid levels. Long-term elevated uric acid levels can trigger renal tubular epithelial cell apoptosis, inflammatory responses, and oxidative stress, leading to cell apoptosis. These damaged renal tubular cells subsequently cause renal tubular epithelial fibrosis through autocrine or paracrine processes. The renal tubular epithelial fibrosis phenotype is a key factor in the progression of hyperuricemia to hyperuricemic nephropathy. However, current drugs for treating renal fibrosis caused by hyperuricemia have serious adverse reactions such as exfoliative dermatitis and liver and kidney damage, and research on the molecular mechanisms of renal fibrosis treatment is still insufficient.

[0003] Exosomes are nanoscale, bilayered phospholipid vesicles with the same topological structure as cells. They contain various substances such as DNA, proteins, and enzymes. After being taken up by recipient cells, they play a role in the treatment of various diseases by altering cell signaling. Exosomes derived from mesenchymal stem cells (MSCs) are considered promising drug delivery carriers due to their low immunogenicity and rich content of regenerative regulatory molecules. However, natural exosomes suffer from poor therapeutic efficacy and targeting due to insufficient targeting ability and short retention time; furthermore, traditional MSCs involve invasive harvesting procedures and a decline in stemness with cell passage. Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) have stronger proliferative capacity, lower immunogenicity, and smaller batch-to-batch variability, allowing for the large-scale production of iPSC-MSCs and avoiding the problems associated with traditional MSCs. Moreover, autologous iPSC-MSCs or their exosomes can be used for treatment without raising ethical issues or immune rejection reactions, showing great therapeutic potential in hyperuricemia and renal fibrosis. Engineered exosomes are natural exosomes that have been precisely modified through genetic engineering, chemical modification, and other advanced biotechnologies to give them entirely new functions. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide an engineered exosome derived from iPSC-MSC. The present invention constructs renal-targeting engineered exosomes by engineering iPSC-MSC-derived exosomes to achieve efficient enrichment and controllable release of the loaded drug in the renal parenchyma, laying a delivery platform for precise intervention in renal fibrosis. The second objective is to provide a method for preparing the engineered exosome derived from iPSC-MSC. The third objective is to provide an application of the engineered exosome derived from iPSC-MSC, with targeted inhibition of renal tubular epithelial cell fibrosis becoming a treatment strategy for the prevention and treatment of HUA and hyperuricemic nephropathy developed from HUA.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an engineered exosome derived from iPSC-MSC, wherein the engineered exosome highly expresses FIH-1 protein on its surface, the surface of the engineered exosome is modified with a targeting peptide, and the engineered exosome markers are CD9, CD63 and TSG101; Preferably, the amino acid sequence of the FIH-1 protein is: MAATAAEAVASGSGEPREEAGALGPAWDESQLRSYSFPTRPIPRLSQSDPRAEELIENEEPVVLTDTNLVYPALKWDLEYLQENIGNGDFSVYSASTHKFLYYDEKKMANFQNFKPRSNREEMKFHEFVEKLQDIQQRGGEERLYLQQTLNDTVGRKIVMDFLGFNWNWINKQQGKRGWGQLTSNLLLIGMEGNVTPAHYDEQQNFFAQIKGYKRCILFPPDQFECLYPYPVHHPCDRQSQVDFDNPDYERFPNFQNVVGYETVVGPGDVLYIPMYWWHHIESLLNGGITITVNFWYKGAPTPKRIEYPLKAHQKVAIMRNIEKMLGEALGNPQEVGPLLNTMIKGRYN (SEQ ID NO:1), and the targeting peptide sequence is: CGRRRRPGK (SEQ ID NO:1). NO:2); Further: The method for preparing engineered exosomes derived from iPSC-MSCs is characterized by the following preparation steps: S1: Cell Culture and Induction: iPSCs were cultured in mTESR1 medium and induced in differentiation medium for 14 days to obtain iPSC-MSCs. S2: Functional protein loading: The iPSC-MSCs obtained in step S1 were infected with lentiviral particles carrying the FIH-1 gene to overexpress the FIH-1 protein. S3: Exosome extraction: The iPSC-MSC cell culture supernatant from step S2 was collected using exosome-free medium, and exosomes were extracted by differential centrifugation. S4: Targeted Modification: The exosomes extracted in step S3 were coupled with cholesterol-modified targeting peptides to obtain targeted engineered exosomes Exos-FIH1-KT.

[0006] Preferably, the differential centrifugation procedure is as follows: Centrifuge the cell culture supernatant at 10,000g for 20 minutes to remove cell debris and large particles; Collect the supernatant and centrifuge at 100,000g for 60 minutes to collect the exosome precipitate; Preferably, the method for preparing the cholesterol-modified targeting peptide is as follows: cholesterol, PEG8, and the targeting peptide are coupled in a PBS solution; Furthermore, the use of the engineered exosomes derived from iPSC-MSC in the preparation of drugs for treating hyperuricemic nephropathy; Furthermore, the engineered exosomes derived from iPSC-MSCs are used in the preparation of drugs for the treatment of hyperuricemic renal fibrosis.

[0007] The beneficial effects of this invention are as follows: 1. Overcoming the limitations of natural exosomes By chemically conjugating kidney-specific targeting peptides, active targeting of exosomes to renal tubular epithelial cells (HK-2) was achieved, solving the problem of low enrichment efficiency of natural exosomes at lesion sites. Through genetic engineering, the therapeutic protein FIH-1 was overexpressed in exosomes (iPSC-MSCs), making exosomes a delivery vector for FIH-1.

[0008] 2. Optimize cell sources to ensure quality and sustainability. Using iPSC-MSCs as the exosome source avoids the problems of limited proliferation capacity, large batch-to-batch variability, and invasive harvesting associated with traditional MSCs. iPSC-MSCs have stronger expansion capacity and a stable phenotype, providing a renewable and high-quality cell source for large-scale, homogeneous production of therapeutic-grade exosomes.

[0009] 3. Precise intervention in the core pathways of the disease Experiments have shown that engineered exosomes can effectively deliver FIH-1 into HK-2 cells. FIH-1 inhibits the transcriptional activity of HIF-α through hydroxylation, thereby inhibiting downstream pathways, reducing the expression of inflammatory factors such as Cleaved-caspase-1, IL-1β, and IL-18, regulating autophagy-related proteins such as LC3 and p62, and restoring autophagy homeostasis.

[0010] 4. Significantly reverses fibrosis In vitro experiments: In a uric acid-stimulated HK-2 cell model, Exos-FIH1-KT significantly reduced the expression of fibrosis markers (α-SMA, Fibronectin, TGF-β1) and reversed the epithelial-mesenchymal transition (EndMT) process.

[0011] In vivo experiments: In a mouse model of hyperuricemic nephropathy, it significantly reduced serum uric acid, creatinine, and blood urea nitrogen levels, improved renal function, significantly reduced collagen deposition in renal tissue (Masson and Sirius Red staining positive areas), alleviated renal tubular interstitial fibrosis, reduced the expression of renal tissue injury markers (TIM-1) and fibrosis markers, and improved mitochondrial ultrastructural damage in renal tubular epithelial cells.

[0012] 5. Clinical translational potential Targeted modification ensured the enrichment of exosomes at the renal lesion site, improving treatment efficiency. Mice treated with Exos-FIH1-KT showed no significant pathological changes in vital organs such as the heart, liver, spleen, and lungs, and blood routine indicators (such as RBC, HGB, and PLT) and liver function indicators (ALT and AST) were not significantly different from the control group. Using autologous iPSC-derived cells and exosomes avoids immune rejection and ethical controversies.

[0013] The "functional protein loading + targeted peptide modification" approach employed in this invention is a modular strategy. By replacing the targeted peptide (targeting other organs or cells) and / or the loaded protein (targeting different disease targets), this platform can be applied to the treatment of other fibrotic diseases (such as liver and lung fibrosis) or inflammatory diseases, showing broad application prospects.

