Engineered platelets, preparation method thereof and application of engineered platelets in type 1 diabetes mellitus

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to engineered platelets, a preparation method thereof and application of the engineered platelets in type 1 diabetes mellitus. According to the engineering blood platelet, PD-L1 protein, PD-L2 protein, BTLA protein and Galectin-9 protein are overexpressed, and the engineering blood platelet is prepared by adopting a cell factor IFN-gamma to stimulate megakaryocyte. Experiments find that engineered platelets over-expressing PD-L1, PD-L2, BTLA and Galectin-9 molecules can accumulate in inflamed pancreatic tissues and inhibit the activity of pancreatic autoreactive T cells, so that beta-cells producing insulin are protected from being attacked, the beta-cells can be rescued by intervening an immune system, insulin secretion is recovered, and the effect of preventing and treating insulin is achieved. Therefore, the blood sugar of the NOD mouse is kept at a normal level, the treatment of newborn type 1 diabetes is realized, and the medicine has a very good treatment effect when being applied to the treatment of diabetes.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an engineered platelet, a preparation method thereof, and an application thereof in type 1 diabetes. Background Art

[0002] Type 1 diabetes mellitus (T1DM) is a chronic disease in which the immune system attacks the beta cells in the pancreas, resulting in insufficient insulin. It usually occurs in children and adolescents, but can also be seen in other age groups. According to data from 2007 to 2019, the incidence of T1DM in the United States and European countries is approximately 26 to 60 / 100,000 people, while in China it is approximately 2.2 / 100,000 people. Given the large population, there are approximately 13,000 new cases of T1DM in China each year. Currently, the diagnosis of T1DM mainly relies on detecting the presence of pancreatic autoimmune antibodies in the blood. Once diagnosed, patients need lifelong insulin injections to maintain blood sugar levels. Although insulin injections can effectively control blood sugar, patients are still at risk of insulin resistance, cardiovascular disease, kidney disease, and other serious complications.

[0003] Currently, the main treatment for T1DM is long-term insulin injection to control blood sugar. Restoring the function of autologous pancreatic islet cells or transplanting pancreatic islet cells is the ultimate goal of treating advanced T1DM and achieving cure. However, pancreatic β-cell transplantation faces challenges such as donor shortage and severe toxic side effects of immunosuppressants (such as cyclosporine and CD3 monoclonal antibody drugs), which limits its application in the clinical treatment of T1DM. Immunomodulatory strategies inhibit autoimmune attacks by reconstructing the pancreatic immune tolerance microenvironment. Among them, immune tolerance strategies that limit the activity of immune cells and prevent T lymphocytes from attacking β cells can inhibit the progression of T1DM. For patients with T1DM who have developed to the late stage, a combination of cell transplantation and immune tolerance is required. Immunomodulatory factors such as IL-10 and TGF-β have been used to induce regulatory T cells to treat T1D.

[0004] Another ideal treatment method is the immune tolerance strategy, which prevents the development of T1D by limiting the attack of immune cells on β-cells. For T1D that has developed to the late stage, most of the patient's β-cells have been damaged, so a combination of β-cell transplantation and immune tolerance therapy is required. At present, the most effective treatment method in clinical practice is bone marrow transplantation to rebuild the body's immune system and achieve immune tolerance to pancreatic islets. However, due to safety considerations and the risks of bone marrow transplantation, this method has not been widely used in clinical treatment. Immunomodulatory cells, such as regulatory T cells (Treg), suppressor DC cells, M2 macrophages, etc., can also be used to rebuild the body's immune tolerance. However, in clinical trials, the inhibitory effect of patients' regulatory T cells (Treg) on ​​T cells is relatively limited. Another option is to apply immunosuppressive factors, such as the combined use of IL-10 and vitamin D3 to increase regulatory T cells in the blood circulation. In addition, TGF-β can effectively induce CD4 + FoxP3 + Regulatory T cells, but this approach has systemic nonspecific effects.

[0005] Therefore, it is necessary to develop a more specific immunotherapy approach. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide an engineered platelet.

[0007] The purpose of the second aspect of the present invention is to provide a method for preparing the engineered platelets of the first aspect of the present invention.

[0008] The purpose of the third aspect of the present invention is to provide the application of the engineered platelets described in the first aspect of the present invention.

[0009] The fourth aspect of the present invention aims to provide a medicine.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0011] The purpose of the first aspect of the present invention is to provide an engineered platelet, wherein the engineered platelet overexpresses an immunosuppressive molecule.

[0012] In some embodiments of the present invention, the immunosuppressive molecules include PD-L1 protein, PD-L2 protein, BTLA protein and Galectin-9 protein.

[0013] In some embodiments of the invention, the immunosuppressive molecule is increased relative to a reference level; the reference level is the wild-type level.

