IL11 siRNA (at) EVs multi-stage stent, preparation method and application in preparation of kidney defect repair product

Abstract

The invention discloses a preparation method of an IL11 siRNA (at) EVs multi-stage stent and application of the IL11 siRNA (at) EVs multi-stage stent in preparation of kidney defect repair products, and belongs to the technical field of medicine products. By combining ultracentrifugation with an electroporation technology, IL11 siRNA is efficiently loaded in the stem cell-derived extracellular vesicles, and the encapsulation efficiency is high. GelMA / HAMA is used as biological ink, Alg fibers are added, elution is performed after 3D printing, and the macroscopic and microchannel communicated multistage channel network stent is constructed. The stent simulates a kidney structure, the mechanical property is matched with that of a natural kidney, and compared with a traditional hydrogel stent, cell infiltration and neovascularization are effectively guided. The channel structure and mechanical properties of the multi-stage stent prepared by simple physical mixing and DLP 3D printing are not affected, and IL11 siRNA (at) EVs are uniformly distributed in the stent and can be continuously released in the damaged kidney for more than 14 days. Compared with systemic administration, the stent has the advantages that renal injury and fibrosis after partial renal resection can be remarkably relieved, endogenous repair is accelerated, and an ideal scheme is provided for repair after partial renal resection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical products, in particular to an IL11 siRNA@EVs multi-level scaffold, a preparation method and application in preparation of a kidney defect repair product. BACKGROUND

[0002] Partial nephrectomy (PN) is widely recognized as the gold standard for the treatment of renal tumors, traffic accidents and war injuries caused by kidney contusion. However, patients undergoing this surgery inevitably suffer from a decrease in renal parenchymal volume, resulting in the loss of functional nephrons. Clinical studies have shown that most patients retain histological lesions in the renal parenchyma, of which nearly half will progress to chronic kidney disease (CKD). At the edge of the damaged kidney, some renal tubular epithelial cells (TECs) undergo epithelial-to-mesenchymal transition (EMT), accompanied by fibroblast activation and abnormal deposition of extracellular matrix (ECM). These pathological processes collectively drive the progression of renal fibrosis, ultimately leading to CKD or even end-stage renal disease. Notably, current clinical prevention and treatment of postoperative renal injury and fibrosis after PN mainly rely on intraoperative optimization of kidney suturing techniques to reduce wound exposure, and through early postoperative volume management, blood pressure control and other supportive therapies. However, these methods can only indirectly reduce the risk of secondary injury and fibrosis of the renal parenchyma. For patients who have progressed to CKD before surgery, the commonly used sodium-glucose co-transporter 2 inhibitor drugs can delay the progression of early renal fibrosis by regulating renal hemodynamics and improving glomerular hyperfiltration state, but they only act on the level of hemodynamic regulation, have no clear therapeutic target, cannot target and inhibit core fibrosis mechanisms such as EMT, and are prone to cause hypovolemia-related hypotension, hyponatremia and hypokalemia and other side effects during treatment, leading to accelerated progression of CKD. Current kidney replacement therapies for advanced CKD include dialysis and kidney transplantation, which have heavy economic burdens and cannot effectively inhibit the progression of fibrosis. Therefore, identifying key therapeutic targets for PN-related renal fibrosis and developing precise gene silencing strategies and targeted delivery systems are of great significance for delaying fibrosis progression, restoring kidney function and improving the clinical prognosis of PN patients.

[0003] Interleukin 11 (IL11) is a member of the interleukin 6 (IL6) cytokine family. In recent years, it has attracted much attention due to its role in pro-inflammatory diseases through autocrine and paracrine signaling. IL11 is highly expressed in parenchymal and interstitial cells, and has been shown to be involved in the fibrosis of organs such as the heart, lung, and liver. It is a potential target for anti-renal fibrosis therapy. Although preliminary studies have found that IL11 expression increases after kidney injury, its role in renal fibrosis caused by PN is still unclear. In addition, IL11 neutralizing antibodies have shown therapeutic potential, but their poor tissue penetration, high immunogenicity, and the need for repeated systemic administration have limited their clinical application and may cause secondary complications in patients treated with PN. Small interfering RNAs (siRNAs) are an attractive gene therapy option that can specifically target and degrade IL11 mRNA, achieving precise gene silencing with high sequence fidelity. Several siRNA drugs have been approved for marketing or are in late-stage clinical evaluation, showing good prospects for clinical application. However, due to poor in vivo stability, off-target effects, and low kidney enrichment efficiency, efficient delivery of siRNA to injured kidneys remains a major challenge, and therefore there is an urgent need to develop an efficient and biocompatible delivery system to improve the therapeutic effect of IL11 siRNA. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have become an ideal carrier for the delivery of therapeutic molecules. Their lipid bilayer structure provides protection and has very low immunogenicity. Electroporation technology applies a transient current pulse to EVs through an external electric field, increasing the permeability of the EV membrane surface to achieve efficient loading of siRNA. This method is simple to operate and causes minimal damage to EVs, making it highly valuable for clinical translation. MSC-EVs are not only effective carriers for siRNA delivery, but also have intrinsic antioxidant, anti-inflammatory, and anti-fibrotic activities that can effectively delay the progression of kidney disease. However, intravenous injection of MSC-EVs loaded with siRNA is rapidly cleared by the liver and spleen, limiting the enrichment and retention of MSC-EVs and IL11 siRNA in the injured kidney and affecting their therapeutic effect on PN.

[0004] Hydrogel-based bioactive scaffolds have been used for local delivery of therapeutic drugs, small-molecule nucleic acids, and so on to overcome the various limitations of post-PN renal fibrosis therapies. Among them, gelatin methacryloyl (GelMA) and hyaluronic acid methacryloyl (HAMA) composite hydrogels have attracted much attention due to their excellent biocompatibility, adjustable mechanical properties, and inherent bioactivity. The synergistic combination of the two can highly mimic the mechanical properties and composition of the natural extracellular matrix (ECM), making them ideal candidates for kidney support and repair. However, most existing GelMA and HAMA hydrogel systems rely on bulk gelation and simple microporous structures formed by ice crystal leaching, which cannot reproduce the anisotropy and zonal structure of the kidney. The natural kidney has radially distributed nephrons, hierarchical vascular networks, and a unique physiological microenvironment, which are crucial for the filtration and reabsorption functions of the kidney. Without precise spatiotemporal control, traditional scaffolds cannot effectively guide cell infiltration and vascularization, nor can they support the ordered regeneration of nephrons, ultimately limiting their application in post-PN kidney defect repair.

[0005] Digital light processing (DLP) 3D printing provides a customizable porous scaffold preparation strategy that can achieve high-resolution, high-precision printing of complex structures while simultaneously adjusting the mechanical properties of the scaffold, making it an ideal method for preparing porous biological scaffolds and providing ideal carriers and mechanical support for post-PN kidney regeneration. However, there is a contradiction between DLP 3D printing efficiency and structural accuracy. In high-resolution printing mode, the single forming size is limited, and the complex porous structure of the simulated kidney is prone to internal stress-induced scaffold delamination and misplacement, which limits the application of porous biomimetic scaffolds in PN. SUMMARY

[0006] The purpose of the present application is to overcome the technical defects in the prior art and provide a preparation method of IL11 siRNA@EVs multi-level scaffold.

[0007] Another aspect of the present application provides an IL11 siRNA@EVs multi-level scaffold obtained by the above preparation method.

[0008] Another aspect of the present application provides the use of the above IL11 siRNA@EVs multi-level scaffold in the preparation of kidney defect repair products.

[0009] The technical scheme adopted to achieve the purpose of the present application is: A preparation method of an IL11 siRNA@EVs multi-level scaffold, comprising the following steps: Step 1, extracting stem cell-derived extracellular vesicles by ultracentrifugation method; Step 2, preparation of IL11 siRNA-loaded extracellular vesicles IL11 siRNA@EVs: adding IL11 siRNA to the extracellular vesicles extracted in step 1, mixing uniformly, performing electroporation, centrifuging, and obtaining IL11 siRNA-loaded extracellular vesicles IL11 siRNA@EVs; Step 3, preparation of IL11 siRNA@EVs multistage scaffold HCS-IL11 siRNA@EVs: Step 3.1, melting and mixing GelMA lyophilized block and HAMA lyophilized block, adding a photoinitiator, filtering, and obtaining GH bioink; Step 3.2, adding alginate sodium fiber (alginate Alg fiber) to the GH bioink, mixing uniformly to obtain GHA bioink; Step 3.3, directly adding IL11 siRNA@EVs obtained in step 2 to the GHA bioink, mixing uniformly, performing 3D printing, after printing, eluting alginate sodium fiber, and forming IL11 siRNA@EVs multistage scaffold with large channels and microchannels interpenetrating multistage channel network.

[0010] In the above technical solution, in step 1, the process of extracting stem cell-derived extracellular vesicles is as follows: Human umbilical cord-derived mesenchymal stem cells are cultured, when the mesenchymal stem cells MSCs are in the logarithmic growth phase, the culture medium is aspirated, washed multiple times, the complete culture medium without extracellular vesicles is added, and the culture is continued to obtain the condition culture medium rich in extracellular vesicles; the condition culture medium is centrifuged multiple times, filtered, ultracentrifuged again, the tube bottom precipitate is resuspended with PBS, and the extracellular vesicles are obtained.

[0011] In the above technical solution, in step 1, the temperature of the ultracentrifugation is 4℃, the centrifugal force is 100000-150000 g, and the centrifugation time is 2-12 h.

[0012] In the above technical solution, in step 2, IL11 siRNA is added to extracellular vesicles, followed by PBS, and the mixture is thoroughly mixed to obtain a solution. The solution is then transferred to a Bio-Rad electroporation cuvette and electroporated using the Bio-Rad GenePulserXcell electroporation system. The mixture is kept at room temperature to restore the extracellular vesicle membrane structure. After centrifugation, the solution is transferred to an ultracentrifuge tube, and the supernatant is removed to obtain extracellular vesicles loaded with IL11 siRNA (IL11 siRNA@EVs), which are then resuspended in PBS. The electroporation voltage is 400-450 V, the capacitance is 125-250 μF, and the resistance is 1000-+∞ Ω.

[0013] In the above technical solution, in step 2, the mass ratio of IL11 siRNA to extracellular vesicles is (0.5~1):(1~50), preferably, the mass ratio is 1:1.

[0014] In the above technical solution, in step 2, the IL11 siRNA includes IL11 siRNA-1, IL11 siRNA-2, or IL11 siRNA-3, preferably IL11 siRNA-1. The sense strand of the IL11 siRNA-1 sequence is as shown in SEQ ID NO.1, and the antisense strand is as shown in SEQ ID NO.2. The sense strand of the IL11 siRNA-2 sequence is as shown in SEQ ID NO.3, and the antisense strand is as shown in SEQ ID NO.4. The sense strand of the IL11 siRNA-3 sequence is as shown in SEQ ID NO.5, and the antisense strand is as shown in SEQ ID NO.6.

[0015] In the above technical solution, in step 3.1, the mass ratio of the GelMA freeze-dried block to the HAMA freeze-dried block is (3~7):(3~7), preferably, the mass ratio is 7:3, and the photoinitiator is a mixed solution of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (Lithium Acylphosphinate, LAP) and water.

[0016] In the above technical solution, in step 3.1, the printing parameters of the printer are: printing light intensity of 10~20 mV / cm. 2 The layer height is set to 100~200 μm, the exposure time is 15~20 s, the peeling distance is 2~5 mm, the peeling speed is 3~10 mm / s, and the peeling recovery speed is 20~200 mm / s.

[0017] In the above technical solution, in step 3.2, 0.5~5 wt% Alg fiber is added to the GH bio-ink, preferably 1 wt% Alg fiber.

[0018] In the above technical solution, in step 3.3, after printing, the scaffold is peeled off into a solution of disodium ethylenediaminetetraacetic acid (EDTA) and eluted on a shaker to obtain an IL11siRNA@EVs multilevel scaffold.

[0019] Another aspect of the present invention includes the IL11 siRNA@EVs multilevel scaffold obtained by the preparation method.

[0020] Another aspect of the present invention includes the application of the IL11 siRNA@EVs multilevel scaffold in the preparation of renal defect repair products.

[0021] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs ultracentrifugation combined with electroporation to achieve highly efficient loading of IL11 siRNA without altering the morphology, particle size, or marker protein expression of extracellular vesicles derived from mesenchymal stem cells. This results in an extracellular vesicle nanosystem loaded with IL11 siRNA, achieving an encapsulation efficiency of up to 34.21%. This system protects the siRNA through the extracellular vesicle (EV) membrane structure, significantly enhancing its stability in storage and body fluid environments, thus integrating drug therapy and gene therapy. 2. The Alg fibers of this invention are natural polysaccharides extracted from brown algae, possessing both excellent and mild ionic cross-linking properties and biocompatibility, making them an ideal natural substrate for pore formation in bio-scaffold fabrication. Through adjustable wet spinning and pulverization processes, precise control over the pore size, porosity, and connectivity of the Alg fibers can be achieved. The Alg fibers can be removed by immersion in EDTA, a simple and gentle process with good biosafety. The anisotropic multichannel structure formed after elution effectively promotes cell infiltration and angiogenesis, facilitating nutrient transport and exchange, while maintaining the scaffold's good mechanical properties, thus meeting the needs of kidney repair after PN (nephrolithiasis). 3. Compared with the sodium alginate microsphere pore-forming technology in current patents, the sodium alginate fiber pore-forming technology used in this invention achieves superior continuity and uniformity in pore structure. Sodium alginate microspheres, formed through aggregation, often result in discrete gaps with wide pore size distribution and poor connectivity. Furthermore, microsphere aggregation can easily lead to localized channel blockage, severely impacting material transport efficiency. In contrast, sodium alginate fibers, with their tubular channel structure, form a continuous network of pores within the scaffold, significantly improving connectivity compared to microsphere-based pore-forming technology. This fundamentally solves the core limitation of impaired material transport in traditional pore-forming technologies. Moreover, the high specific surface area of ​​sodium alginate fibers is particularly prominent, reaching 2-3 times that of microsphere materials, greatly enhancing renal cell infiltration and vascularization. 4. This invention uses GelMA and HAMA as the base bio-inks and adds 1 wt% Alg fibers to construct a biocompatible and biodegradable GHA bio-ink. The scaffold structure is precisely controlled using DLP 3D printing technology, and pores are created by eluting Alg sacrificial fibers, constructing a multi-level channel network with precisely arranged macroscopic channels and anisotropic microchannels interconnected, forming a kidney-like biomimetic structure with mechanical properties matching those of a natural kidney. The multi-level channel design guides rapid infiltration of surrounding tissue cells and promotes the directional migration of vascular endothelial cells and vascular network formation. Compared with solid W / O channel (conventional) scaffolds, HCS significantly enhances cell infiltration by 478.37 times and angiogenesis by 22.54 times. Furthermore, HCS can achieve rapid hemostasis, making it an ideal carrier for kidney repair after nephrectomy (PN). 5. IL11 siRNA@EVs can be loaded into HCS through gentle and simple physical mixing and DLP 3D printing without affecting the multi-level channel structure and mechanical properties of HCS. IL11 siRNA@EVs are uniformly distributed in HCS and can be slowly released continuously for 14 days, while the released IL11 siRNA@EVs still retain their original biological activity; 6. HCS-IL11 siRNA@EVs, as a sustained-release carrier of IL11 siRNA@EVs, can achieve sustained release of IL11 siRNA@EVs at the kidney defect site for up to 14 days after in situ implantation during the perioperative window of PN. Compared with tail vein injection of IL11 siRNA@EVs, HCS-IL11 siRNA@EVs significantly increased the accumulation of IL11 siRNA@EVs in the kidney by 4.81 times on day 7. HCS-IL11 siRNA@EVs targeted and inhibited the IL11-mediated EMT process, restoring the kidney weight-to-body weight ratio by 1.53 times and collagen fiber deposition by 3.91 times after PN, significantly inhibiting the atrophy and fibrosis of the defective kidney, repairing the renal microvascular system, regulating the immune microenvironment, promoting endogenous repair of the defective kidney after PN, and restoring kidney function to normal levels. 7. This invention efficiently loads IL11 siRNA into extracted extracellular vesicles (MSCs-EVs) via electroporation, then uses DLP 3D printing to create a multi-level scaffold. This combines structural guidance with localized anti-fibrotic gene silencing, and integrates with Alg sacrificial fibers during 3D printing, resulting in a precisely controlled multi-scale channel network of IL11 siRNA@EVs multi-level scaffold. Simultaneously, the DLP 3D printing generates ordered macroscopic channels arranged along the x, y, and z axes, while the Alg fibers added as sacrificial phase are subsequently removed to create randomly distributed secondary microchannels, thus forming a hierarchical structure with structural guidance and enhanced substance transport functions. This invention utilizes the perioperative window after partial nephrectomy to provide structural guidance for renal tissue repair, while simultaneously achieving in-situ, highly efficient, sustained release of the therapeutic drugs MSCs-EVs and IL11 siRNA, integrating drug therapy and gene therapy to effectively promote significant recovery of renal function. Attached Figure Description

[0022] Figure 1 In the diagram, A is a schematic diagram of the mouse PN model; B shows the expression level and statistical analysis of IL11 in the kidneys with defects at different days after PN, detected by immunofluorescence staining, with white arrows representing IL11 located in TECs and renal interstitium; C shows the expression level and statistical analysis of IL11RA in the kidneys with defects at different days after PN, detected by immunohistochemical staining; D shows the correlation between IL11RA and IL11; E shows the expression level and statistical analysis of α-SMA in the kidneys with defects at different days after PN, detected by immunofluorescence staining; F shows the correlation between α-SMA and IL11; G shows the expression level and statistical analysis of Vimentin in the kidneys with defects at different days after PN, detected by immunofluorescence staining; H shows the correlation between Vimentin and IL11; I shows the expression level and statistical analysis of E-Cadherin in the kidneys with defects at different days after PN, detected by immunohistochemical staining; and J shows the correlation between E-Cadherin and IL11.

[0023] Figure 2 In this diagram, A shows the screening of IL11 siRNA by qRT-PCR; B is a schematic diagram of loading IL11 siRNA into MSC-EVs by electroporation; and C is the electroporation system with the highest loading efficiency.

[0024] Figure 3 In the figure, A is the TEM observation of MSC-EVs and IL11 siRNA@EVs; B is the particle size distribution of MSC-EVs and IL11 siRNA@EVs; C is the Zeta potential of MSC-EVs and IL11 siRNA@EVs; and D is the Western blot identification of marker proteins of MSC-EVs and IL11 siRNA@EVs.

[0025] Figure 4 In the figure, A is an agarose gel electrophoresis image of IL11 siRNA@EVs after treatment with RNase for different times; B is an agarose gel electrophoresis image of IL11 siRNA@EVs after treatment in PBS at 4℃ for 8 h and in FBS at 37℃ for 8 h.

[0026] Figure 5 To observe and statistically analyze the efficiency of IL11 siRNA and IL11 siRNA@EVs internalization in CLSM.

[0027] Figure 6 The mRNA levels of IL11 and IL11RA in different treatment groups were detected by qRT-PCR.

[0028] Figure 7 A shows the mRNA levels of α-SMA, Vimentin, and E-Cadherin in different treatment groups detected by qRT-PCR; B shows representative images of the protein expression levels of α-SMA, Vimentin, and E-Cadherin in different treatment groups detected by Western blot; C shows the statistical analysis of α-SMA, Vimentin, and E-Cadherin in different treatment groups detected by Western blot; D shows the expression level of α-SMA in different treatment groups detected by immunofluorescence staining and its statistical analysis; E shows the expression level of Vimentin in different treatment groups detected by immunofluorescence staining and its statistical analysis.

[0029] Figure 8 To detect the survival of TCMK-1 cells on GH bio-ink after different number of days and to perform statistical analysis.

[0030] Figure 9 Among them, A is a representative image of biological scaffolds with different numbers of channels observed under an optical microscope and the channel condition after red dye perfusion; B is the channel condition of different scaffolds observed by SEM; C is a representative image of H&E staining, DAPI staining and CD31 staining after subcutaneous implantation of different scaffolds; D is a statistical analysis of DAPI staining after subcutaneous implantation of different scaffolds; E is a statistical analysis of CD31 staining after subcutaneous implantation of different scaffolds.

[0031] Figure 10 Representative images and statistical analysis of CD206 / F4 / 80 and iNOS / F4 / 80 staining after subcutaneous implantation of different scaffolds.

[0032] Figure 11 The mechanical properties of different supports.

[0033] Figure 12Where A represents the coagulation index of blood alone, commercial calcium carbonate hemostatic powder, commercial gelatin sponge, and HCS multilevel stent; B represents the erythrocyte adsorption rate of commercial calcium carbonate hemostatic powder, commercial gelatin sponge, and HCS multilevel stent; and C represents the platelet adsorption rate of commercial calcium carbonate hemostatic powder, commercial gelatin sponge, and HCS.

[0034] Figure 13 The hemolysis assay was used to test the blood compatibility of different stents.

[0035] Figure 14 Force-displacement curves of IL11 siRNA@EVs multilevel scaffolds in different embodiments.

[0036] Figure 15 Rheological diagrams of different proportions of GelMA and HAMA in hydrogel state and after curing under 405 nm UV light.

[0037] Figure 16 In the figures, A is a SEM image of the HCS multilevel scaffold and the HCS-IL11 siRNA@EVs of the present invention; B is a statistical analysis of the macroscopic channel diameter in the HCS multilevel scaffold and the HCS-IL11 siRNA@EVs of the present invention; and C is a statistical analysis of the microchannel diameter in the HCS multilevel scaffold and the HCS-IL11 siRNA@EVs of the present invention.

[0038] Figure 17 The in vitro release curves of the multi-level scaffolds of IL11 siRNA@EVs in Example 1 and the four sets of scaffolds in Comparative Example 2 were detected by BCA method.

[0039] Figure 18 In the figures, A is a schematic diagram of IL11 siRNA@EVs loaded into an HCS multilevel scaffold; B is a three-dimensional reconstructed image of the distribution of DiI-labeled IL11 siRNA@EVs in the scaffold; C is the in vitro release curve of HCS-IL11 siRNA@EVs detected by the BCA method; D is a representative image and statistical analysis of the in vitro release of HCS-IL11 siRNA@EVs at different time points detected by the imaging system; E is a schematic diagram of the internalization of IL11 siRNA@EVs released by HCS-IL11 siRNA@EVs in TCMK-1 cells detected by the Transwell method; and F is a representative image of the internalization of IL11 siRNA@EVs released by HCS-IL11 siRNA@EVs in TCMK-1 cells detected by the Transwell method.

[0040] Figure 19In the figure, A is a schematic diagram of HCS-IL11 siRNA@EVs treatment of PN mice; B is a representative image of HCS-IL11 siRNA@EVs used for in vivo treatment of PN mice; C is a representative image of the kidney scaffold at different days after treatment; D is the ratio of defective kidney to body weight in different treatment groups at 14 and 56 days after treatment; E is the serum creatinine and blood urea nitrogen levels in different treatment groups at 56 days; F is the statistical analysis of HE staining and Masson staining of defective kidney sections in different treatment groups at 14 and 56 days after treatment; G is a representative image of HE staining and Masson staining of defective kidney sections in different treatment groups at 14 and 56 days after treatment.

[0041] Figure 20 In the figure, A is a representative image of the expression levels of IL11 and IL11RA in the defective kidney on day 56 after treatment; B is a statistical analysis of the expression levels of IL11 and IL11RA in the defective kidney on day 56 after treatment; C is a representative image of the expression levels of Vimentin and E-Cadherin in the defective kidney on day 56 after treatment; and D is a statistical analysis of the expression levels of Vimentin and E-Cadherin in the defective kidney on day 56 after treatment.

[0042] Figure 21 In this study, A represents the clustering and GO enrichment analysis of differentially expressed genes among the PN group, HCS group, and HCS-IL11 siRNA@EVs group after transcriptome sequencing; B represents the GSEA analysis of the HCS-IL11 siRNA@EVs group.

[0043] Figure 22 In this table, A represents the expression level and statistics of α-SMA in each treatment group as detected by immunofluorescence; B represents the expression level and statistics of CD31 in each treatment group as detected by immunofluorescence; C represents the expression level and statistics of Cyclin D1 in each treatment group as detected by immunohistochemistry; D represents the expression level and statistics of SOX9 in each treatment group as detected by immunofluorescence; and E represents the expression level and statistics of CD4 in each treatment group as detected by immunofluorescence.

[0044] Figure 23In this table, A represents in vivo imaging of PN mice at different time points after treatment with HCS-Cy5-IL11 siRNA@EVs and Cy5-IL11 siRNA@EVs; B represents statistical analysis of in vivo PN mice at different time points after treatment with HCS-Cy5-IL11 siRNA@EVs and Cy5-IL11 siRNA@EVs; C represents ex vivo imaging of major tissues and organs of PN mice at different time points after treatment with HCS-Cy5-IL11 siRNA@EVs and Cy5-IL11 siRNA@EVs; D represents statistical analysis of ex vivo imaging of major tissues and organs of PN mice at different time points after treatment with HCS-Cy5-IL11 siRNA@EVs and Cy5-IL11 siRNA@EVs; and E represents CLSM observation of HCS-Cy5-IL11 siRNA@EVs and Cy5-IL11 siRNA@EVs. Representative images of Cy5 signaling in the defective kidney at 24 h and 7 d after treatment in the siRNA@EVs group; F represents the statistical analysis of Cy5 signaling in the defective kidney at 24 h and 7 d after treatment in the HCS-Cy5-IL11 siRNA@EVs group and the Cy5-IL11 siRNA@EVs group.

[0045] In this specific implementation, cell culture medium, antibiotics, trypsin, and other reagents were all purchased from Gibco; cell culture consumables were all purchased from Corning. The serum required for cell culture was fetal bovine serum (FBS) with extracellular vesicles removed, purchased from BI. The processing steps were as follows: 130,000 g of FBS was placed in an ultracentrifuge tube and centrifuged at 4°C for 12 h. The supernatant was collected in a clean bench and filtered through a 0.22 μm syringe filter to obtain the serum for cell culture. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0047] Example 1 A method for preparing an IL11 siRNA@EVs multilevel scaffold includes the following steps: Step 1, extraction of extracellular vesicles derived from stem cells, specifically includes the following steps: Step 1.1: Collect conditioned medium containing human umbilical cord-derived mesenchymal stem cells (hUC-MSCs): At 75cm 2Human umbilical cord-derived mesenchymal stem cells were cultured in a culture medium in a cell culture flask. When the mesenchymal stem cells (MSCs) were in the logarithmic growth phase and the cell confluence reached 60%, the culture medium was aspirated, the cells were washed twice with PBS, and then 10 ml of complete culture medium containing 10% FBS without extracellular vesicles was added to the cell culture flask. After culturing for another 24 hours, the culture medium was collected into a centrifuge tube. This culture medium was a conditioned medium rich in extracellular vesicles.

[0048] Step 1.2, ultracentrifugation to isolate and extract extracellular vesicles: The culture medium obtained in Step 1 was centrifuged at 500g for 10 min at 4℃ to remove dead cells; the supernatant was centrifuged at 2000g for 20 min at 4℃ to remove cell debris; the supernatant was centrifuged at 10000g for 30 min at 4℃ to remove apoptotic bodies; the supernatant was filtered through a 0.22μm needle filter to remove microvesicles with a diameter greater than 220nm; the filtered supernatant was placed in an ultracentrifuge tube and centrifuged at 130000g for 2 h at 4℃, the supernatant was discarded, and an appropriate amount of PBS was added to resuspend the precipitate at the bottom of the tube to obtain extracellular vesicles MSC-EVs.

[0049] Step 2, Preparation of extracellular vesicles loaded with IL11 siRNA: IL11 siRNA@EVs like Figure 2 As shown in Figure B, the preparation of extracellular vesicles IL11 siRNA@EVs loaded with IL11 siRNA: 100 µg of IL11 siRNA was added to 100 µg of extracellular vesicles MSC-EVs obtained in step 2, and the volume was increased to 400 µl with PBS. The mixture was thoroughly mixed, avoiding the formation of air bubbles, to obtain a solution. The solution was transferred to a 4 mm Bio-Rad electroporation cuvette and electroporated using a Bio-Rad GenePulserXcell electroporation system at 400 V, 125 µF, +∞ Ω. The mixture was then placed at room temperature for 1 h to restore the extracellular vesicle membrane structure. The solution was then transferred to an ultracentrifuge tube, filled with PBS, and centrifuged at 4 °C, 130000 g for 2 h. The supernatant was removed to obtain extracellular vesicles IL11 siRNA@EVs loaded with IL11 siRNA. The IL11 siRNA@EVs were then resuspended in PBS. The mass ratio of siRNA to extracellular vesicles (MSCs-EVs) was 1:1.

[0050] like Figure 18 As shown in A, step 3, preparation of IL11 siRNA@EVs multilevel scaffold HCS-IL11 siRNA@EVs: Step 3.1: Mix GelMA lyophilized blocks and HAMA lyophilized blocks at a mass ratio of 7:3, vortex at 37°C until completely melted and mixed, add 0.1% LAP solution (a mixed solution of LAP and water) as a photoinitiator, and filter in a clean bench using a 0.22μm needle filter to obtain GH bio-ink.

[0051] Step 3.2: Add 1 wt% sodium alginate fiber (Alg fiber) to GH bio-ink and mix well to obtain GHA bio-ink.

[0052] Step 3.3: The obtained GHA bio-ink was directly dropped onto extracellular vesicles IL11siRNA@EVs loaded with IL11 siRNA, vortexed to mix, and then added to the printer's feed hopper. The multi-level scaffold model was then imported for DLP 3D printing. After printing, the scaffold was peeled off from the deposition platform into a 0.05% EDTA solution and eluted with Alg fibers on a shaker at 37°C for 12 hours to obtain the IL11siRNA@EVs multi-level scaffold, denoted as HCS-IL11 siRNA@EVs. The DLP 3D printer parameters were set as follows: printing light intensity was set to 15 mV / cm². 2 The layer height was set to 100μm, the exposure time to 17s, the peeling distance to 2mm, the peeling speed to 5mm / s, and the peeling recovery speed to 100mm / s.

[0053] Example 2 This embodiment provides a method for preparing an IL11 siRNA@EVs multilevel scaffold. Compared with Example 1, the mass ratio of IL11 siRNA to extracellular vesicle MSC-EVs in this embodiment is 0.5:1, and the remaining steps are the same.

[0054] Example 3 This embodiment provides a method for preparing an IL11 siRNA@EVs multilevel scaffold. Compared with Example 1, the mass ratio of IL11 siRNA to extracellular vesicle MSC-EVs is 1:10, and the remaining steps are the same.

[0055] Example 4 This embodiment provides a method for preparing an IL11 siRNA@EVs multilevel scaffold. Compared with Example 1, the mass ratio of IL11 siRNA to extracellular vesicles MSC-EVs is 1:50, and the remaining steps are the same.

[0056] Example 5 This embodiment provides a method for preparing an IL11 siRNA@EVs multilevel scaffold. Compared with Example 1, the preparation method provided in this embodiment is the same except that in step 3.2, 0.5 wt% Alg fiber is added to GH bio-ink.

[0057] Example 6 This embodiment provides a method for preparing an IL11 siRNA@EVs multilevel scaffold. Compared with Example 1, the preparation method provided in this embodiment is the same except that in step 3.2, 5 wt% Alg fiber is added to GH bio-ink.

[0058] Example 7 This embodiment provides a method for preparing an IL11 siRNA@EVs multilevel scaffold. Compared with Example 1, the preparation method provided in this embodiment has the same steps except that in step 3.1, the mass ratio of GelMA lyophilized block to HAMA lyophilized block is 5:5.

[0059] Example 8 This embodiment provides a method for preparing an IL11 siRNA@EVs multilevel scaffold. Compared with Example 1, the preparation method provided in this embodiment has the same steps except that in step 3.1, the mass ratio of GelMA lyophilized block to HAMA lyophilized block is 3:7.

[0060] Comparative Example 1 This embodiment provides a method for preparing a multi-level scaffold. Compared with the method in embodiment 1, the method provided in this embodiment has the same steps except that in step 3.1, the mass ratio of GelMA freeze-dried block to HAMA freeze-dried block is 1:0.

[0061] Comparative Example 2 This comparative example provides methods for preparing solid scaffolds, uniaxial scaffolds, biaxial scaffolds, and triaxial scaffolds of IL11 siRNA@EVs. The preparation methods in this comparative example differ from those in Example 1 in the following ways: After obtaining GH bio-ink in step 3.1, in step 3.2, the obtained GH bio-ink is directly dropped onto extracellular vesicles of IL11 siRNA@EVs loaded with IL11 siRNA, vortexed to mix, and then added to the printer's feed hopper. Solid scaffold models, uniaxial scaffold models, biaxial scaffold models, and triaxial scaffolds are then printed. After printing, the scaffolds are peeled from the deposition platform into a 0.05% EDTA solution and eluted with Alg fibers on a shaker at 37°C for 12 hours to prepare solid scaffolds of IL11 siRNA@EVs, denoted as W / O-IL11 siRNA@EVs; uniaxial scaffolds of IL11 siRNA@EVs are denoted as 1D-IL11. IL11 siRNA@EVs); a biaxial scaffold for IL11 siRNA@EVs is denoted as 2D-IL11 siRNA@EVs; a triaxial scaffold for IL11 siRNA@EVs is denoted as 3D-IL11 siRNA@EVs.

[0062] Comparison Example A method for fabricating a DLP 3D-printed HCS multilevel scaffold includes the following steps: Step 1: Mix GelMA lyophilized blocks and HAMA lyophilized blocks at a mass ratio of 7:3, vortex at 37°C until completely melted and mixed, add 0.1% LAP solution (a mixed solution of LAP and water) as a photoinitiator, and filter in a clean bench using a 0.22μm needle filter to obtain HCS bio-ink.

[0063] Step 2: Add 1 wt% Alg fiber to GH bio-ink and mix well to obtain GHA bio-ink.

[0064] Step 3, digitally model the shape of the multi-level support structure, specifically including the following steps: Step 3.1, set the parameters for the digital light processing 3D printer: set the printing light intensity to 15mv / cm. 2 The layer height was set to 100μm, the exposure time to 17s, the peeling distance to 2mm, the peeling speed to 5mm / s, and the peeling recovery speed to 100mm / s.

[0065] Step 3.2: Add the obtained GH bio-ink solution to the printer's feed tank and import it into the multi-stage scaffold model for printing; after printing, the scaffold is obtained, and the scaffold is peeled from the deposition platform into a 0.05% EDTA solution and eluted on a shaker at 37°C for 12 hours to obtain the HCS multi-stage scaffold.

[0066] Application Example 1 like Figure 1 As shown, the partial nephrectomy model in mice was established as follows: Multiple 6-8 week old C57BL / 6 mice were anesthetized with isoflurane, their back hair was removed, and the area was disinfected with iodine. The skin and mucous membranes on the back were carefully cut open to expose the left kidney. The renal artery was clamped using a miniature arterial clamp, and a 2.5×2×1.3 mm section was surgically removed. 3 The kidney tissue was removed and sutured with 9-0 sutures; the clamping time was controlled within 10 minutes, the microartery was removed, and the wound was sutured in layers with 5-0 sutures. The wound was disinfected with iodine, and the surgery was completed, resulting in a partial nephrectomy model in control mice (e.g., Figure 1 (As shown in A in the figure). A mouse partial nephrectomy model was sacrificed at different number of days (0 days, 3 days, 7 days, 14 days, 28 days and 56 days) to obtain 6 groups of defective kidneys.

[0067] Six groups of defective kidneys were removed from mice, embedded in paraffin, and sectioned for the following tests: IL11 levels in six groups of defective kidneys were detected by immunofluorescence and immunohistochemical staining (e.g., ...). Figure 1 (as shown in B) and IL11RA (as shown in B) Figure 1 The expression levels of IL11 (as shown in C) were analyzed, and the correlation between IL11 and IL11RA was analyzed (e.g., ...). Figure 1 (As shown in D in the figure). The degree of fibrosis in the defective kidney was determined by immunofluorescence staining of α-SMA (e.g., ...). Figure 1 As shown in E), the correlation between α-SMA and IL11 was analyzed (e.g. Figure 1 (as shown by F in the diagram).

[0068] like Figure 1 G in Figure 1 H in Figure 1 The I in Figure 1 As shown in J, the expression levels of Vimentin and E-Cadherin, key biomarkers of EMT in six groups of defective kidneys, were detected to clarify the progression of EMT in defective kidneys, and the correlation between IL11 and EMT was analyzed.

[0069] Depend on Figure 1It was found that the expression of IL11 and its receptor IL11RA in defective kidneys was significantly upregulated with the progression of fibrosis. IL11 is initially secreted by the renal tubules and co-localized with AQP1. Subsequently, it is gradually secreted into the interstitium, where it exerts a paracrine effect. Correlation analysis with EMT markers showed that IL11 can mediate post-nephropathy EMT, thereby promoting fibrosis progression.

[0070] Application Example 2 Screening for siRNAs specifically targeting IL11: Based on the IL11 siRNA sequence, three IL11 siRNAs were designed using Prime 5 software and named IL11siRNA-1, IL11siRNA-2, and IL11 siRNA-3, respectively.

[0071] The sense strand of the IL11 siRNA-1 sequence is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2. SEQ ID NO.1: UUCUCCGAACGUGUCACGUTT; SEQ ID NO.2: ACGUGACACGUUCGGAGAATT.

[0072] The sense strand of the IL11 siRNA-2 sequence is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4. SEQ ID NO.3: GAGUAGACUUGAUGUCCUACCTT; SEQ ID NO.4: GGUAGGACAUCAAGUCUACUCTT.

[0073] The sense strand of the IL11 siRNA-3 sequence is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.6: SEQ ID NO.5: GCUGUUAAAGACUCGACUTT; SEQ ID NO.6: AGUCGAGUCUUUAACAACAGCTT.

[0074] Mouse renal tubular epithelial cells TCMK-1 were cultured in medium for 30 h, serving as the Ctrl group. TCMK-1 cells were also cultured in medium for 6 h, then the medium was replaced with medium containing 10 ng / mL transforming growth factor-β (TGF-β), and cultured for another 24 h, serving as the TGF-β group. TCMK-1 cells were also cultured in medium and transfected with IL11 siRNA-1, IL11 siRNA-2, and IL11 siRNA-3 using lipo2000, respectively. After 6 h, the medium was replaced with medium containing 10 ng / mL transforming growth factor-β (TGF-β), and cultured for another 24 h, serving as the IL11 siRNA-1, IL11 siRNA-2, and IL11 siRNA-3 groups, respectively. Mouse renal tubular epithelial cells TCMK-1 were cultured in a medium and transfected with negative siRNA (Scr siRNA) using lipo2000. After 6 h, the medium was replaced with medium containing 10 ng / mL transforming growth factor-β (TGF-β), and cultured for another 24 h. This group was designated as the siRNA group and denoted as Scr siRNA@EVs.

[0075] RNA was collected from cells in each treatment group and reverse transcribed into cDNA. The IL11 mRNA content in each treatment group was detected by qRT-PCR using IL11 siRNA primers.

[0076] Depend on Figure 2 As shown in A, TGF-β treatment significantly increased IL11 mRNA levels. Knockdown with IL11 siRNA significantly reduced IL11 mRNA levels, with IL11 siRNA-1 showing the highest knockout efficiency. Sequence comparison of the three IL11 siRNAs revealed that the binding site of IL11 siRNA-1 is located at the 5' UTR-CDS junction of IL11 mRNA, where the mRNA secondary structure is loose, resulting in the highest binding free energy between IL11 siRNA-1 and IL11 mRNA. All embodiments of this invention and subsequent application examples used IL11 siRNA-1 in their experiments.

[0077] Application Example 3 like Figure 2As shown in C, qRT-PCR was performed using stem-loop primers with different concentrations of IL11 siRNA, and a standard curve was plotted comparing the Ct value with the IL11 siRNA concentration. IL11 siRNA@EVs prepared in Examples 1-4 were subjected to qRT-PCR, and the corresponding IL11 siRNA loading efficiency was calculated based on the standard curve. The loading efficiency was highest, reaching 34.21%, when the mass ratio of IL11 siRNA to MSC-EVs was 1:1.

[0078] Application Example 4 Methods for identifying the obtained extracellular vesicle MSC-EVs and extracellular vesicle IL11 siRNA@EVs loaded with IL11 siRNA: Morphology of extracellular vesicles MSC-EVs obtained in Example 1 was observed using transmission electron microscopy: IL11 siRNA@EVs and MSC-EVs were respectively dropped onto a 200-mesh copper grid and allowed to stand at room temperature for 2 min. Excess liquid was then blotted dry with filter paper. 20 mg / mL uranium acetate solution was added to the copper grid, and the grid was allowed to stand at room temperature for 1 min to perform negative staining. Excess liquid was then blotted dry with filter paper, and the copper grid was allowed to air dry before being observed under a transmission electron microscope and photographed. Figure 3 As shown in A, both IL11 siRNA@EVs and MSC-EVs have the classic bilayer membrane cup vesicle structure, indicating that electroporation does not change the morphology of IL11 siRNA@EVs, which has a diameter of about 120 nm.

[0079] like Figure 3 In the A-cell extracellular vesicles MSC-EVs and IL11 siRNA@EVs loaded with IL11 siRNA, both exhibit the classic bilayer membrane cup vesicle structure, indicating that electroporation does not alter the morphology of IL11 siRNA@EVs.

[0080] 2. Dynamic light scattering detection of extracellular vesicle size and zeta potential: The MSC-EVs and IL11 siRNA@EVs obtained in Example 1 were diluted with double-distilled water and added to the sample cell of a Malvern nanoparticle size potentiostat for detection. As described in B and C of section 3, the particle size distribution and zeta potential of MSC-EVs did not change significantly after loading with IL11 siRNA.

[0081] Western blot was used to detect marker proteins of extracellular vesicles MSC-EVs and extracellular vesicles loaded with IL11 siRNA@EVs (e.g., IL11 siRNA@EVs). Figure 3 (D in the middle).

[0082] RIPA lysis buffer was added to the MSC-EVs and IL11 siRNA@EVs obtained in Example 1 for lysis. Additionally, RIPA lysis buffer was added to the MSCs obtained in Example 1 as a control group for lysis. Subsequently, the protein samples from each group were subjected to polyacrylamide electrophoresis and transduced, followed by incubation with CD9, CD63, Alix, TSG101, and Calnexin antibodies.

[0083] Depend on Figure 3 The D in the figure indicates that electroporation affects the biological characteristics of MSC-EVs, and the levels of extracellular vesicle markers of IL11 siRNA@EVs are not significantly different from those of MSC-EVs.

[0084] Application Example 5 The stability of the IL11 siRNA@EVs obtained in Example 1 was tested: The IL11 siRNA@EVs obtained in Example 1 were mixed with DNA loading buffer to form the IL11 siRNA@EVs group; naked IL11 siRNA without EV loading was mixed with DNA loading buffer to form the Naked siRNA group; and IL11 siRNA@EVs were treated with the membrane-breaking agent Triton X-100 and then mixed with DNA loading buffer to form the IL11 siRNA@EVs+Triton group, resulting in three groups of samples.

[0085] The three groups of samples were co-incubated with RNase A for different times (0 min, 15 min, 30 min, 60 min, and 120 min), respectively. The incubated samples were collected and subjected to agarose gel electrophoresis. After 20 min of electrophoresis, the retention of siRNA in the three groups was observed under ultraviolet light. Figure 4 As shown in A, IL11 siRNA@EVs exhibits strong stability and is resistant to RNase degradation, indicating that extracellular vesicle EVs have a protective effect on IL11 siRNA.

[0086] The three groups of samples were placed in phosphate-buffered saline (PBS) at 4°C for 8 hours or in 50% serum-free saline (FBS) at 37°C for 8 hours, and then subjected to agarose gel electrophoresis. Figure 4 As shown in B, IL11 siRNA@EVs exhibited significant stability under both simulated preservation and body fluid conditions.

[0087] Application Example 6 After labeling IL11 siRNA with a commercially available Cy5 fluorescent probe, Cy5-IL11 siRNA@EVs were prepared according to the method described in Example 1. The Cy5-IL11 siRNA@EVs were then added to TCMK-1 culture medium and incubated for 24 h. IL11 siRNA labeled with a Cy5 fluorescent probe served as a control group. Figure 5 As shown, the efficiency of IL11 siRNA@EVs being internalized in TCMK-1 cells was significantly higher than that of IL11 siRNA by approximately 1.52 times.

[0088] Application Example 7 Detection of IL11 siRNA@EVs obtained in Example 1 inhibiting IL11 expression and EMT: TCMK-1 cells were cultured for 24 h in TGF-β-free medium (control group, Ctrl); TCMK-1 cells were cultured for 24 h in TGF-β-containing medium (TGF-β group); TCMK-1 cells were transfected with IL11 siRNA for 6 h in TGF-β-free medium, then cultured for 24 h in TGF-β-containing medium (IL11 siRNA group); IL11 siRNA@EVs obtained in Example 1 were co-incubated with TCMK-1 cells and cultured in TGF-β-containing medium for 24 h (IL11 siRNA@EVs group). Four cell groups were obtained, and RNA was collected from each group. The mRNA levels of IL11 and IL11RA were detected by qRT-PCR. Figure 6 As shown, IL11 siRNA@EVs can significantly inhibit the expression of IL11 and its receptor IL11RA, increasing the knockdown efficiency of IL11 to 93.21%.

[0089] The mRNA levels of α-SMA, Vimentin, and E-Cadherin in the four groups of cells were detected by qRT-PCR. Figure 7 As shown in A, IL11 siRNA@EVs can significantly downregulate the mRNA levels of α-SMA, Vimentin and E-Cadherin, thereby inhibiting TCMK-1 EMT.

[0090] Proteins were collected from four groups of cells, and Western blot analysis was performed to detect the protein levels of α-SMA, Vimentin, and E-Cadherin. Figure 7 As shown in B and C, IL11 siRNA@EVs significantly downregulated the protein levels of α-SMA, Vimentin, and E-Cadherin.

[0091] Immunofluorescence staining of four groups of cells for α-SMA and Vimentin was performed. Figure 7 As shown in D and E, IL11 siRNA@EVs significantly downregulated the protein levels of α-SMA and Vimentin, and inhibited TCMK-1 EMT.

[0092] Application Example 8 GH bio-ink was added to the culture medium of TCMK-1 cells and cultured for 1, 3, and 5 days. The survival rate of TCMK-1 cells was then assessed by cell viability staining. Figure 8 As shown, TCMK-1 exhibited high cell viability at different culture days, thus GH bioink has excellent cell compatibility.

[0093] The macroscopic and microscopic structures of multilevel channels in solid scaffolds (denoted as W / O channel (conventional)), uniaxial scaffolds (denoted as 1D macro-channel), biaxial scaffolds (2D macro-channel), triaxial scaffolds (3D macro-channel), and control HCS multilevel scaffolds (denoted as 3D macro-microchannel (HCS)) were observed using optical microscopy and SEM. Figure 9 As shown in Figure A, the HCS has an interconnected channel network, which includes large channels with a diameter of 200 μm and micro channels with a diameter of 30 μm.

[0094] Application Example 9 Multiple 6-8 week old C57BL / 6 mice were anesthetized with isoflurane, their back hair was removed, and the area was disinfected with iodine. The skin and mucous membranes on the back were carefully cut open to expose the left kidney. The renal artery was clamped using a miniature arterial clamp, and a 2.5×2×1.3 mm section was surgically removed. 3 Kidney tissue was used to fill the renal defect with solid scaffolds (W / O channel (conventional)), uniaxial scaffolds (1D macro-channel), biaxial scaffolds (2D macro-channel), triaxial scaffolds (3D macro-channel), and the control HCS multilevel scaffold (3D macro-microchannel (HCS)). The sutures were then made with 9-0 sutures. The clamping time was controlled within 10 minutes. The microartery clamps were removed, and the wound was sutured in layers with 5-0 sutures. The wound was disinfected with iodine, and the surgery was completed, resulting in a partial nephrectomy model in mice. These mice were cultured for 14 and 28 days, then sacrificed. The five sets of scaffolds encased in the subcutaneous sac were removed and soaked in 4% paraformaldehyde for 24 hours. Five sets of frozen sections were prepared, and these sections were stained with H&E, DAPI, and CD31.Figure 9 As shown in Figure B, the 3D macro-microchannel (HCS) group of scaffolds exhibits clearly interconnected hierarchical channels, and cell infiltration within these channels is more pronounced compared to other scaffold groups. Figure 9 As shown in Figures C, D, and E, H&E staining revealed that the internal channels were well preserved in different scaffolds, and the number of cells infiltrating the scaffolds increased. DAPI staining further showed that cell infiltration gradually increased with the increase of channel number, reaching the highest level in the HCS multilevel scaffold. The HCS multilevel scaffold significantly promoted rapid cell infiltration within the scaffold and promoted its proliferation and migration. Immunofluorescence of CD31 showed that CD31 expression was significantly upregulated in the HCS multilevel scaffold compared with other scaffolds, indicating that HCS can promote the proliferation of vascular endothelial cells and promote vascularization.

[0095] Application Example 10 Immunofluorescence staining was performed on the five groups of frozen sections from Example 9 to identify macrophage markers. M1 macrophages were stained with iNOS and F4 / 80, and M2 macrophages were stained with CD206 and F4 / 80. Figure 10 It can be seen that HCS significantly promotes the proliferation of M2 anti-inflammatory macrophages without causing the proliferation of M1 macrophages, indicating that the HCS multilevel scaffold forms an immunosuppressive microenvironment, which is suitable for use in the treatment of PN.

[0096] Application Example 11 Mechanical performance tests were conducted on five groups of stents: solid stents (W / O channel (conventional)), uniaxial stents (1D macro-channel), biaxial stents (2D macro-channel), triaxial stents (3D macro-channel), and the control example HCS multi-stage stent (3D macro-microchannel (HCS)). Power on the electronic universal testing machine and preheat for 30 minutes to allow the load and displacement sensors to stabilize and prevent temperature drift. Perform load zero-point calibration and displacement zero-point calibration. Place the five sets of supports on the sample stage, start the machine, and set the loading rate to 1 mm / min and the sampling frequency to 200 Hz. Stop the machine when the support reaches 80% of its maximum load. Collect force and displacement data during the test and plot the force-displacement curve, as shown below. Figure 11 As shown. By Figure 11 It is known that the HCS multi-level stent has mechanical properties that match those of a natural kidney, making it suitable for the treatment of PN.

[0097] Application Example 12 Gelatin sponge and calcium carbonate hemostatic powder (CaCO3) were used as control group stents, and HCS multilevel stents were used as experimental group stents to test the hemostatic ability of HCS multilevel stents: 1. Coagulation index test: Blood from the tail vein of 8-week-old male SD rats was added to a heparin anticoagulation tube to obtain rat anticoagulated whole blood. 50 μL of rat anticoagulated whole blood was dropped onto the experimental group stent or the control group stent, and then 5 μL of 0.2 MCaCl2 solution was immediately dropped onto the surface of the experimental group stent or the control group stent. Every 1 minute, the experimental group stent or the control group stent was placed in 10 ml of deionized water, and the absorbance of the supernatant at 540 nm was measured.

[0098] 5 μL of 0.2M CaCl2 was directly added to 50 μL of anticoagulated whole blood from rats as a control group, denoted as bloodonly. Figure 12 As shown in A, the HCS multilevel stent has a significant hemostatic effect, rapidly coagulating blood within 5 minutes. Its hemostatic ability is not significantly different from that of commercially available gelatin sponge and calcium carbonate hemostatic powder (CaCO3).

[0099] 2. Red blood cell adsorption rate and platelet adsorption rate test: Take 1 mL of anticoagulated whole blood from rats and dilute it to 10 mL with physiological saline. Place either the control group scaffold or the experimental group scaffold into the diluted anticoagulated whole blood and incubate at 37°C with a shaker at 100 rpm for 30 min. After adsorption is complete, remove all scaffolds and place them in erythrocyte lysis buffer, continuing to shake at 100 rpm for 30 min. Measure the absorbance of the supernatant at 540 nm. Separately, place the control group scaffold and the experimental group scaffold into diluted anticoagulated whole blood, incubate at 37°C with a shaker at 100 rpm for 30 min, remove all scaffolds, and incubate at 200 rpm for 30 min. Measure the absorbance of the supernatant at 650 nm. Figure 12 As shown in B and C, the HCS multilevel scaffold can promote the adsorption of red blood cells and platelets, thereby improving coagulation ability.

[0100] 3. Hemolysis test: 1 mL of anticoagulated whole blood from rats was diluted to 10 mL with physiological saline. Solid scaffolds (W / O channel (conventional)), uniaxial scaffolds (1D macro-channel), biaxial scaffolds (2D macro-channel), triaxial scaffolds (3D macro-channel), and the control HCS multilevel scaffold (3D macro-microchannel (HCS)) were respectively placed in the diluted anticoagulated whole blood and incubated in a 37 ℃ constant temperature shaker at 100 rpm for 30 min. Red blood cells were lysed using Triton X-100 as the positive group, and phosphate-buffered saline (PBS) as the negative group. Figure 13 As shown, the HCS multilevel stent does not cause hemolysis and has strong biocompatibility.

[0101] Application Example 13 Following the method of Application Example 11, the mechanical properties of the IL11 siRNA@EVs multilevel scaffolds prepared in Examples 1, 5, and 6 were measured, and force-displacement curves were plotted, as shown below. Figure 14 As shown, the IL11siRNA@EVs multilevel scaffold with 1% Alg fiber added in Example 1 exhibits the best mechanical properties.

[0102] Application Example 14 The rheological properties of hydrogels formed by mixing GelMA lyophilized blocks and HAMA lyophilized blocks at different mass ratios in Examples 1, 7-8, and the hydrogels prepared in the comparative examples were tested using a rotational rheometer. The results are as follows: Figure 15 As shown, the GelMA / HAMA hydrogel of Example 1 exhibits optimal flowability and viscosity, and can be cured into a gel under 405 nm UV light. Comparative Example 1, without the presence of HAMA lyophilized blocks, could not be gelled.

[0103] Application Example 15 The HCS-IL11 siRNA@EVs of this invention, when loaded with IL11 siRNA@EVs, do not affect the multilevel channel structure of the HCS multilevel scaffold: the macroscopic and microscopic structures of the multilevel channels of HCS-IL11 siRNA@EVs were observed using optical microscopy and SEM. For example... Figure 16 As shown, HCS-siL11@EVs still possess macroscopic channels arranged along the x, y, and z axes and uniformly distributed microchannels. For example... Figure 16 As shown in Figure A, SEM results show that the macroscopic and microchannel diameters of HCS-siL11@EVs are not significantly different from those of the HCS multilevel scaffold, indicating that loading IL11 siRNA@EVs with HCS-IL11 siRNA does not affect the multilevel channel structure of the HCS multilevel scaffold.

[0104] Application Example 16 BCA assay was used to detect the in vitro release curves of the multilevel scaffolds of IL11 siRNA@EVs in Example 1 and the four groups of scaffolds in Comparative Example 2: The four scaffolds prepared in Comparative Example 2 and the multi-level scaffold (HCS-IL11siRNA@EVs) of Example 1 containing IL11 siRNA@EVs were placed in sterile PBS and incubated at 37 °C with a shaker to release IL11 siRNA@EVs from the scaffolds. 10 μL of supernatant was collected daily, and the protein content in the supernatant was determined by BCA assay to evaluate the ability of each scaffold to release IL11 siRNA@EVs in vitro. Figure 17 As shown, the multi-level channel network of HCS-IL11 siRNA@EVs promotes material exchange within the scaffold, enabling nearly 90% release of the loaded IL11 siRNA@EVs. Other scaffolds, lacking this channel network structure, cannot achieve effective material exchange; traditional solid scaffolds can only release 60% of the IL11 siRNA@EVs.

[0105] Application Example 17 In vitro release rate of HCS-IL11 siRNA@EVs In Example 1, IL11 siRNA was labeled with a Cy5 fluorescent probe to prepare a multilevel scaffold of IL11 siRNA@EVs, denoted as HCS-Cy5-IL11 siRNA@EVs; HCS-Cy5-IL11 siRNA@EVs were observed under a fluorescence microscope, as shown... Figure 18 As shown in B, IL11 siRNA@EVs are uniformly dispersed throughout the scaffold, indicating that HCS can effectively load IL11 siRNA@EVs.

[0106] HCS-IL11 siRNA@EVs were observed using an IVIS imaging system. Figure 18 As shown in D, the fluorescence intensity within the scaffold gradually decreases, indicating that the release process was well controlled.

[0107] HCS-IL11 siRNA@EVs were placed in sterile PBS and incubated at 37 °C with a shaker to release IL11 siRNA@EVs from the multilevel scaffold. 10 μL of supernatant was collected daily, and the protein content in the supernatant was determined by BCA assay to evaluate the in vitro release rate of HCS-IL11 siRNA@EVs. Figure 18 As shown in C, HCS-IL11 siRNA@EVs can effectively prolong the release of IL11 siRNA@EVs for up to 14 days.

[0108] Furthermore, the bioactivity of IL11 siRNA@EVs released from HCS-IL11 siRNA@EVs was assessed using Transwell assay. Figure 18 As shown in Figure E, HCS-Cy5-IL11 siRNA@EVs were placed in the upper layer of a Transwell, and TCMK-1 cells were cultured in the lower layer. After incubating this system for 24 h, the TCMK-1 cells in the lower layer were observed using a fluorescence microscope. Figure 18 As shown in F, the IL11 siRNA@EVs released by HCS-IL11siRNA@EVs can be effectively internalized by TCMK-1 cells, demonstrating that HCS-IL11siRNA@EVs can maintain the biological activity of IL11 siRNA@EVs.

[0109] Application Example 18 Methods for detecting the therapeutic effect of HCS-IL11 siRNA@EVs on PN mice Six groups of mice were created: a control mouse partial nephrectomy model obtained in Example 1, denoted as PN; siScr@EVs prepared in Example 2 were loaded into an HCS multilevel scaffold (HCS-siScr@EVs) according to the modeling method of Example 9, filling the kidney defect site, denoted as HCS-siScr@EVs; HCS-IL11 siRNA@EVs were filled into the kidney defect site according to the modeling method of Example 9, denoted as HCS-IL11 siRNA@EVs; IL11 siRNA@EVs obtained in Example 1 were injected into the tail vein of the control mouse partial nephrectomy model obtained in Example 1, denoted as IL11siRNA@EVs; mice in the sham surgery group, where only the back skin was cut and then directly sutured without any operation on the kidney, served as the control group, denoted as Sham.

[0110] To evaluate the repair of damaged tissue, tissue samples were collected from the six groups of mice at 14 and 56 days after partial nephrectomy. The weight of the missing kidney was weighed and recorded. Serum creatinine and blood urea nitrogen levels were measured to characterize renal function. Figure 19 As shown in D, E, and F, the HCS-IL11 siRNA@EVs group significantly reduced renal atrophy after PN and had a significant effect on improving renal function.

[0111] Kidney defects from six groups of mice were removed from the body, embedded in paraffin, and sectioned. Hematoxylin and eosin (HE) staining was performed to evaluate the damage and repair of kidney tissue. HCS-IL11 siRNA@EVs significantly inhibited renal tubular necrosis and vacuolation, reduced renal tubular dilation, decreased renal interstitial cell infiltration, and significantly promoted renal tissue repair.

[0112] Kidney defects from six groups of mice were removed from the body, embedded in paraffin, sectioned, and stained with Masson's red and Sirius red to evaluate the degree of renal fibrosis. Figure 19 As shown in BC, HCS-IL11 siRNA@EVs can reduce fibrosis in defective kidneys and decrease the formation of abnormal scar tissue. Immunofluorescence staining of a-SMA further demonstrates that HCS-IL11 siRNA@EVs can effectively inhibit the renal fibrosis process after PN.

[0113] Evaluation of renal IL11 and IL11RA expression: Immunofluorescence staining was used to detect the expression of IL11 and IL11RA in kidney samples taken on day 56. Figure 20 As shown, HCS-IL11 siRNA@EVs can target and inhibit IL11-related molecules, significantly reducing the expression levels of IL11 and IL11RA in defective kidneys.

[0114] To evaluate the expression of EMT-related indicators in the defective kidneys: Vimentin expression was measured in paraffin-embedded kidney sections taken on day 56 of immunofluorescence staining from six groups of mice, and E-Cadherin expression levels were detected by immunohistochemical staining. Figure 20 As shown, HCS-IL11 siRNA@EVs can significantly reduce the expression level of the mesenchymal marker Vimentin and significantly increase the expression of the epithelial cell marker E-Cadherin, thereby reducing the EMT degree of TEC.

[0115] Application Example 19 like Figure 21 As shown, bioinformatics methods were used to detect the therapeutic effects of different scaffolds on PN mice: Three groups of mice were established: a control mouse model of partial nephrectomy obtained in Example 1, denoted as PN; a HCS multilevel scaffold filled into the kidney defect site using the modeling method in Example 9, denoted as HCS; and HCS-IL11 siRNA@EVs filled into the kidney defect site using the modeling method in Example 9, denoted as HCS-IL11 siRNA@EVs. After 56 days of feeding, transcriptomic sequencing was performed on the defective kidney tissues of the three groups of mice. Differentially expressed genes among the PN, HCS, and HCS-IL11 siRNA@EVs groups were screened, and GO enrichment analysis revealed four distinct clusters, each with unique functional characteristics and biological significance. Cluster 1 represents the set of genes common to both HCS and HCS-IL11 siRNA@EVs, which are mainly involved in angiogenesis, cell adhesion, and coagulation processes. Genes in Cluster 2 showed significantly reduced expression in HCS-IL11 siRNA@EVs, involved in fibrosis and EMT processes. Cluster 3 contained genes specifically upregulated in HCS-IL11 siRNA@EVs, involved in the tricarboxylic acid cycle and kidney development. Cluster 4 contained genes with significantly reduced expression in HCS-IL11 siRNA@EVs, related to the regulation of immune responses. Furthermore, gene set enrichment analysis confirmed that HCS-IL11 siRNA@EVs treatment significantly upregulated genes associated with angiogenesis, cell adhesion, and kidney development.