[0014] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 A schematic diagram illustrating the preparation process and application of Exos-FIH1-KT engineered exosomes; Figure 2 Features of Exos-FIH1-KT: (A) Schematic diagram of the collection and isolation of FIH1-overexpressing exosomes and their binding to cholesterol-targeting peptides; (B-D) mRNA and protein levels of FIH1 in iMSC, iMSC + LV-NC, and iMSC + LV-NC-FIH1 detected by PCR and Western Blot; (E) TEM images of Exos, Exos-FIH1, and Exos-FIH1-KT. (F) Size distribution of Exos, Exos-FIH1 and Exos-FIH1-KT captured by NTA system; (G) Protein levels of FIH1 and extracellular vesicle marker proteins (such as CD9, CD63, CD81 and TSG101) in iMSC, Exos, Exos-FIH1 and Exos-FIH1-KT detected by Western blotting; (H) Confirmation of the binding of Exos-FIH1 to cholesterol-targeting peptides by confocal microscopy, scale bars 100 and 20 micrometers. Figure 3 The anti-fibrotic effect of Exos-FIH1-KT: (A) Schematic diagram of Exos-FIH1 inhibiting the transformation of HK-2 phenotype to fibrotic phenotype; (B) Changes in cell viability under different uric acid concentrations and treatment times; (C–D) qRT-PCR and WB analysis showing the mRNA and protein expression levels of FN1, α-SMA, and TGF-β1 in HK-2 under different uric acid concentrations; (E–F) qRT-PCR and WB analysis showing the mRNA and protein expression levels of FN1, α-SMA, and TGF-β1 in HK-2 under PBS, UA, UA+Exos, UA+Exos-FIH1, or UA+Exos-FIH1-KT treatments. Figure 4 To investigate the inhibitory effect of Exos-FIH1-KT on NF-κB in in vitro iMSCs: (BD) qRT-PCR and WB analysis showed the expression of inflammatory pathway signaling molecules (NF-κB, NLRP3, Cleaved-casp-1, IL-18 and IL-1β) in HK-2 after treatment with PBS, UA, UA+Exos, UA+Exos-FIH1, and UA+Exos-FIH1-KT; Figure 5To investigate the inhibitory effect of Exos-FIH1-KT on autophagosomes in in vitro iMSCs: (BD) qRT-PCR and WB analysis showed the expression of autophagy (LC-3 and P62) pathway signaling molecules in HK-2 cells after treatment with PBS, UA, UA+Exos, UA+Exos-FIH1, and UA+Exos-FIH1-KT. (EF) Statistical analysis of autophagosome-like vesicles in HK-2 cells treated with PBS, UA, UA+Exos, UA+Exos-FIH1, and UA+Exos-FIH1-KT was performed under transmission electron microscopy. Scale bar: 1 μm. Figure 6 The therapeutic effects of Exos-FIH1-KT in C57BL / 6J hyperuricemic mice: (A) Schematic diagram of Exos-FIH1-KT treatment in mice with renal tubular epithelial cell fibrosis; (B) Survival rate of mice with renal tubular epithelial cell fibrosis during treatment; (CE) Changes in renal function over time in the PBS group, Exos group, Exos-FIH1 group, and Exos-FIH1-KT group; (F) HE, PAS, Masson, and Sirius Red staining of kidney sections in each group at the end of treatment, with a scale bar of 50 micrometers; (G, H) Renal tubular injury scores of kidney sections in each group at the end of treatment based on HE and PAS staining; (I, J) Renal sections in each group at the end of treatment, based on Masson and Sirius Red staining. The positive areas of Red staining, (KP) at the end of treatment, FIH, early renal injury marker TIM-1 and renal fibrosis markers FN1 and α-SMA in each group were fluorescently stained and the mean relative fluorescence intensity was analyzed, (Q) the aspect ratio, roundness and sphericity of mitochondria in each group were compared under transmission electron microscopy, (R, S) qRT-PCR and WB analysis showed the expression of mRNA and protein of FN1, α-SMA and TGF-β1 in each group; Figure 7 Microscopic image (A) and linear graph (B) of FIH1 overexpression on exosomes achieved by lentivirus transfection carrying FIH1; Figure 8 The trend of body weight change during injection treatment in each experimental group of PBS, Exos, Exos-FIH1 and Exos-HIF1-KT; Figure 9 Changes in serum RBC, HGB, PLT, ALT, AST, and serum WBC in each experimental group (PBS, Exos, Exos-FIH1, and Exos-HIF1-KT). Figure 10 The visceral morphology and structural characteristics of each experimental group including PBS, Exos, Exos-FIH1, and Exos-HIF1-KT. Detailed Implementation