[0014] In some embodiments of the present invention, the PD-L1 protein, PD-L2 protein, BTLA protein and Galectin-9 protein exist in the form of a complex. It is understood that the PD-L1 protein, PD-L2 protein, BTLA protein and Galectin-9 protein can be loaded on the engineered platelets in a mosaic manner, or wrapped inside the engineered platelets in the form of contents. Immune checkpoint ligands PD-L1 molecules, PD-L2 molecules, BTLA molecules and Galectin-9 molecules can be used as immunosuppressive molecules to inhibit autoantigen-reactive immune cells for the treatment of type 1 diabetes.

[0015] In some embodiments of the present invention, the engineered platelets have a particle size of 1000 nm;

[0016] In some preferred embodiments of the present invention, the particle size of the activated engineered platelets is 100 nm.

[0017] In some embodiments of the present invention, the Zeta potential of the engineered platelets is -11 to -15 mV.

[0018] In some preferred embodiments of the present invention, the Zeta potential of the engineered platelets is -11 to -15 mV.

[0019] The second aspect of the present invention provides a method for preparing the engineered platelets according to the first aspect of the present invention, comprising the following steps:

[0020] Megakaryocytes were treated with interferon-γ and phorbol 12-myristate 13-acetate, and platelets were collected.

[0021] In some embodiments of the present invention, the megakaryocytes are derived from at least one of mice and humans.

[0022] Preferably, the mouse-derived megakaryocytes include at least one of L8057 (CMK cell line), MK-1 cell line, MKS-2 cell line, D1 cell line, and Mega 1 cell line.

[0023] Preferably, the human-derived megakaryocytes include at least one of the Meg-01 cell line and the HEL cell line.

[0024] Preferably, the concentrations of interferon-γ and phorbol 12-myristate 13-acetate are 0.4-0.6 nM and 80-120 ng / mL, respectively.

[0025] In some embodiments of the present invention, the treatment time is 2 to 4 days.

[0026] In some embodiments of the present invention, the immune system can be intervened to rescue β-cells and restore insulin secretion by stimulating mouse megakaryocytes L8057 to overexpress PD-L1 protein, PD-L2 protein, BTLA protein and Galectin-9 protein using cytokine interferon-γ to produce PD-L1-PD-L2-BTLA-Galectin-9-L8057 engineered platelets.

[0027] The third aspect of the present invention provides use of the engineered platelets described in the first aspect of the present invention in the preparation of products for treating and / or preventing diabetes.

[0028] In some embodiments of the invention, the diabetes comprises type 1 diabetes.

[0029] In some embodiments of the present invention, the use of the product includes at least one of A) to P):

[0030] A) Lowering blood sugar levels;

[0031] B) Increase serum C-peptide levels;

[0032] C) Increase the level of IL-10 in the blood;

[0033] D) reduce the amount of IFN-γ in the blood;

[0034] E) Increase insulin or decrease glucagon levels;

[0035] F) Reduction of pancreatic infiltration of CD8 + T cell content;

[0036] G) Reduce IFN-γ in the pancreas + CD8 + The proportion of T cells;

[0037] H) Reduce GzmB in the pancreas + CD8 + The proportion of T cells;

[0038] I) Reduction of GzmB in the pancreas + CD4 + The proportion of T cells;

[0039] J) Increase CD25 in the pancreas + Foxp3 + The proportion of Tregs;

[0040] K) Reduce F4 / 80 in the pancreas + CD11B + The proportion of macrophages;

[0041] L) Reduction of CD86 in the pancreas + F4 / 80 + The proportion of macrophages;

[0042] M) Increase CD206 in the pancreas + F4 / 80 + The proportion of macrophages;

[0043] N) increases the proliferation of beta cells in the pancreas;

[0044] O) reduce apoptosis of β cells in the pancreas;

[0045] P) Increases the proliferation of blood vessels in the pancreas.

[0046] The fourth aspect of the present invention provides a drug, which includes the engineered platelets described in the first aspect of the present invention.

[0047] Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsule preparations, lyophilized powder injection preparations, injections, suppositories, sprays, powders, soft capsules, and sustained-release preparations.

[0048] Preferably, the drug includes a pharmaceutically acceptable excipient, which is generally recognized for this purpose and as an inactive ingredient of the medicament. A compilation of pharmaceutically acceptable excipients can be found in reference books such as the Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Gess, London, 1994); and the Pharmacopoeia of the People's Republic of China - Catalogue of Pharmaceutical Excipients.

[0049] The present invention provides an engineered platelet having the following advantages:

[0050] (1) It has an excellent therapeutic effect on type 1 diabetes. The engineered platelets overexpressing PD-L1, PD-L2, BTLA and Galectin-9 molecules of the present invention can accumulate in inflamed pancreatic tissue and inhibit the activity of pancreatic autoreactive T cells, thereby protecting insulin-producing β-cells from attack. The engineered platelets can rescue β-cells and restore insulin secretion by intervening in the immune system, thereby maintaining the blood sugar of NOD mice at a normal level and achieving the treatment of newly diagnosed type 1 diabetes.