[0116] Application Example 20 Validation of HCS-IL11 siRNA@EVs improving kidney damage in PN mice: After feeding the six groups of mice prepared in Example 17 for 56 days, the defective kidneys of the six groups of mice were removed from the body, embedded in paraffin, and sectioned. The six sections were evaluated as follows: To assess the degree of fibrosis in the defective kidney, immunofluorescence was used to detect the expression of the fibrosis marker α-SMA. For example... Figure 22 As shown, HCS-IL11 siRNA@EVs significantly downregulated the expression level of α-SMA, with no significant difference compared to the Sham group, and inhibited fibrosis in defective kidneys. However, the PN group, HCS group, HCS-Scr siRNA@EVs group, and IL11 siRNA@EVs group maintained high α-SMA expression levels.

[0117] To evaluate the vascularization of the defective kidney, immunofluorescence was used to detect the expression of the vascular endothelial marker CD31. For example... Figure 22As shown, HCS-IL11 siRNA@EVs significantly upregulated CD31 expression levels, with no significant difference compared to the Sham group, promoting vascularization of the defective kidney. However, CD31 expression levels remained low in the PN group, HCS group, HCS-Scr siRNA@EVs group, and IL11 siRNA@EVs group.

[0118] Evaluation of kidney regeneration: Immunofluorescence staining was used to detect the expression of Cyclin D1 and SOX9 in paraffin sections of kidney tissue taken on day 56. Figure 22 The results showed that HCS-IL11 siRNA@EVs upregulated the expression levels of Cyclin D1 and SOX9 in defective kidneys, significantly promoting endogenous regeneration of the kidneys. In contrast, the expression levels of Cyclin D1 and SOX9 in the PN group, HCS group, HCS-Scr siRNA@EVs group, and IL11 siRNA@EVs group were all lower than those in the HCS-IL11 siRNA@EVs group.

[0119] Evaluation of T cell activation in the defective kidney: Immunofluorescence assay of CD4 expression in paraffin sections of the kidney taken on day 56. Figure 22 As shown, HCS-IL11 siRNA@EVs significantly downregulated the number of CD4+ T cells, with no significant difference compared to the Sham group, thereby suppressing the immune response. Meanwhile, the PN group, HCS group, HCS-Scr siRNA@EVs group, and IL11 siRNA@EVs group maintained high CD4 expression levels.

[0120] Application Example 21 Detection of HCS-IL11 siRNA@EVs increasing renal retention in PN mice: In Example 1, IL11 siRNA was labeled with a Cy5 fluorescent probe to prepare an IL11siRNA@EVs multilevel scaffold, denoted as HCS-Cy5-IL11 siRNA@EVs; similarly, extracellular vesicles loaded with Cy5-labeled IL11 siRNA were prepared, denoted as Cy5-IL11 siRNA@EVs.

[0121] Following the modeling method in Application Example 9, HCS-Cy5-IL11 siRNA@EVs were inserted into the kidney defect site, denoted as HCS-Cy5-IL11 siRNA@EVs, serving as the experimental group, denoted as HCS-Cy5-IL11 siRNA@EVs. In mice, after kidney defects were directly sutured, the kidney defect wound was closed, and Cy5-IL11 siRNA@EVs was injected via the tail vein after skin suturing, serving as the control group, denoted as Cy5-IL11 siRNA@EVs. In vivo imaging was performed on mice in the experimental and control groups at different time points (2h, 24h, 72h, 7d, and 14d). Mice were then sacrificed at selected time points (2h, 24h, 72h, 7d, and 14d) for in vitro imaging of major tissues and organs to monitor the dynamic changes of EVs and IL11 siRNA in vivo. Figure 23 As shown, the orthotopically implanted IL11siRNA@EVs multilevel scaffold can continuously release IL11 siRNA@EVs for up to 14 days, significantly increasing the signal of IL11-loaded extracellular vesicles in the kidney by about 4 times compared with tail vein injection.

[0122] After sectioning kidney tissues at selected time points (24h and 7d), the renal tubular marker AQP1 was stained, and the co-localization of IL11 siRNA@EVs signal with AQP1 was observed under a fluorescence microscope. The results showed that the fluorescence signal of HCS-Cy5-IL11 siRNA@EVs was higher than that of Cy5-IL11 siRNA@EVs, and the signal of IL11 siRNA@EVs co-localized with AQP1, indicating that IL11 siRNA@EVs were mainly internalized in the renal tubules.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A preparation method of an IL11 siRNA@EVs multi-level scaffold, characterized in that, Comprising the following steps: Step 1, extracting stem cell-derived extracellular vesicles by ultracentrifugation method; Step 2, preparation of extracellular vesicles loaded with IL11 siRNA IL11 siRNA@EVs: adding IL11 siRNA to the extracellular vesicles extracted in step 1, mixing uniformly, performing electroporation, centrifuging to obtain extracellular vesicles loaded with IL11 siRNA IL11 siRNA@EVs; Step 3, preparation of IL11 siRNA@EVs multistage scaffold HCS-IL11 siRNA@EVs: Step 3.1, melting and mixing GelMA lyophilized block and HAMA lyophilized block, adding a photoinitiator, filtering to obtain GH bio-ink; Step 3.2, adding sodium alginate fibers to the GH bio-ink, mixing uniformly to obtain GHA bio-ink; Step 3.3, adding the extracellular vesicles IL11 siRNA@EVs obtained in step 2 to the GHA bio-ink, mixing uniformly, performing 3D printing, after printing, eluting the sodium alginate fibers to form the IL11 siRNA@EVs multistage scaffold with a large channel and a microchannel interpenetrating multichannel network.

2. The production method according to claim 1, characterized by, In the step 1, the process of extracting stem cell-derived extracellular vesicles is as follows: The human umbilical cord-derived mesenchymal stem cells are cultured, when the mesenchymal stem cells MSCs are in the logarithmic growth phase, the culture medium is aspirated, washed multiple times, the complete culture medium without extracellular vesicles is added, and the culture is continued to obtain the condition medium rich in extracellular vesicles, the condition medium is centrifuged multiple times, filtered, ultracentrifuged again, the PBS resuspension tube bottom precipitate is added, and the extracellular vesicles are obtained.

3. The preparation method according to claim 1, characterized in that, In the step 1, the temperature of the ultracentrifugation is 4-10℃, the centrifugal force is 100000-150000 g, and the centrifugation time is 2-12 h.

4. The method of claim 1, wherein, In the step 2, IL11 siRNA is added to the extracellular vesicles, and then PBS is added to mix thoroughly, to obtain a mixture, the mixture is moved into a Bio-Rad electric shock cup, and a Bio-Rad GenePulser Xcell electroporation system is used for electroporation, and the extracellular vesicles are placed at room temperature to recover the membrane structure; the mixture is moved into an ultracentrifuge tube, centrifuged to remove the supernatant, and the extracellular vesicles loaded with IL11 siRNA IL11 siRNA@EVs are obtained and resuspended with PBS; The voltage of the electroporation is 400-450 V, the capacitance is 125-250 μF, and the resistance is 1000-+∞ Ω; The mass ratio of IL11 siRNA to extracellular vesicles is (0.5~1):(1~50).

5. The preparation method according to claim 1, characterized in that, In the step 2, the IL11 siRNA comprises IL11 siRNA-1, IL11 siRNA-2 or IL11 siRNA-3, wherein the sense strand of the sequence of IL11 siRNA-1 is as shown in SEQ ID NO. 1, and the antisense strand is as shown in SEQ ID NO. 2, the sense strand of the sequence of IL11 siRNA-2 is as shown in SEQ ID NO. 3, and the antisense strand is as shown in SEQ ID NO. 4, the sense strand of the sequence of IL11 siRNA-3 is as shown in SEQ ID NO. 5, and the antisense strand is as shown in SEQ ID NO.

6.

6. The preparation method according to claim 1, characterized in that, In the step 3.1, the mass ratio of the GelMA lyophilized block and the HAMA lyophilized block is (3~7):(3~7), and the photo initiator is a mixture of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and water.

7. The preparation method according to claim 1, characterized in that, In the step 3.2, 0.5~5 wt% of sodium alginate fibers are added to the GH bio-ink.

8. The method of claim 1, wherein, In step 3.3, the printing parameters are: printing light intensity of 10-20 mv / cm 2 , layer height of 100-200 μm, exposure time of 15-20 s, peeling distance of 2-5 mm, peeling speed of 3-10 mm / s, peeling recovery speed of 20-200 mm / s, after printing, peeling into ethylenediamine tetraacetic acid disodium solution, eluting sodium alginate fiber on a shaker to obtain IL11 siRNA@EVs multi-level scaffold.

9. The IL11 siRNA@EVs multi-level scaffold obtained by the preparation method according to any one of claims 1~8.

10. The IL11 siRNA@EVs multi-level scaffold according to claim 9 is used for preparing a kidney defect repair product.

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