[0016] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0017] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0018] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0019] Example 1: Preparation and Characterization of Engineered Exosomes 1. Culture of iPSC and HK-2 cells and induction of iPSC differentiation, establishment of mesenchymal stem cells overexpressing FIH-1 Induced pluripotent stem cells (iPSCs) were cultured using ESC-certified matrix gel as the substrate and mTESR1 medium to maintain cell growth. Following the kit instructions, cells were cultured in differentiation medium for 14 days, followed by digestion with 0.25% trypsin-EDTA. Subsequently, cells were cultured at 5 × 10⁶ cells / day. 4Cells were reseeded at a density of [density] mL in 0.1% gelatin-coated 25 cm² culture flasks and cultured in a medium specifically for induced mesenchymal stem cells (iMSCs). The first confluence of cells was recorded as passage 1. Typically, by passage 3, cells exhibit typical fibroblast-like morphology, at which point they can be confirmed as successfully induced mesenchymal stem cells (iMSCs). Human Kidney-2 (HK-2) renal tubular epithelial cells were purchased from the Chinese Academy of Sciences. iPSC-MSCs and HK-2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. The medium was changed every 2-3 days, and cells were passaged when they reached 80%-90% confluence. Cells were digested with 0.25% trypsin-EDTA at a 1:3 ratio before passage. Cells from passages 5-10 were used for experiments to ensure stable biological characteristics.

[0020] iPSCs were cultured in DMEM medium containing 10% FBS and passaged when they reached 80%-90% confluence. iPSCs were then infected with the constructed FIH-1 lentiviral particles, with the addition of polybrene (8 μg / mL) to enhance infection efficiency. Forty-eight hours post-infection, the expression of green fluorescent protein (GFP) was observed using a fluorescence microscope to assess transfection efficiency. The overexpression of the FIH-1 gene and protein was detected by RT-PCR and Western blotting to confirm successful FIH-1 overexpression.

[0021] 2. Isolation and characterization of FIH-1 engineered exosomes, and targeted modification of FIH-1 engineered exosomes. When the iPSC density reached approximately 50% to 60%, the conditioned medium was replaced with exosome-free medium (OriCell, Australia) and cultured for 48 hours, followed by collection. FIH-1 engineered exosomes were extracted from the iPSC supernatant by differential centrifugation. The supernatant was then washed with PBS. Finally, the FIH-1 engineered exosomes were resuspended in PBS and stored at -80°C. The ultrastructural morphology of the FIH-1 engineered exosomes was examined using transmission electron microscopy (TEM) (HT 7800, HITACHI). The size of the FIH-1 engineered exosomes was tracked using nanoparticle tracking analysis (NTA) (Nanosight NS 300, Malvern), and the results were analyzed using GraphPadPrism 10 software. Western blotting was performed to validate representative markers of the FIH-1 engineered exosomes (CD9, CD63, and TSG101).

[0022] To enhance the targeting of exosomes to HK-2 cells, a cholesterol-targeting peptide was modified on the surface of FIH-1 engineered exosomes using a chemical coupling method. Cholesterol anchoring motifs and HK-2 targeting peptides were designed, and cholesterol-peptide conjugates were synthesized via solid-phase synthesis. The exosomes and cholesterol-peptide conjugates were mixed in PBS, and a catalyst was added to activate the coupling reaction between carboxyl and amino groups. Unbound peptide conjugates and catalysts were then removed by ultrafiltration centrifugation.

[0023] Figure 1 This paper describes a schematic diagram of the preparation process of extracting FIH1-overexpressing Exos from iMSCs overexpressing FIH1, and then coupling it with a cholesterol-modified targeting peptide to generate Exos-FIH1-KT. First, iPSCs were induced to differentiate into iMSCs, and the successful differentiation was confirmed by optical microscopy and specific staining markers. Then, FIH1 overexpression was achieved in exosomes by transfection with lentiviruses carrying FIH1, and the expression was observed under a microscope. Figure 7 The optimal multiple of infection (MOI) was determined to be 50 plaque-forming units per cell. At the optimal MOI, compared with the untreated group and the lentiviral empty vector transfection group, the results were obtained by PCR and WB (microscopic examination). Figure 2 (B, 2C, and 2D) assess transfection efficiency. FIH1 expression increased by 10.51 ± 2.06 times after transfection with lentivirus.