[0051] (2) Low toxicity and side effects. Small molecule immunosuppressants and CD3 antibodies are common drugs for the treatment of newly diagnosed type 1 diabetes. Cyclosporine A, as a small molecule immunosuppressant, was first used as an immune preparation to treat type 1 diabetes. Although cyclosporine A can protect β-cells, it has severe nephrotoxicity. Anti-CD3 monoclonal antibody drugs Teplizimab and Otelixizumab effectively rescued β cells and maintained insulin production in clinical trials, but these antigen-nonspecific broad-spectrum T cell and B cell clearance treatments have serious side effects and safety hazards. The engineered platelets overexpressing PD-L1, PD-L2, BTLA and Galectin-9 molecules of the present invention contain proteins, miRNA and DNA from parent cells, which are themselves important media for long-distance communication between cells. They have good biocompatibility and stability, and have lower toxicity and side effects than the direct use of chemical small molecule immunosuppressants and CD3 antibodies.

[0052] (3) The method for preparing the engineered platelets of the present invention is simple and low in cost. The engineered platelets prepared by the method of the present invention can bind to PD-1 protein, HVEM protein and Tim-3 protein, and promote CD4 + It can induce apoptosis of T cells and promote the differentiation of splenic T cells into Treg cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0054] Figure 1 for Figure 1The process of producing engineered platelets of the present invention, wherein: A is detecting the expression of CD41 in L8057 cells by immunofluorescence staining; B is detecting the expression of CD41 in L8057 cells by flow cytometry; C is detecting CD42 in L8057 cells treated with 500nM PMA by immunofluorescence staining; D is detecting CD42 in L8057 cells treated with 500nM PMA by flow cytometry; E is detecting the expression of programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), B and T lymphocyte attenuator (BTLA, also known as CD272) and galectin-9, transforming growth factor-β (TGF-β), nucleotidase (CD73), adhesion molecule-2 (Nectin-2), signal regulatory protein α (SIRP-α), V-domain Ig suppressor of T in L8057 cells before and after IFN-γ stimulation by Western blot (WB). cellactivation (VTCN1) and indoleamine 2,3-dioxygenase (IDO) expression; F is the expression of PD-L1 in L8057 cells before and after IFN-γ stimulation detected by immunofluorescence staining; G is the expression of PD-L1 in L8057 cells before and after IFN-γ stimulation detected by flow cytometry; H is the observation of L8057 cells undergoing different stages of maturation and differentiation by biological transmission electron microscopy: I) budding of proplatelets; II) release of proplatelets; I is the observation of L8057 cells undergoing different stages of maturation and differentiation by immunofluorescence staining: I) mature L8057 cells; II) extension of proplatelets; III) release of proplatelets. J is the size distribution of IFN-γplatelet detected by DLS; K is the Zeta potential of IFN-γplatelet and Free platelet.

[0055] Figure 2The in vitro biological characteristics of the platelets of the present invention are characterized, wherein: A is the analysis of platelets before and after interferon γ (IFN-γ) stimulation using Western blot (WB) technology to determine the changes in the expression levels of PD-L1, PD-L2, BTLA, Galectin-9, TGF-β, CD73, Nectin-2, SIRP-α, VTCN1 and IDO; B is the expression level of PD-L1 in 1 mg of platelets detected by enzyme-linked immunosorbent assay (ELISA). (n=3); C is the expression level of PD-L2 in 1 mg of platelets detected by ELISA (n=3); D is the expression level of BTLA in 1 mg of platelets detected by ELISA. (n=3); E is the expression level of Galectin-9 in 1 mg of platelets detected by ELISA (n=3); F is the expression of PD-L1 in platelets before and after IFN-γ stimulation detected by flow cytometry; G is a transmission electron microscopy image showing free platelets, IFN-γ platelets, and platelet cutting observed by biological transmission electron microscopy; H is a transmission electron microscopy image of activated platelets and released platelet microparticles (PMPs); I is the observation of unactivated platelets and activated platelets by laser confocal microscopy; J is the size distribution of PMPs detected by DLS.

[0056] Figure 3 The in vitro biological characteristics of platelets, where A is the observation of CI.LY1 by laser scanning confocal microscopy + 2 - / 9 cells and Free platelet cultured in vitro for 6 hours with confocal microscopy 3D image; B is CI.LY1 observed by laser scanning confocal microscopy + 2 - Confocal microscopy 3D images of CI.LY1 / 9 cells cultured with IFN-γplatelet for 6 hours in vitro; C, D, and E are co-immunoprecipitation assays to investigate the expression of PD-L1, BTLA, and Gal-9 on IFN-γplatelet and PD-1, HVEM, and Tim-3 on CI.LY1 + 2 - / 9 cells; F is the interaction between platelets and CI.LY1 in different treatment groups analyzed by flow cytometry + 2 - Figure 2 shows a representative picture of cell apoptosis after co-incubation with CI.LY1 / 9 cells for 24 hours (n=3); G shows the expression of platelets and CI.LY1 in different treatment groups by flow cytometry analysis. + 2 - After 24 h of co-incubation with 1:1 / 9 cells, the quantitative results of apoptosis are shown (n=3).

[0057] