An interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system and a construction method and application thereof

Abstract

The application discloses an interleukin-1 receptor antagonist protein-extrusion nano vesicle delivery system and a construction method and application thereof. The interleukin-1 receptor antagonist protein-extrusion nano vesicle delivery system is prepared by extruding human umbilical cord mesenchymal stem cells overexpressing a junction protein-interleukin-1 receptor antagonist fusion protein. The delivery system has the characteristics of convenient preparation, high preparation yield and good target receptor binding capacity. By using the delivery system and method, myocardial cell pyroptosis can be down-regulated, heart function can be recovered, and the treatment effect on acute myeloid leukemia related tumor cardiomyopathy can be achieved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to an interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system, its construction method, and its application. Background Technology

[0002] Reports and clinical trials indicate that patients with acute myeloid leukemia (AML) have an increased risk of cardiovascular disease. On the one hand, AML and cardiovascular disease share common systemic pathogenic pathways and mechanisms, such as inflammation, metabolic changes, clonal hematopoiesis, altered angiogenesis, changes in the extracellular matrix, and abnormal cell biological behavior, making AML directly related to cardiac damage. On the other hand, anticancer drugs generally have cardiotoxicity. Anthracyclines commonly used to treat AML, such as daunorubicin and doxorubicin, can cause myocarditis, arrhythmias, acute heart failure, and anthracycline-related left ventricular dysfunction; cytarabine can cause pericarditis, bradycardia, and angina.

[0003] It has been reported that during the progression and treatment of acute myeloid leukemia (AML), leukemia cells continuously secrete interleukin-1 (IL-1) to promote their own survival and proliferation. Simultaneously, chemotherapy drugs also act on leukemia cells, vascular endothelial cells, macrophages, etc., promoting the release of IL-1. IL-1 participates in several important stages of cardiovascular disease development, including cardiomyocyte pyroptosis, leading to cardiomyocyte loss and cardiac tissue inflammation, ultimately progressing to fibrosis, severe arrhythmias, and heart failure. Interleukin-1 receptor antagonist proteins are endogenous proteins belonging to the IL-1 family. After competitively binding to the IL-1R1 receptor, they do not trigger activation of its intracellular TIR domain, thereby inhibiting the IL-1 signaling pathway.

[0004] Currently, nanovesicles have become a new generation of drug delivery platforms, and extrusion is an effective technology for producing large quantities of nanovesicles. Studies have shown that extruded nanovesicles derived from human umbilical cord mesenchymal stem cells can promote cardiac repair and improve cardiac function. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide an interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system.

[0006] Another object of the present invention is to provide a method for constructing the above-mentioned interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system.

[0007] Another object of the present invention is to provide the application of the above-described interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing an interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system includes the following steps:

[0010] (1) The nucleotide sequence encoding the human calcium-linked protein transcript, the linker peptide, and the nucleotide sequence of the human interleukin-1 receptor antagonist are linked in sequence to obtain the insertion fragment of the fusion protein. Then, the fragment is inserted into the vector to obtain a vector carrying the fusion protein sequence.

[0011] (2) The vector carrying the fusion protein sequence was co-transfected with the helper plasmid into HEK293T cells. After culturing, the supernatant was collected, centrifuged, the precipitate was collected, resuspended, and purified to obtain the purified virus solution.

[0012] (3) Human umbilical cord mesenchymal stem cells were cultured in a culture dish, and then infected with viral fluid to obtain cells expressing fusion protein;

[0013] (4) The cells expressing the fusion protein were digested, resuspended, and extruded through a polycarbonate membrane using an extruder to obtain vesicles. After centrifugation, the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system was obtained.

[0014] The nucleotide sequence of the human calcium-linked protein transcript described in step (1) is accessed in the NCBI database with the accession number NM_001746.4.

[0015] The nucleotide sequence of the human interleukin-1 receptor antagonist described in step (1) is registered in the NCBI database with the sequence number BC009745.2.

[0016] The linker peptide mentioned in step (1) is 3×GGGGS or 3×EAAAK.

[0017] The nucleotide sequence of the insert fragment of the fusion protein described in step (1) is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0018] The vector described in step (1) is LV-pEFS-MCS-pCMV-EGFP / T2A / Puro.

[0019] The nucleotide sequence of the vector carrying the fusion protein sequence described in step (1) is shown in SEQ ID NO.3.

[0020] The helper plasmids mentioned in step (2) are pMD2.G and psPAX2.

[0021] The culture described in step (2) is carried out at room temperature for 36 to 60 hours.

[0022] The centrifugation conditions described in step (2) are 1500-2500g for 5-15 minutes.

[0023] The resuspension in step (2) is to resuspend using HBSS buffer.

[0024] The purification described in step (2) is performed using sucrose density gradient centrifugation.

[0025] The cultivation in step (3) is to cultivate until the culture adheres to the wall, with a density of 60-80%.

[0026] The titer of the virus solution in step (3) is 4 to 6 × 10⁻⁶. 9 TU / mL.

[0027] The infection conditions described in step (3) are 37°C and 5% CO2 environment for 36 to 60 hours.

[0028] The digestion described in step (4) is digestion using trypsin.

[0029] The density of cells expressing the fusion protein in step (4) is 8 × 10⁻⁶. 7 per mL.

[0030] The specific steps of extrusion described in step (4) are as follows:

[0031] Nitrogen gas was introduced using a tank-type pneumatic liposome extruder to force cells to pass through a 10 μm track etched polycarbonate membrane. This was repeated 3 times. Subsequently, a 5 μm track was used to etch the polycarbonate membrane, and so on, forcing cells to pass through 10, 5, 1, and 0.4 μm tracks etched polycarbonate membranes in sequence.

[0032] The centrifugation in step (4) involves centrifuging at 1000g, 3000g, and 10000g respectively to remove fragments, followed by ultra-high-speed centrifugation at 120000g.

[0033] An interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system was prepared by the above-described method.

[0034] The application of the above-mentioned interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system in the preparation of IL-1 antagonists.

[0035] The application of the above-mentioned interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system in the preparation of therapeutic drugs for acute myeloid leukemia-related tumor heart disease.

[0036] The aforementioned acute myeloid leukemia-related oncological cardiomyopathy includes cardiotoxicity resulting from the progression of acute myeloid leukemia and / or cardiotoxicity resulting from chemotherapy drugs for acute myeloid leukemia.

[0037] The chemotherapy drugs for acute myeloid leukemia include daunorubicin, cytarabine, and etoposide.

[0038] The present invention has the following advantages and effects compared with the prior art:

[0039] This invention relates to an interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system prepared by extruding human umbilical cord mesenchymal stem cells overexpressing a calcium-linked protein-interleukin-1 receptor antagonist fusion protein. This delivery system is characterized by its ease of preparation, high yield, and good binding affinity to the target receptor. Using the delivery system and method of this invention, cardiomyocyte pyroptosis can be downregulated, cardiac function restored, and therapeutic effects achieved on acute myeloid leukemia-related tumor-related cardiac diseases can be achieved. Attached Figure Description

[0040] Figure 1 To enrich the interleukin-1 receptor antagonist protein on the surface of the extruded nanovesicle delivery system obtained in Example 1 by the nano-gold immunolabeling detection in Example 1;

[0041] Figure 2 The binding of the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system obtained in Example 1 to the IL-1R receptor was detected by the nano-gold immunolabeling assay in Example 1.

[0042] Figure 3 The number of interleukin-1 receptor antagonist protein molecules in the extruded nanovesicle delivery system obtained in Example 1, as detected by ELISA in Example 1, represents the number of such molecules.

[0043] Figure 4 The SPR assay in Example 1 was used to determine the affinity of the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system obtained in Example 1 for IL-1R.

[0044] Figure 5 The content of IL-1β, LDH, and ATP in the conditioned medium of differentiated AC16 cells after treatment with DNR and the interleukin-1 receptor antagonist protein-extrusion nanovesicle delivery system obtained in Example 1 was detected by ELISA in Example 2.

[0045] Figure 6The immunostaining experiment in Example 2 was used to detect the expression of GSDMD in differentiated AC16 cells after treatment with DNR and the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system obtained in Example 1.

[0046] Figure 7 The Western blotting experiment in Example 2 was used to detect the expression and activation of the NF-κB-inflammator-GSDMD pathway in differentiated AC16 cells after treatment with DNR and the interleukin-1 receptor antagonist protein-extrusion nanovesicle delivery system obtained in Example 1.

[0047] Figure 8 In Example 2, echocardiography was used to detect cardiac function in mice with acute myeloid leukemia-related tumors after treatment with DNR and the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system obtained in Example 1.

[0048] Figure 9 In Example 2, the serum proANP and proBNP levels of acute myeloid leukemia-related tumor-related cardiac mice were detected by ELISA after treatment with the interleukin-1 receptor antagonist protein-extrusion nanovesicle delivery system obtained in Example 1 and by DNR.

[0049] Figure 10 The immunostaining experiment in Example 2 was used to detect the activation level of GSDMD in the heart of acute myeloid leukemia-related tumor heart mice after treatment with DNR and the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system obtained in Example 1;

[0050] Figure 11 This is an example of the duration of cardiac electrical signals and the direction of cardiac conduction in isolated heart perfusion assays of acute myeloid leukemia-related tumor-related cardiac mice after treatment with DNR and the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system obtained in Example 1. Detailed Implementation

[0051] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0052] Unless otherwise specified, the experimental conditions in the following implementation plan are generally as per standard experimental conditions or the experimental conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available. All cell experiments, unless otherwise specified, shall be repeated at least three times or have at least three replicates per group; animal experiments shall include at least five animals per group.

[0053] Example 1: Construction of an interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system

[0054] 1.1 Construction of a callinkin-interleukin-1 receptor antagonist fusion protein vector

[0055] The nucleotide sequences encoding the human calcium-linked protein transcript (NCBI RefSeq: NM_001746.4), the linker peptide (3×GGGGS), and the human interleukin-1 receptor antagonist (GenBank: BC009745.2) were sequentially linked to obtain the fusion protein insert fragment, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0056] Subsequently, the insertion fragment of the above fusion protein was inserted into the multiple cloning site (MCS) of the plasmid vector LV-pEFS-MCS-pCMV-EGFP / T2A / Puro using a homologous recombination rapid cloning kit to obtain a plasmid vector expressing the calcium-linked protein-interleukin-1 receptor antagonist fusion protein. The complete sequence of the vector is shown in SEQ ID NO.3.

[0057] 1.2 Lentiviral Packaging

[0058] The plasmid expressing the calcitonin-interleukin-1 receptor antagonist fusion protein, along with the helper plasmids pMD2.G and psPAX2, was co-transfected into HEK293T cells via polyethyleneimine (MCE: HY-K2014). Forty-eight hours after transfection, the culture supernatant was harvested. The cells were centrifuged at 2000g for 10 min, and the supernatant was collected. 50% (w / v) PEG6000 was added to a final concentration of 10% (w / v), and the cells were incubated overnight at 4°C. The cells were then centrifuged at 3000×g for 15 min, the supernatant was discarded, and the precipitate was resuspended in HBSS buffer to obtain the viral stock solution. 2 ml of 20% (w / v) sucrose solution was added to an ultracentrifuge tube (Beckman: #344058), and the collected viral stock solution was added to the top layer. The mixture was balanced with HBSS buffer and ultracentrifuged at 83000g for 2.5 h at 4°C. The supernatant was discarded, and the bottom precipitate was resuspended in HBSS buffer to obtain the purified viral solution.

[0059] 1.3 Lentiviral transfection and expression

[0060] Seed 2×10⁶ cells into a cell culture dish 5Personal umbilical cord mesenchymal stem cells were cultured at 37°C and 5% CO2 using complete umbilical cord mesenchymal stem cell culture medium (Cyagen: HUXUC-90011) to achieve adhesion in culture dishes at a density of approximately 70%. Subsequently, 10 μg / mL polybrene and lentivirus containing a fusion protein sequence encoding a calcium-linked protein-interleukin-1 receptor antagonist were added, with a lentivirus titer of approximately 4.80 × 10⁻⁶. 9 The virus was inoculated at TU / mL at 37°C and 5% CO2 for 48 hours. The viral load was then removed, and the cells were cultured again using complete umbilical cord mesenchymal stem cell culture medium.

[0061] 1.4 Preparation of Nanovesicles

[0062] Human umbilical cord mesenchymal stem cells stably overexpressing the calcium-linked protein-interleukin-1 receptor antagonist fusion protein were expanded to 8 × 10⁸. 7 Cells were digested with trypsin and resuspended in 25 mL of PBS. Using a tank-type pneumatic liposome extruder (GJE-100mL), nitrogen gas was introduced to force cells through a 10 μm track etched into a polycarbonate membrane, repeated three times. Subsequently, a 5 μm track was used to etch the polycarbonate membrane. This process was repeated, forcing cells to sequentially pass through polycarbonate membranes with 10, 5, 1, and 0.4 μm tracks (Cytiva: 10 μm: 10418412; 5 μm: 10417412, 1 μm: 10418712, 0.4 μm: 10417112). The vesicles were then centrifuged sequentially at 1000 g, 3000 g, and 10000 g for 10 min to remove debris, followed by ultracentrifugation at 120000 g for 2 h to obtain extruded nanovesicles enriched with interleukin-1 receptor antagonists, which were then resuspended in PBS buffer.

[0063] Compare with Example 1

[0064] The vesicle delivery system was constructed according to the method of Example 1, except that an empty carrier was added in 1.2, which did not include the calcium-linked protein-interleukin-1 receptor antagonist fusion protein. The resulting extruded nanovesicles did not enrich the interleukin-1 receptor antagonist protein.

[0065] Compare with Example 2

[0066] The vesicle delivery system was constructed according to the method of Example 1, except that in 1.2, the carrier only carries the sequence of interleukin-1 receptor antagonist protein. The resulting extruded nanovesicles do not enrich the interleukin-1 receptor antagonist protein on their membrane surface.

[0067] Compare with Example 3

[0068] The vesicle delivery system was constructed according to the method of Example 1, except that in the construction of the fusion protein in 1.1, the linker peptide (3×GGGGS) was replaced with the linker peptide (3×EAAAK) to construct the insert fragment of the fusion protein. The remaining steps were the same, and its nucleotide sequence is shown in SEQ ID NO.2.

[0069] Example 2 Characterization of the calc-linked protein-interleukin-1 receptor antagonist fusion protein carrier

[0070] 2.1 Detection of enrichment effect on the surface of extruded nanovesicle membrane by nano-gold immunolabeling

[0071] (1) Take 5×10 6 Extruded nanovesicles were fixed with 4% paraformaldehyde and then dropped onto a copper grid for detection, and left to stand for 20 minutes.

[0072] (2) Discard the droplets, rinse the copper mesh with PBS 3 times, and then block it with 5% BSA (prepared with PBS) blocking solution for 1 hour.

[0073] (3) Discard the droplet and drop the 1:20 diluted recombinant Anti-IL-1RA antibody (Abcam, ab303490) onto the detection copper grid and incubate overnight.

[0074] (4) Discard the droplets and rinse the copper mesh 6 times with 0.1% BSA (prepared with PBS).

[0075] (5) Drop the 1:20 diluted colloidal gold-labeled Anti-Rabbit antibody (Bioss, bs-0295G-Gold) onto the detection copper grid and incubate for 1 hour.

[0076] (6) Discard the droplet, rinse the copper grid with PBS 6 times, and then incubate with 1% glutaraldehyde for 5 minutes.

[0077] (7) Discard the droplet, rinse the copper mesh with deionized water 8 times, then stain it with uranium acetate for 90 seconds, and then use TEM for detection.

[0078] The results are as follows Figure 1 As shown, the extruded nanovesicles in Example 1 and Comparative Example 3 were labeled with colloidal gold, showing that their surfaces were enriched with interleukin-1 receptor antagonist protein, while the extruded nanovesicles in Comparative Examples 1 and 2 were not labeled with colloidal gold.

[0079] 2.2 Immunolabeling of gold nanoparticles to detect the binding effect of extruded nanovesicles on interleukin-1 receptor antagonist protein and IL-1R receptor

[0080] (1) Take 5×10 6Each nanovesicle was extruded and 1 μg of His-labeled recombinant human IL-1R1 protein (SinoBiological, 10126-H08H) was added, and incubated at 4°C for 1 hour.

[0081] (2) The subsequent steps are the same as the nano-gold immunolabeling steps in 2.1, except that the first antibody is Anti-His-Tag monoclonal antibody (Proteintech, 66005-1-lg), and the second antibody is colloidal gold-labeled Anti-Mouse antibody (bs-0296G-gold).

[0082] The results are as follows Figure 2 As shown, the extruded nanovesicles in Example 1 were labeled with colloidal gold, demonstrating their binding to the IL-1R receptor, while the extruded nanovesicles in Control Examples 1-3 were not labeled with colloidal gold, proving that they could not effectively bind to the IL-1R receptor.

[0083] 2.3 ELISA was used to determine the average number of interleukin-1 receptor antagonist proteins per vesicle.

[0084] (1) Preparation of pyrolysis solution

[0085] Prepare a solution using deionized water containing 100 mM pH 7.4 Tris-base, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 1 mM PMSF.

[0086] (2) Pyrolysis and detection

[0087] Take 1×10 8 Extruded nanovesicles were added and diluted to 100 μl with lysis buffer. The vesicles were then lysed and disrupted using an ultrasonic disruptor. Subsequently, the interleukin-1 receptor antagonist protein was detected using an ELISA kit (Jianglai Biotechnology, JL19264).

[0088] Test results as follows Figure 3 As shown, the interleukin-1 receptor antagonist protein-extruded nanovesicles prepared in Example 1 contain an average of 95 interleukin-1 receptor antagonist protein molecules per vesicle, compared to 18 molecules in Control Example 1 and 85 molecules in Control Example 3.

[0089] 2.4 SPR Detection of Affinity between Interleukin-1 Receptor Antagonist Protein-Extruded Nanovesicle Delivery System and Target Receptor

[0090] (1) Sensor Chip L1 (Cytiva, 29104993) was activated in a biomolecular interaction instrument using 20 mM 3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, followed by injection of vesicle samples at 5 μL / min for 20 minutes, for a total of 1×10 9 An extruded nanovesicle is used to couple it to a chip.

[0091] (2) The running buffer for the compound is 1×PBS-P+ containing 5% (v / v) DMSO. Therefore, solvent correction is performed beforehand using 4.5% and 5.8% DMSO.

[0092] (3) Prepare 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 μM IL-1R1 protein (SinoBiological: 10126-H08H) interaction buffers using PBS buffer supplemented with 0.1 mM benzyl sulfonyl fluoride (Solarbio: P8340). Pass the buffers through the chip from low to high concentrations to couple them with the vesicles. The flow rate is 30 μL / min and the duration is 150 seconds. After each concentration point, regenerate the chip with 10 mM HEPES solution for 2 minutes. Repeat this process until all corresponding concentrations of protein interaction buffers have been processed.

[0093] (4) Use Biacore Insight evaluation software to globally fit the data to a 1:1 Langmuir binding model to obtain the binding and dissociation constants.

[0094] The results are as follows Figure 4 As shown, the dissociation constant between the extruded nanovesicles and the IL-1R1 receptor in Example 1 is 3.25 × 10⁻⁶. 7 The binding rate constant is 2.42 × 10⁻⁶. 4 The dissociation rate constant is 7.87 × 10⁻⁶. 3 The results showed that the interleukin-1 receptor antagonist protein-extruded nanovesicles had a good binding ability with the IL-1R receptor; while although control example 3 was prepared using a similar fusion protein and could be labeled with colloidal gold, it could not bind well with the IL-1R receptor, and the binding response value was close to 0, proving that the vesicles prepared by this method could not achieve the expected effect.

[0095] Example 3 Cell Experiment

[0096] This experiment aimed to evaluate the efficacy of an interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system in improving cardiac outcomes associated with acute myeloid leukemia-related tumors. The study included in vitro assays to detect pyroptosis in cells simultaneously treated with chemotherapy drugs and the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system.

[0097] 3.1 Cell co-culture experiment

[0098] Daunorubicin (DNR) and interleukin-1 receptor antagonist protein-extruded nanovesicles were co-cultured with differentiated human cardiomyocyte AC-16 cell line. The activation of the NF-κB-inflammatory body-GSDMD pathway, as well as the amounts of lactate dehydrogenase, ATP, and IL-1β released into the conditioned medium after pyroptosis, were detected qualitatively and quantitatively. The specific steps were as follows:

[0099] (1) AC16 cells were differentiated using shSV40 and horse serum culture medium.

[0100] (2) After differentiation, add 1.5 μM DNR and 6 × 10 8 The extruded nanovesicles in Example 1 or Comparative Example 1 were processed for 18 hours.

[0101] (3) Collect the conditioned medium after culture, filter it with a 0.22 μm needle filter, and then use the Anti-IL-1β ELISA kit, LDH detection kit and ATP detection kit to detect the conditioned medium.

[0102] The results are as follows Figure 5 As shown, after differentiation, AC16 cells treated with DNR and the extruded nanovesicles in Example 1 had significantly lower levels of IL-1β, LDH, and ATP in their conditioned medium compared to the control group and the control group (which only had an equal volume of PBS without nanovesicles added), indicating a lower degree of pyroptosis.

[0103] 3.2 Detection of Pyroptosis Effect

[0104] Cells cultured in step 3.1 were lysed using RIPA to extract proteins. A portion of the protein was fixed with paraformaldehyde, washed three times with PBS, and then incubated with 0.5% Triton X-100 for 20 minutes. Following this, 5% BSA was added for blocking for 1 hour. Incubation with the primary antibody was then performed overnight. Cells were then washed three times with PBST and incubated with the secondary antibody for 2 hours. After washing three times with PBST, cells were stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes and observed under a laser scanning confocal microscope.

[0105] The primary antibodies used in this experiment were GSDMD Rabbit pAb antibody (Abclonal, A18281) and α-Actinin Mouse mAb antibody (CST, #69758), and the secondary antibodies were Goat Anti-Rabbit IgG H&L Alexa Fluor® 594 (Abcam, ab150080) and Goat Anti-Mouse IgG H&L Alexa Fluor® 488 (Abcam, ab150113).

[0106] The results are as follows Figure 6 As shown, after treatment with DNR and extruded nanovesicles in Example 1, differentiated AC16 cells showed lower GSDMD expression than the control group, indicating a lower degree of pyroptosis.

[0107] 3.3 Western Blot Experiment

[0108] The proteins extracted in step 3.2 were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 80V, followed by transfer to a PVDF membrane at 300mA. Blocking was performed for 1 hour using commercial Western blotting buffer (Beyotime, P0252), followed by overnight incubation with the primary antibody. After three washes with TBST, the membrane was incubated with the secondary antibody for 2 hours. Following three washes with TBST, the membrane was developed using ECL chemiluminescence buffer.

[0109] The primary antibodies used were Anti-ACTC1 antibody (Abcam, ab46805), Phospho-NF-kB p65 / RelA-S536 Rabbit mAb (Abclonal, AP1294), NFKB p65 Recombinant Rabbit mAb (Bsm-52305R), GAPDH Monoclonal antibody (Proteintech, 60004-1-Ig), Beta TubulinRecombinant monoclonal antibody (Proteintech, 80713-1-RR), Anti-TMS1 / ASCantibody (Abcam, ab309497), Anti-NLRP3 antibody (Abcam, ab283819), Anti-proCaspase-1 + p10 + p12 antibody (Abcam, ab179515), Caspase-1 p20 Rabbit pAb (Bioss, bs-10743R), GSDMD Rabbit pAb Antibody (Abclonal, A18281), Anti-cleaved N-terminal GSDMD antibody (Abcam, ab215203); the second antibodies used were Goat Anti-Rabbit IgG H&L antibody (Bioss, bs-0295G) and Goat Anti-Mouse IgG H&L antibody (Bioss, bs-0296G).

[0110] The results are as follows Figure 7 As shown, after treatment with DNR and extruded nanovesicles in Example 1, differentiated AC16 cells exhibited lower levels of NF-κB p65 activation, expression levels of various inflammasome components, Caspase-1 activation, and GSDMD expression compared to Control Group 1 and the control group. This indicates that NF-κB pathway activation is inhibited by extruded nanovesicles in Example 1, subsequently preventing the expression and activation of downstream inflammasomes, and ultimately inhibiting pyroptosis.

[0111] Example 4 Animal Experiment

[0112] This experiment used NSG mice (NOD scid gamma mice) to establish an animal model of acute myeloid leukemia-related tumor-related heart disease. An interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system was administered intravenously. The therapeutic effect of the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system on acute myeloid leukemia-related tumor-related heart disease was evaluated using echocardiography, ELISA, immunohistochemical staining, and isolated heart perfusion.

[0113] 4.1 Establishment of an animal model of acute myeloid leukemia-related tumor-cardiac disease

[0114] NSG mice were intraperitoneally injected with busulfan at a dose of 20 mg / kg body weight. One day later (Day 0), they were injected with 2 × 10 mg / kg body weight via the tail vein. 6 HL-60 cells. Subsequently, on days 7-8, DNR was administered intraperitoneally at 4 mg / kg body weight, simultaneously via tail vein injection of 1×10⁻⁶ cells. 9 The mice were extruded nanovesicles in Example 1 or Control Example 1, while the control group was injected with an equal volume of physiological saline. Subsequently, on Day 10, the mice were anesthetized by inhalation of 2% isoflurane, followed by enucleation to collect blood, and then sacrificed and their hearts were sectioned.

[0115] 4.2 Echocardiography

[0116] Echocardiography was performed on days 0, 7, and 10 of the animal model establishment process described above. Mice were anesthetized with 2% isoflurane at a flow rate of 0.8 L / min, maintaining a heart rate of approximately 350-400 bpm. A bubble-free ultrasound coupling agent was applied to the mouse chest, and short-axis images of the heart near the sternum were acquired using a small animal ultrasound system (FUJIFILM, Vevo® 3100 LT). The images were then analyzed using Vevo LAB analysis software.

[0117] The results are as follows Figure 8 As shown, mice with acute myeloid leukemia-associated tumor-related heart disease exhibited centripetal remodeling characteristics, manifested as decreased left ventricular diameter at end-diastole (LVID;d), left ventricular volume at end-diastole (LVEDV), and cardiac output (CO), while increasing left ventricular posterior wall thickness at end-diastole (LVPW;d). After treatment with extruded nanovesicles in Example 1, compared with Control 1 and the control group, the centripetal remodeling characteristics were weakened, suggesting improved cardiac function.

[0118] 4.3 ELISA detection of serum proANP and proBNP levels

[0119] After blood collection from mice, the blood was allowed to stand at room temperature for 30 minutes, followed by centrifugation (1000 rpm, 15 minutes) to obtain serum. The serum was diluted 5-fold with PBS containing 1% BSA. ProANP and proBNP were then detected using an ELISA kit to assess the degree of heart failure.

[0120] The results are as follows Figure 9 As shown, in mice with acute myeloid leukemia-associated tumor-related heart disease treated with extruded nanovesicles in Example 1, the serum levels of proANP and proBNP were significantly lower than those in Control Example 1 and the control group, indicating a lower degree of heart failure.

[0121] 4.4 Western Blotting to assess GSDMD expression levels

[0122] Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 80V, followed by transfer to a PVDF membrane at 300mA. Blocking was performed for 1 hour using commercial Western blotting buffer (Beyotime, P0252), followed by overnight incubation with the primary antibody. After three washes with TBST, the secondary antibody was added and incubated for 2 hours. This was followed by three washes with TBST and development using ECL chemiluminescence buffer. The primary antibodies used were GAPDH monoclonal antibody (Proteintech, 60004-1-Ig), GSDMD polyclonal antibody (Proteintech, 20770-1-AP), and Cleaved N-terminal GSDMD (MCE, YA5850).

[0123] The results are as follows Figure 10 As shown, mice with acute myeloid leukemia-associated tumors and heart disease treated with extruded nanovesicles in Example 1 showed lower levels of GSDMD and GSDMD-NT expression compared to Control Group 1 and the control group.

[0124] 4.5 Detection of Cardiac Conductivity Using Ex vivo Heart Perfusion and Langendorff Ex vivo Heart Perfusion System

[0125] Mice were intraperitoneally injected with heparin at a dose of 5000 U / kg body weight. The hearts were then harvested and fixed to perfusion needles, which were subsequently connected to the Langendorff ex vivo heart perfusion system. Electrodes were used to detect cardiac electrocardiograms and the direction of current conduction in the ventricles.

[0126] The results are as follows Figure 11As shown, in mice with acute myeloid leukemia-associated tumor-related heart disease treated with extruded nanovesicles in Example 1, the direction of cardiac conduction and the duration of electrical signals were more normal compared with those in Control Example 1 and the control group.

