Application of dendritic cell-derived nano-vesicles in treatment of type 1 diabetes mellitus

Abstract

The invention relates to application of dendritic cell-derived nano-vesicles in treatment of type 1 diabetes mellitus, and belongs to the technical field of biological medicines. Artificial tolerance dendritic cells are obtained through in-vitro induced differentiation and incubated together with insulin, then low-temperature and high-pressure homogenization is conducted, the tolerance dendritic cell-derived nano-vesicles are obtained, the particle size is 90-140 nanometers, and the tolerance dendritic cell-derived nano-vesicles can be used for preparing the medicine for treating type 1 diabetes mellitus. In-vitro immunosuppression experiments prove that the tolerant dendritic cell-derived nano-vesicles can effectively inhibit T cell immune response and promote immune tolerance. Mouse in-vivo experiments prove that the tolerant dendritic cell-derived nano vesicles can target pancreas to drain lymph nodes, relieve the occurrence of type 1 diabetes mellitus in mice and stabilize the blood glucose level, have good application prospects, and provide a brand new direction for treatment of type 1 diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of dendritic cell-derived nanovesicles in the treatment of type 1 diabetes. Background Art

[0002] Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic islet β cells. According to statistics from the International Diabetes Federation (IDF), the number of global T1D patients has exceeded 9 million, and the incidence rate is increasing at a rate of 3% per year, becoming a major challenge in the field of public health in the 21st century. The core of the T1D pathogenesis is immune response disorder, mainly due to autoreactive T cells attacking pancreatic islet β cells, inducing pancreatitis, and ultimately leading to β cell destruction, resulting in severe insulin deficiency, elevated blood glucose levels, and various complications. Traditional treatment strategies are centered around insulin replacement therapy, combined with lifestyle interventions and blood glucose monitoring. However, these methods often face challenges such as insufficient blood glucose control, hypoglycemia risk, and poor compliance, and also require lifelong dependence on insulin therapy, greatly increasing the medical cost.

[0003] Immunotherapy is a treatment method aimed at restoring the function of pancreatic islet β cells fundamentally by preventing the immune system from abnormally attacking them. In recent years, the role of tolerogenic dendritic cells in the potential treatment of T1D has received extensive attention. A large number of studies have shown that tolerogenic dendritic cells can present self-antigens in a non-inflammatory manner, regulate the immune system to promote central and peripheral tolerance without damaging the broader immune system, and prevent autoimmune attacks on pancreatic islet β cells.

[0004] Although immunotherapy based on tolerogenic dendritic cells has proven its efficacy and safety in delaying or suppressing T1D in several clinical trials, there are still limitations. First, the isolation, large-scale expansion, quality identification, and personalized requirements of tolerogenic dendritic cells are very complex and difficult to ensure. Second, existing cryopreservation techniques may damage the cell viability and the stability of the tolerogenic phenotype, thus having an adverse effect. Third, these dendritic cells need to extract autologous peripheral blood CD14 + cells from patients for in vitro culture, and the tolerance of patients and medical ethics also need to be taken into consideration. Summary of the Invention

[0005] To solve the above technical problems, the present invention obtains artificial tolerogenic dendritic cells by in vitro induction and differentiation, co-incubates them with insulin, and then performs cryogenic high-pressure homogenization to obtain tolerogenic dendritic cell-derived nanovesicles with a particle size of 90 - 140 nanometers, which can be used for the preparation of drugs for the treatment of type 1 diabetes.

[0006] The first object of the present invention is to provide the use of dendritic cell-derived nanovesicles in the preparation of a therapeutic drug for type 1 diabetes, wherein the dendritic cells are tolerogenic dendritic cells, and the tolerogenic dendritic cells are loaded with insulin antigen.

[0007] Cell-derived extracellular vesicles from dendritic cells are rich in antigens, co-stimulatory molecules, and other immunomodulatory factors, and play an important role in regulating immune tolerance and immune responses. Compared with cells, nanoscale vesicles have significant advantages such as lower immunogenicity, strong tissue penetration ability, and high stability. They can be stored as lyophilized powder at room temperature or in solution at -80°C for a long time, improving patient compliance and reducing the treatment burden.