[0024] Subsequently, Exos-FIH1 was isolated from the exosome-free supernatant of iMSCs-FIH1 cultured by differential centrifugation. To enhance the targeting ability of Exos-FIH1 to renal tubular epithelial cells, we modified Exos-FIH1 with a cholesterol-modified targeting peptide (cholesterol-PEG8-cGrrrrpGk), ultimately generating Exos-FIH1-KT. TEM was used to analyze the results. Figure 2 E), NTA ( Figure 2 F) and WB ( Figure 2 G) Exos-FIH1-KT, Exos-FIH1, and Exos from untreated iMSCs were characterized. No significant differences were observed in shape, size, or concentration distribution among Exos-FIH1-KT, Exos-FIH1, and Exos. Typical Exos biomarkers (CD63, TSG101, and CD81) were confirmed, while the negative biomarker (Calnexin) was absent in all Exos preparations. Furthermore, significantly higher levels of FIH1 protein were observed in Exos-FIH1-KT and Exos-FIH1 compared to Exos and untreated cells, confirming successful loading of the FIH1 protein. To investigate the binding of cholesterol-modified targeting peptides to Exos-FIH1, Exos-FIH1 was labeled with PKH67 green fluorescent dye, while the cholesterol-modified targeting peptides were labeled with Cy5 dye (Cy5-KT). The appearance of combined yellow fluorescence resulting from the superposition of green and red fluorescence confirmed that Cy5-KT was successfully coupled to the Exos-FIH1 surface. Figure 2 H).

[0025] Example 2: In vitro anti-renal fibrosis effect of engineered exosomes 1. In vitro FIH-1 engineered exosome uptake detection iPSC-MSC cell culture supernatant was collected using exosome-free medium, centrifuged at 10,000g at 4°C for 20 minutes to discard the large vesicle pellet, and then extracted exosomes by ultracentrifugation at 100,000g at 4°C for 60 minutes. Cholesterol peptide was then added to the extracted exosome suspension at a concentration of 10 μg / mL in a 100-200 μL system and incubated at room temperature for 30 minutes. DiL dye (pre-diluted with anhydrous ethanol) was added to the mixture to a final concentration of 10 μg / mL, and the mixture was gently mixed again and incubated for another 30 minutes in the dark to prevent dye quenching. DiL-labeled exosomes were then extracted by ultracentrifugation at 100,000g at 4°C for 60 minutes. The isolated FIH-1 engineered exosomes were then incubated with HK-2 for 12 hours. Twelve hours later, unbound FIH-1 engineered exosomes were washed away with PBS (Gibco, USA), fixed with 4% paraformaldehyde (PFA) for 10 minutes, and stained with DAPI for 5 minutes. The uptake of FIH-1 engineered exosomes by HK-2 cells was observed under a laser confocal microscope (LSM880, Germany), and the uptake efficiency of exosomes was quantitatively analyzed using flow cytometry to evaluate the effectiveness of targeted modification and the cellular uptake capacity of exosomes.

[0026] 2. Immunoprecipitation and ChIP detection of HIF-α and NF-κB / NLRP3 inflammatory pathways Cells were washed twice with PBS, and lysed for 30 minutes with lysis buffer containing protease inhibitors. The cells were then centrifuged at 12,000 × g for 10 minutes at 4 °C, and the supernatant was collected. After pretreating magnetic beads, HIF-α antibody was added, and the mixture was stirred at room temperature for 15 minutes. Cells were washed three times with buffer, and then incubated overnight at 4 °C with cell lysis buffer. Cells were washed three times with buffer. 1× SDS-PAGE loading buffer was added, and the mixture was stirred thoroughly. The mixture was heated at 95 °C for 5 minutes, centrifuged at 10,000 × g for 10 minutes at room temperature, and the supernatant was collected for Western blot analysis.

[0027] Cells were cross-linked with 1% formaldehyde at room temperature for 10 min, and the cross-linking reaction was terminated with glycine for 5 min. Cells were washed twice with pre-chilled PBS, collected in centrifuge tubes, and centrifuged at 4 °C, 1500 r / min for 5 min. The supernatant was discarded, and the cell pellet was collected. The cells were incubated on ice for 10 min with cell lysis buffer containing protease inhibitors, and the nuclear pellet was collected. After nuclear lysis, chromatin was sheared to 200-1000 bp fragments using an ultrasonic homogenizer. Immunoprecipitation was then performed, with samples divided into an input group and an immunoprecipitation (IP) group. The IP group was incubated overnight at 4 °C with either HIF-α antibody or IgG control antibody, and then Protein A / G magnetic beads were added to capture the antibody-chromatin complex. After washing with low-salt buffer, high-salt buffer, lithium chloride buffer, and TE buffer, the DNA fragments were then decrosslinked overnight at 65°C with SDS elution buffer (1% SDS + 1M NaHCO3), and the proteins were digested with protease. Subsequently, the DNA fragments were purified by phenol-chloroform extraction or by a kit. Primers for the NLRP3 promoter region were designed, and the enrichment folds of HIF-α and NF-κB-bound DNA were detected.