[0127] 4.6 Results and Discussion

[0128] The above experimental results demonstrate that this invention, based on the characteristic of interleukin-1 receptor antagonist protein inhibiting the IL-1 signaling pathway, can block leukemia cells from promoting their own survival and proliferation through the self-secretion of IL-1 when acting on leukemia cells. This, in turn, synergizes with chemotherapy drugs to enhance the killing ability against leukemia cells. Furthermore, when acting on cardiomyocytes, it inhibits the IL-1 signaling pathway and its downstream NF-κB signaling pathway, thereby downregulating the activation of the NLRP3 inflammasome, inhibiting cardiomyocyte pyroptosis, cardiomyocyte loss, and cardiac tissue inflammation. Simultaneously, the extruded nanovesicles derived from human umbilical cord mesenchymal stem cells contain RNA and protein expression profiles similar to those of human umbilical cord mesenchymal stem cells, and can downregulate peroxide levels in cardiomyocytes. After internalization by cardiomyocytes, the extruded nanovesicles derived from human umbilical cord mesenchymal stem cells help promote cardiac repair and improve cardiac function. Therefore, the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system has the potential for therapeutic application in acute myeloid leukemia-related tumor-related cardiac diseases.

[0129] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system, characterized in that... Includes the following steps: (1) The nucleotide sequence encoding the human calcium-linked protein transcript, the linker peptide, and the nucleotide sequence of the human interleukin-1 receptor antagonist are linked in sequence to obtain the insertion fragment of the fusion protein. Then, the fragment is inserted into the vector to obtain a vector carrying the fusion protein sequence. (2) The vector carrying the fusion protein sequence was co-transfected with the helper plasmid into HEK293T cells. After culturing, the supernatant was collected, centrifuged, the precipitate was collected, resuspended, and purified to obtain the purified virus solution. (3) Human umbilical cord mesenchymal stem cells were cultured in a culture dish, and then infected with viral fluid to obtain cells expressing fusion protein; (4) The cells expressing the fusion protein were digested, resuspended, and extruded through a polycarbonate membrane using an extruder to obtain vesicles. After centrifugation, the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system was obtained.

2. The method for preparing the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system according to claim 1, characterized in that: The nucleotide sequence of the human calcium-linked protein transcript described in step (1) has the NCBI database accession number NM_001746.4; The nucleotide sequence of the human interleukin-1 receptor antagonist described in step (1) has the NCBI database accession number BC009745.2; The linker peptide mentioned in step (1) is 3×GGGGS.

3. The method for preparing the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system according to claim 1, characterized in that: The nucleotide sequence of the insert fragment of the fusion protein described in step (1) is shown in SEQ ID NO.1; The vector mentioned in step (1) is LV-pEFS-MCS-pCMV-EGFP / T2A / Puro; The nucleotide sequence of the vector carrying the fusion protein sequence described in step (1) is shown in SEQ ID NO.

3.

4. The method for preparing the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system according to claim 1, characterized in that: The helper plasmids mentioned in step (2) are pMD2.G and PSPAX2; The culture described in step (2) is carried out at room temperature for 36–60 h; The centrifugation conditions described in step (2) are 1500–2500 g for 5–15 min; The resuspension mentioned in step (2) is resuspension using HBSS buffer; The purification described in step (2) is performed using sucrose density gradient centrifugation.

5. The method for preparing the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system according to claim 1, characterized in that: The cultivation described in step (3) involves culturing until the plants adhere to the walls, with a density of 60-80%. The titer of the virus solution in step (3) is 4 to 6 × 10⁻⁶. 9 TU / mL; The infection conditions described in step (3) are 37°C and 5% CO2 environment for 36 to 60 hours.

6. The method for preparing the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system according to claim 1, characterized in that: The digestion described in step (4) is performed using trypsin digestion; The density of cells expressing the fusion protein in step (4) is 8 × 10⁻⁶. 7 cells / mL; The centrifugation in step (4) involves centrifuging at 1000g, 3000g, and 10000g for 10 minutes to remove fragments, followed by ultra-high speed centrifugation at 120000g for 2 hours.

7. The method for preparing the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system according to claim 1, characterized in that: The specific steps of extrusion described in step (4) are as follows: Nitrogen gas was introduced using a tank-type pneumatic liposome extruder to force cells to pass through a 10 μm track etched polycarbonate membrane. This was repeated 3 times. Subsequently, a 5 μm track was used to etch the polycarbonate membrane, and so on, forcing cells to pass through 10, 5, 1, and 0.4 μm tracks etched polycarbonate membranes in sequence.

8. An interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system, prepared by any one of the preparation methods described in claims 1 to 7.

9. The use of the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system according to claim 8 in the preparation of IL-1 antagonists.

10. The use of the interleukin-1 receptor antagonist protein-extruded nanovesicle delivery system of claim 8 in the preparation of a therapeutic drug for acute myeloid leukemia-related tumor-related heart disease.

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