[0008] Further, every 6×10 5 tolerogenic dendritic cells are loaded with 10 - 15 μg of insulin antigen.

[0009] Preferably, every 6×10 5 tolerogenic dendritic cells are loaded with 10 μg of insulin antigen.

[0010] Further, the tolerogenic dendritic cells are obtained by in vitro induction with dexamethasone.

[0011] Further, the tolerogenic dendritic cells are co-incubated with lipopolysaccharide.

[0012] The addition of lipopolysaccharide promotes the maturation of tolerogenic dendritic cells.

[0013] Further, the method for preparing the dendritic cell-derived nanovesicles comprises the following steps:

[0014] Step S1: Extract bone marrow cells, obtain tolerogenic dendritic cells by in vitro induction, and co-incubate with insulin;

[0015] Step S2: Wash the cells collected in Step S1, resuspend them in a buffer, perform low-temperature high-pressure homogenization, centrifuge and collect the supernatant. The working pressure of the low-temperature high-pressure homogenization is 300 - 500 bar, the working temperature is 3 - 6°C, and the number of working times is 2 - 3 times;

[0016] Step S3: Centrifuge the supernatant in Step S2, collect the precipitate, which is the dendritic cell-derived nanovesicles.

[0017] In an embodiment of the present invention, in Step S2, the rotation speed of the centrifugation is 3000 rpm.

[0018] In an embodiment of the present invention, in Step S3, the rotation speed of the centrifugation is 14800 rpm.

[0019] Further, the prepared dendritic cell-derived nanovesicles are freeze-dried to prepare dendritic cell-derived nanovesicle powder.

[0020] The second object of the present invention is to provide a drug for treating type 1 diabetes, wherein the drug for treating type 1 diabetes contains dendritic cell-derived nanovesicles, the dendritic cells are tolerogenic dendritic cells, and the tolerogenic dendritic cells are loaded with insulin antigen.

[0021] Further, every 6×10 5 of the tolerogenic dendritic cells are loaded with 10 - 15 μg of insulin antigen.

[0022] Preferably, every 6×10 5 of the tolerogenic dendritic cells are loaded with 10 μg of insulin antigen.

[0023] Further, the administration method of the drug for treating type 1 diabetes is intravenous administration.

[0024] The third object of the present invention is to provide the application of dendritic cell-derived nanovesicles in the preparation of drugs for promoting the differentiation of regulatory T cells, characterized in that: the dendritic cells are tolerogenic dendritic cells.

[0025] The fourth object of the present invention is to provide the application of dendritic cell-derived nanovesicles in the preparation of drugs for inhibiting T cell immune response, characterized in that the dendritic cells are tolerogenic dendritic cells.

[0026] The beneficial effects of the present invention:

[0027] The tolerogenic dendritic cell-derived nanovesicles provided by the present invention have good biosafety and preparation repeatability, and can be used for mass production; they can be stably stored at room temperature for 1 month after freeze-drying, and are easy to transport and store; it is confirmed by in vitro immunosuppression experiments that they can effectively inhibit T cell immune response and promote immune tolerance; it is confirmed by in vivo experiments in mice that they can target pancreatic draining lymph nodes, produce immunomodulatory effects, inhibit the autoimmune response against pancreatic β cells, relieve the occurrence of type 1 diabetes in vivo, and stabilize blood glucose levels, and have good application prospects, providing a new direction for the treatment of type 1 diabetes. Description of the Drawings

[0028] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention and in combination with the drawings, wherein:

[0029] Figure 1Transmission electron micrograph of nanovesicles derived from artificial tolerogenic dendritic cells in Example 2 of the present invention, where A is the nanovesicles derived from dendritic cells ACDVtolDC, and B is the freeze-dried nanovesicles derived from dendritic cells ACDVtolDC (FD);

[0030] Figure 2 Particle size distribution diagrams of nanovesicles derived from artificial tolerogenic dendritic cells before and after freeze-drying in Example 2 of the present invention;

[0031] Figure 3 Potential distribution diagrams of nanovesicles derived from artificial tolerogenic dendritic cells before and after freeze-drying in Example 2 of the present invention;

[0032] Figure 4 SDS-PAGE gel electrophoresis diagram of nanovesicles derived from artificial tolerogenic dendritic cells in Example 2 of the present invention;

[0033] Figure 5 Relative abundance diagram of in vitro inhibition of T cells by nanovesicles derived from artificial tolerogenic dendritic cells in Example 3 of the present invention;

[0034] Figure 6 Flow cytometry diagram of in vitro inhibition of T cells by nanovesicles derived from artificial tolerogenic dendritic cells in Example 3 of the present invention;

[0035] Figure 7 Gene expression diagram of in vitro inhibition of T cells by nanovesicles derived from artificial tolerogenic dendritic cells in Example 3 of the present invention, where A is the gene expression volcano plot and B is the expression level results of different genes;

[0036] Figure 8 In vitro imaging results of targeting pancreatic draining lymph nodes by nanovesicles derived from artificial tolerogenic dendritic cells in Example 4 of the present invention, where A is the in vitro imaging diagram and B is the statistical result of the average radiation rate of different treatment groups;

[0037] Figure 9 Confocal microscopy of targeting pancreatic draining lymph nodes by nanovesicles derived from artificial tolerogenic dendritic cells in Example 4 of the present invention, where the scale bar is 50 μm;

[0038] Figure 10 Incidence rate diagram of type 1 diabetes in mice after administration of nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention;

[0039] Figure 11 Blood glucose level diagram of type 1 diabetes in mice after administration of nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention;

[0040] Figure 12Confocal microscopy image of mouse pancreatic islet β cells after administration of nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention (scale bar, 100 μm);

[0041] Figure 13 Intraperitoneal glucose tolerance test graph of mice after administration of nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention, where A is the change curve of blood glucose content and B is the statistical result of the area under the curve;

[0042] Figure 14 Result graph of treating T1D diseased mice with nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention, where A is the incidence of type 1 diabetes in mice and B is the change of blood glucose level in mice;

[0043] Figure 15 Result graph of treating NOD diseased mice with nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention, where A is the incidence of type 1 diabetes in mice and B is the change of blood glucose level in mice;

[0044] Figure 16 Confocal microscopy results of pancreatic T cells in treating type I diabetes with nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention, where A is the confocal microscopy image, scale bar 50 μm, B is the statistical result of the proportion of CD4 + T cells, and C is the statistical result of the proportion of CD8 + T cells;

[0045] Figure 17 Flow cytometry analysis graph of pancreatic T cells in treating type 1 diabetes with nanovesicles derived from artificial tolerogenic dendritic cells in Example 5 of the present invention. Detailed implementation manners

[0046] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0047] C57BL / 6 male mice at 6 - 8 weeks of age and NOD / ShiLtJ female mice at 7 - 8 weeks of age were purchased from Changzhou Cavens Experimental Animal Co., Ltd. and Jiangsu GICC Biopharm Technology Co., Ltd. respectively. All mouse experiments were carried out in accordance with the animal experiment protocol approved by the Experimental Animal Center of Soochow University.

[0048] Example 1: Preparation of nanovesicles derived from artificial tolerogenic dendritic cells