[0028] Renal tubular epithelial fibrosis models were established by treating HUA with 0, 250, 500, 750, and 1000 μM UA for 12, 24, 36, and 48 hours, respectively. The model was validated by upregulated expression of fibrotic cell markers FN1 and α-SMA, and upregulated expression of the pro-fibrotic cytokine TGF-β1. After 24 hours of incubation, treatment with only 750 μM UA resulted in cell viability between 70% and 80%. Figure 3 B). qRT-PCR and WB results showed that 24 hours of treatment with 750 μM UA resulted in significant renal tubular epithelial fibrosis. Figure 3 (C and 3D). Therefore, the subsequent renal tubular epithelial cell fibrosis model was established by treatment with 750 μM UA for 24 hours. The efficacy of Exos-FIH1-KT against renal tubular epithelial fibrosis in HK-2 was investigated using PBS, UA, UA+Exos, UA+Exos-FIH1, and UA+Exos-FIH1-KT treatments. qRT-PCR and WB results showed that ( Figure 3 E and 3F) indicated that Exos-FIH1-KT significantly inhibited renal tubular epithelial fibrosis. qRT-PCR results showed that treatment with Exos-FIH1-KT reduced the mRNA levels of FN1, α-SMA, and TGF-β1 in HK-2 cells. Figure 3 E). Similar results were also observed in WB (E). Figure 3F). Furthermore, compared to the untreated group (UA group), Exos-FIH1-KT treatment significantly reduced the number of Vimentin-positive cells and significantly increased the number of VE-cadherin-positive cells, with no statistically significant difference between the Exos-treated group and the untreated group (UA group). Overall, these results collectively demonstrate that Exos-FIH1-KT significantly inhibits EndMT in MAECs in vitro by delivering FIH1.

[0029] Engineered exosomes inhibit inflammatory pathways in HK-2 via qRT-PCR ( Figure 4 B) and WB Figure 4 The expression of FIH1, HIF-1α, and downstream pathway factors (NF-κB, NLRP3, Cleaved-casp-1, IL-18, and IL-1β) in HK-2 was detected by C and 4D. The results confirmed that, compared with the PBS group, FIH1 levels were decreased in the UA group, while the mRNA and protein levels of HIF-1α, NF-κB, NLRP3, Cleaved-casp-1, IL-18, and IL-1β were significantly increased. Furthermore, compared with the UA group, FIH1 levels were increased in the UA+Exoes-FIH-KT group, while the mRNA and protein levels of HIF-1α, NF-κB, NLRP3, Cleaved-casp-1, IL-18, and IL-1β were significantly decreased.

[0030] Engineered exosomes inhibit the autophagy pathway in HK-2. Examples used PCR (…). Figure 5 B) and WB Figure 5 C and Figure 5 D) The effects on autophagy-related proteins (LC3, p62) were assessed. Compared with the PBS group, the autophagy markers LC3 and p62 were significantly increased in the UA group. Compared with the UA group, the autophagy markers (LC3 and p62) were significantly decreased in the UA+Exoes-FIH and UA+Exoes-FIH-KT groups. Statistical analysis of autophagosome-like vesicles was also performed. Figure 5 E, Figure 5 F). It was found that autophagosome vesicles were significantly reduced in the UA+Exoes-FIH and UA+Exoes-FIH-KT groups compared with the UA group.

[0031] Example 3: In vivo anti-fibrotic application of engineered exosomes 1. Establishing an animal model Eight-week-old male C57BL / 6J mice were fed a chronic purine diet (250 mg / kg potassium oxonate and 250 mg / kg hypoxanthine once daily for two weeks) to induce hyperuricemia. Serum uric acid, creatinine, and blood urea nitrogen levels were analyzed using a uric acid assay kit, and the degree of tubulointerstitial fibrosis in kidney sections was assessed using Masson staining. Mice were housed in a controlled environment with a 12-hour light / dark cycle. The animal experimental protocol was reviewed and approved by the Ethics Committee of Army Medical University.

[0032] 2. In vivo anti-fibrosis detection of engineered exosomes To detect HK-2 cell damage in high-altitude hyperuricemia lesions (using HIF-α as a hypoxia marker and α-SMA and Fibronectin as fibrosis markers. ① In mouse kidney tissue, immunohistochemistry and in situ hybridization were used to detect marker expression; ② In isolated HK-2 cells, RT-PCR and Western Blot were used to detect marker expression levels.) HK-2 cells were placed in culture media containing 0, 250, 500, 750, and 1000 μM uric acid for 24, 48, and 72 hours, respectively. The expression of fibrosis markers αSMA and FN1 was monitored by RT-PCR and Western Blot, and the level of oxidative stress damage in HK-2 cells was assessed by ROS immunofluorescence staining.

[0033] Immunohistochemistry was performed by dewaxing paraffin sections (5 μm) of the kidney with xylene and rehydrating with fractionated ethanol. Antigens were extracted by microwave in sodium citrate buffer (pH 6.0). The activity of endogenous tissue peroxidase was quenched with 3% hydrogen peroxide. Sections were treated with blocking buffer for 30 minutes at room temperature. Sections were incubated overnight at 4°C with primary antibody against α-smooth muscle actin (α-SMA) (1:1000), followed by appropriate secondary antibody application. After washing, the immunoreactivity of the slides was detected with diaminobenzidine, followed by hematoxylin and eosin staining. The slides were then observed under a light microscope.

[0034] In situ hybridization experiments were performed by incubating with proteinase K (40 μg / mL) at room temperature for 20 minutes, followed by incubation in pretreatment buffer for 10 minutes. Preliminary hybridization was then carried out at 60°C for 2 hours. The miRNA probe was incubated at 90°C for 5 minutes, followed by overnight hybridization at 60°C. The slides were then mounted at room temperature for 1 hour, incubated overnight with anti-digoxigenin antibody at 4°C, and stained with BCIP / NBT in the dark for 4 to 48 hours. The reaction was terminated with pH 8.0 buffer. Nuclear Fast Red staining was performed again, and the slides were mounted with neutral resin. The final working concentration was 20 nanomolar, and the hybridization temperature was 60°C.

[0035] For protein blotting, cultured HK-2 cells were collected and homogenized in radioimmunoprecipitation assay lysis buffer (RIPA) containing protease / phosphatase inhibitors and phenylmethanesulfonyl fluoride (PMSF). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit. After high-temperature denaturation, gel electrophoresis was performed (80 V, 30 min; 120 V, 60–90 min). Wet transfer was performed (200 mA, 60 min). After transfer, the membrane was washed three times with Tris-buffered saline containing 0.05% Tween 20 for 5 min each time. Blocking was performed with 5% skim milk at room temperature for 1 h, followed by incubation with primary antibody (all antibodies diluted 1:2000) at 4°C overnight (8 h–12 h). On the second day, the membrane was incubated with Tris-buffered saline containing 0.05% Tween 20. After washing the membrane with buffered saline, it was incubated with the corresponding secondary antibody at room temperature for 1 h. After washing the membrane three times with Tris buffered saline containing 0.05% Tween 20, it was exposed to luminescent solution and the grayscale analysis bands were detected using a chemiluminescence system. The expression of the hypoxia marker HIF-α and the fibrosis marker α-SMA was detected by RT-PCR. Total RNA was extracted from tissues or cultured cells using Trizol reagent, and the RNA concentration and purity were determined. 400 ng of RNA was used as a template and reacted with reverse transcriptase at 37°C for 1 hour, followed by reverse transcriptase inactivation at 70°C for 15 minutes. RT-PCR was performed using SYBR Green Master Mix, with a total reaction volume of 20 μL containing 10 μL SYBR Green Master Mix, 2 μL cDNA template, and 0.3 μM primers. Statistical analyses were performed, and data were expressed as mean ± standard error (SEM) of at least three independent experiments. One-way ANOVA was used to assess differences between groups, followed by Tukey's multiple comparison test to determine significant differences between specific groups. All statistical analyses were performed using GraphPad Prism 10.1.1 software. A p-value less than 0.05 was considered statistically significant.

[0036] Effects of Exos-HIF1-KT on a renal tubular epithelial fibrosis model. C57BL / 6J mice were fed a high-purine diet for 8 weeks. Starting from week 9, they were injected twice weekly with PBS, Exos, Exos-FIH1, and Exos-HIF1-KT (1×10^10 particles / mouse) for 4 consecutive weeks. Figure 6 A). The results showed that the body weight was similar in all groups during the treatment period ( Figure 8 However, the levels of uric acid, serum creatinine, and blood urea nitrogen in the treatment group were significantly lower than those in the non-treatment group. Figure 6 C Figure 6 D, and Figure 6 E). Mouse survival rate graph ( Figure 6 (B) The results showed that one mouse died in the PBS group at both the third and fourth weeks of treatment, and one mouse died in the Exos group at the fourth week of treatment. No mouse deaths occurred in the other two groups during the treatment period. Furthermore, HE, PAS, Masson, and SiriusRed staining of the renal tubular epithelium showed that, compared with the Exos-FIH1, Exos, and PBS groups, Exos-HIF1-KT treatment significantly reduced the renal tubular epithelial injury score and the number of fibrosis-positive areas. Figure 6 F, Figure 6 G, Figure 6 H, Figure 6 I and Figure 6 J).