[0049] C57 mice or NOD mice were killed by cervical dislocation, and the femur and tibia were removed. The muscle tissue around the bones was removed, and bone marrow cells were collected under sterile conditions using the established method. 1640 complete medium containing fetal bovine serum and granulocyte-macrophage colony stimulating factor was added and cultured for 7 days. Dexamethasone was added on the third day to induce the differentiation of tolerogenic dendritic cells (tolDCs). Insulin and lipopolysaccharide were added on the sixth day to make the final concentration of insulin 10μg / mL and the final concentration of lipopolysaccharide 100ng / mL, and the cells were collected on the seventh day. The tolDCs were collected by centrifugation at 1500rpm for 3 minutes and then resuspended in homogenization buffer (0.25M sucrose, 1mM EDTA, 20mM Hepes, protease inhibitor cocktail) to a final concentration of 2×10 7 cells / mL. The cell suspension was treated with a high-pressure homogenizer (JN-Mini Pro) at a working pressure of 300-500 bar, a working temperature of 3-6°C, and a working number of 2-3 times. The supernatant was then centrifuged at 3000 rpm to collect the supernatant. The supernatant was centrifuged at 14800 rpm to collect the precipitate, which was the artificial tolerant dendritic cell-derived nanovesicle (ACDVtolDC). The precipitate was resuspended in phosphate buffered saline (PBS), sealed after freeze-drying (FD), and stored at room temperature.

[0050] Example 2: Characterization of artificial tolerogenic dendritic cell-derived nanovesicles

[0051] The morphology of ACDVtolDC and freeze-dried vesicles ACDVtolDC (FD) was characterized by transmission electron microscopy (TEM). Figure 1 As shown in the figure, the particle size of ACDVtolDC before and after freeze-drying is about 90-140nm, all of which are spherical and evenly distributed. Dynamic light scattering (DLS) detection, the results are as follows Figure 2 and Figure 3 As shown in the figure, ACDVtolDC freeze-dried powder can be stably stored at room temperature for at least 1 month, with a potential of -6mV to -10mV, and no significant change. The surface protein was analyzed by SDS-PAGE gel electrophoresis, and the results are shown in the figure. Figure 4 As shown, ACDVtolDC contains the same protein components before and after freeze-drying, and has similarities with the proteins of its source cells, the tolerant dendritic cells, which indicates that it has similar immunosuppressive functions as tolerant dendritic cells.

[0052] Example 3: In vitro immunosuppressive effects of nanovesicles derived from artificially tolerant dendritic cells on T cells

[0053] Mouse inguinal lymph node immune cells were extracted according to the existing method, and T cells were stimulated with mature dendritic cells (mDCs) loaded with insulin antigen. The two were co-incubated at a ratio of 5:1, and the ACDVtolDC freeze-dried powder prepared in the above-mentioned example was added at the same time. After 4 days, the immunosuppressive ability of ACDVtolDC on T cells was evaluated by flow cytometry and RNA-seq, using unstimulated T cells and T cells stimulated with mature dendritic cells alone as controls. As Figure 5 shown, the relative abundances of various T cell subsets except regulatory T cells in the ACDVtolDC group were significantly reduced. As Figure 6 shown, the proportions of CD8 + T cells secreting IFNγ, GzmB and TNFα and the proportions of CD4 + T cells secreting IFNγ and IL17 in the ACDVtolDC group were reduced. At the same time, the level of CD4 + CD49b + type 1 regulatory T cells (Tr1) was significantly increased, and the level of CD4 + Foxp3 + regulatory T cells (Treg) was significantly increased compared with the mDCs control group. These cells play a key role in suppressing T cell responses and maintaining immune tolerance.

[0054] The immunomodulatory effect of ACDVtolDC was further analyzed by RNA-seq. The results were as Figure 7 shown. Compared with the control group, genes related to T cell proliferation and activation were down-regulated after ACDVtolDC treatment, such as the cell proliferation markers Mi67, Mcm2 and the inflammation response-related genes Ifng, Tbx21, Il17a and GzmB. At the same time, genes related to suppressing the inflammatory response and restricting autoreactivity were up-regulated, such as Lilrb4a, Lilrb4b, Nrp1 and Foxp3. These results indicate that ACDVtolDC can effectively inhibit the T cell immune response and promote immune tolerance and immunosuppression.

[0055] Example 4: Analysis of the ability of artificial tolerogenic dendritic cell-derived nanovesicles to target pancreatic draining lymph nodes

[0056] The ACDVtolDC prepared in the above-mentioned example was labeled with cy5.5 and injected into T1D or normal C57BL / 6 mice via the tail vein, using tolDCs labeled with cy5.5 and free cy5.5 dye as the control group. The mice were euthanized 24 h after administration, and the main organs, pancreas and pancreatic draining lymph nodes (pLN) were collected for in vivo fluorescence imaging. As Figure 8As shown, compared with the control group, ACDVtolDC was more likely to enter and target pancreatic draining lymph nodes. In addition, ACDVtolDC exhibited the characteristic of preferentially homing to the pancreatic draining lymph nodes of diabetic mice, and this homing effect was not observed in healthy mice. As Figure 9 shown, confocal imaging further confirmed these results, finding that in all experimental groups, the cy5.5 signal was the weakest in the pancreatic draining lymph nodes of healthy mice, while obvious ACDVtolDC signals were detected in diabetic mice. This homing effect may be due to the binding of integrin αMβ2 (Mac-1) on ACDVtolDC to intercellular adhesion molecule-1 (ICAM-1) in the lymph nodes. Especially under the inflammatory conditions associated with diabetes, the increase in ICAM-1 in the lymph nodes made the homing ability of the vesicles more obvious.

[0057] Example 5: Analysis of the therapeutic effect of artificial tolerogenic dendritic cell-derived nanovesicles

[0058] Use the method provided in Example 1 to prepare artificial mature dendritic cell-derived vesicles ACDVmDC, which is different from Example 1 in that dexamethasone is not added for induced differentiation.

[0059] Establish a STZ-induced type 1 diabetes model in mice according to the existing method, and start treatment intervention on the first day of modeling. Inject PBS, artificial mature dendritic cell-derived vesicles (ACDVmDC), tolerogenic dendritic cells tolDC or ACDVtolDC via the tail vein. The dose of the vesicles (ACDVmDC, ACDVtolDC) is 100 μg / time, and the dose of tolDC is 1×10 6 cells / time. Administer the drug once every 5 days for a total of 8 times. Monitor the blood glucose of the mice, which is the glucose content measured from the tail vein blood of the mice at any time. A random blood glucose > 13.8 mmol / L is determined as diabetes. As Figure 10 and Figure 11 shown, ACDVtolDC can delay the occurrence and development of diabetes in mice. Compared with the control group, the blood glucose level remained stable within 50 days after ACDVtolDC treatment.

[0060] Evaluate the effect of ACDVtolDC on the number and function of pancreatic islet β cells in mice by confocal imaging technology. The results are as Figure 12 shown, after ACDVtolDC treatment, the number of β cells with normal insulin secretion function in the pancreas of mice increased significantly.

[0061] Conduct an intraperitoneal glucose tolerance test (IPGTT) on the mice. The results are as Figure 13As shown, compared with the control group, the blood glucose level in the ACDVtolDC-treated mice recovered faster, and the ability of the body to regulate glucose was significantly enhanced. The analysis of the area under the curve (AUC) of the IPGTT further confirmed that ACDVtolDC could preserve the function of β cells and enhance glucose tolerance. These data indicate that in addition to effectively protecting the quantity and function of β cells, ACDVtolDC can slow down the occurrence and development of T1D.

[0062] To further evaluate the potential of ACDVtolDC to improve or reverse diabetes, we tested the incidence and blood glucose level in STZ-induced confirmed T1D mice and non-obese diabetic (NOD) mice. In the STZ-induced model, administration started 7 days after the first STZ injection. The doses of ACDVtolDC and ACDVmDC were 100 μg per injection, and the dose of tolDC was 1×10 6 cells per injection, designated as day 0, and administered once every 5 days for a total of 8 times. The test results are as Figure 14 shown. In the control group, the blood glucose level of all mice increased significantly starting from day 4 and were diagnosed with diabetes on day 9. After ACDVtolDC treatment, the blood glucose of approximately 1 / 3 of the mice remained normal, and the blood glucose level of the remaining diabetic mice was better controlled compared with the untreated mice. Grouped administration started after the first NOD mouse spontaneously developed the disease. The test results are as Figure 15 shown. During the 84-day blood glucose monitoring period, none of the mice treated with ACDVtolDC developed diabetes and maintained a healthy blood glucose level, while in the untreated mice, approximately half of the mice were diagnosed with diabetes, accompanied by a sharp increase in blood glucose level. These results indicate that ACDVtolDC can improve blood glucose regulation and slow down the progression of type 1 diabetes in diabetic patients, supporting its potential as a treatment strategy for type 1 diabetes management.