[0037] The therapeutic effect of Exos-HIF1-KT on renal tubular epithelial fibrosis was investigated by detecting the expression levels of FIH1 and renal injury markers in renal tubular epithelial cells using FIH1, TIM-1, FN1, and α-SMA staining. Consistent with previous results, staining showed that the Exos-HIF1-KT group had the highest FIH1 expression level. Figure 6 K, Figure 6 L) and the levels of early renal injury markers TIM-1 and renal fibrosis markers FN1 and α-SMA were all the lowest ( Figure 6 M, Figure 6 N、 Figure 6 O、 Figure 6 P). Further observation of mitochondrial damage in renal tubular epithelial cells using transmission electron microscopy revealed that the Exos-HIF1-KT group had the largest mitochondrial aspect ratio and the smallest roundness and sphericity. Figure 6 Q). Furthermore, the expression levels of FN1, α-SMA, and TGF-β were quantitatively detected by PCR and Western blotting. Compared to the Exos-FIH1, Exos, and PBS groups, the Exos-HIF1-KT group showed decreased levels of FN1, α-SMA, and TGF-β. Figure 6 R, Figure 6 These results demonstrate that Exos-HIF1-KT successfully delivered FIH1 to renal tubular epithelial cells via tail vein injection and effectively alleviated renal tubular epithelial cell fibrosis induced by hyperuricemia in C57BL / 6J mice. To test whether systemic administration of Exos affects in vivo biosafety, mouse viscera (heart, liver, spleen, and lungs) were examined using HE staining. No significant differences were found in visceral morphology and structural characteristics among all groups. Figure 10 Furthermore, serum RBC, HGB, PLT, ALT, and AST levels were similar across all groups, while serum WBC levels were slightly lower in the treatment group than in the PBS and Exos groups. Figure 9 ). Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Engineered exosomes derived from iPSC-MSC, characterized by: The engineered exosomes highly express FIH-1 protein on their surface, the engineered exosomes are modified with targeting peptides, and the engineered exosomes are marked with CD9, CD63 and TSG101.

2. The engineered exosomes derived from iPSC-MSCs according to claim 1, characterized in that: The amino acid sequence of the FIH-1 protein is shown in SEQ ID NO:1, and the target peptide sequence is shown in SEQ ID NO:

2.

3. The method for preparing engineered exosomes derived from iPSC-MSCs as described in claim 1 or 2, characterized in that, Its preparation method is as follows: S1: Cell Culture and Induction: iPSCs were cultured in mTESR1 medium and induced in differentiation medium for 14 days to obtain iPSC-MSCs. S2: Functional protein loading: The iPSC-MSCs obtained in step S1 were infected with lentiviral particles carrying the FIH-1 gene to overexpress the FIH-1 protein. S3: Exosome extraction: The iPSC-MSC cell culture supernatant from step S2 was collected using exosome-free medium, and exosomes were extracted by differential centrifugation. S4: Targeted Modification: The exosomes extracted in step S3 were coupled with cholesterol-modified targeting peptides to obtain targeted engineered exosomes Exos-FIH1-KT.

4. The preparation method according to claim 3, characterized in that: The differential centrifugation procedure is as follows: Centrifuge the cell culture supernatant at 10,000g for 20 minutes to remove cell debris and large particles; Collect the supernatant and centrifuge at 100,000g for 60 minutes to collect the exosome precipitate.

5. The preparation method according to claim 3, characterized in that: The method for preparing the cholesterol-modified targeting peptide is as follows: cholesterol, PEG8, and the targeting peptide are coupled in PBS solution.

6. The use of engineered exosomes derived from iPSC-MSC as described in claim 1 or 2 in the preparation of a medicament for treating hyperuricemic nephropathy.

7. The use of engineered exosomes derived from iPSC-MSC as described in claim 1 or 2 in the preparation of a medicament for the treatment of hyperuricemic renal fibrosis.

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