[0063] Analysis of immune cell infiltration in the pancreatic microenvironment showed results as Figure 16 and Figure 17 shown. Compared with other control groups, the ACDVtolDC treatment group was able to significantly reduce pancreatic immune cell infiltration and simultaneously significantly reduce the proportion of CD8 + T cells and CD4 + T cells in the pancreas, significantly improving the pancreatic autoimmune microenvironment. ACDVtolDC treatment blocked CD4 + and CD8 +The expression of IFNγ in T cells simultaneously promotes the increase of Treg and Tr1 cells and secretes more IL10, thereby reducing the damage of immune cells to pancreatic β cells and delaying and alleviating the occurrence and development of type 1 diabetes. This further indicates that the artificial tolerogenic dendritic cell-derived nanovesicles can be used as a therapeutic drug for type 1 diabetes.

[0064] In summary, the artificial tolerogenic dendritic cell-derived nanovesicles of the present invention, as an anti-type 1 diabetes drug, target the pancreatic draining lymph nodes, improve the immune infiltration in the pancreas of type 1 diabetes patients, inhibit autoreactive T cells, promote the differentiation of regulatory T cells, reduce the damage of immune cells to β cells, slow down the occurrence and development of type 1 diabetes, and stabilize blood glucose levels.

[0065] Obviously, the above examples are only for clear illustration and not a limitation of the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Application of dendritic cell-derived nanovesicles in the preparation of a drug for the treatment of type 1 diabetes, characterized in that: The dendritic cells are tolerant dendritic cells, and the tolerant dendritic cells are loaded with insulin antigens.

2. The use according to claim 1, characterized in that: Each 6×10 5 Each of the tolerogenic dendritic cells was loaded with 10-15 μg of insulin antigen.

3. The use according to claim 1, characterized in that: The tolerant dendritic cells are induced in vitro by dexamethasone.

4. The use according to claim 1, characterized in that: The tolerogenic dendritic cells are co-incubated with lipopolysaccharide.

5. The use according to claim 1, characterized in that: The method for preparing the dendritic cell-derived nanovesicles comprises the following steps: Step S1, extracting bone marrow cells, inducing in vitro to obtain tolerant dendritic cells, and incubating them with insulin; Step S2, washing the cells collected in step S1, resuspending them in a buffer, performing low-temperature high-pressure homogenization, and collecting the supernatant after centrifugation, wherein the low-temperature high-pressure mean working pressure is 300-500 bar, the working temperature is 3-6° C., and the number of operations is 2-3 times; Step S3, centrifuging the supernatant of step S2, and collecting the precipitate, which is the dendritic cell-derived nanovesicles.

6. A drug for treating type 1 diabetes, characterized in that: The type 1 diabetes treatment drug contains dendritic cell-derived nanovesicles, the dendritic cells are tolerant dendritic cells, and the tolerant dendritic cells are loaded with insulin antigens.

7. The drug for treating type 1 diabetes according to claim 6, characterized in that: Each 6×10 5 Each dendritic cell was loaded with 10-15 μg insulin antigen.

8. The drug for treating type 1 diabetes according to claim 6, characterized in that: The administration method of the drug for treating type 1 diabetes is intravenous administration.

9. Use of dendritic cell-derived nanovesicles in the preparation of a drug for promoting regulatory T cell differentiation, characterized in that: The dendritic cells are tolerogenic dendritic cells.

10. Use of dendritic cell-derived nanovesicles in the preparation of a drug for inhibiting T cell immune response, characterized in that: The dendritic cells are tolerogenic dendritic cells.

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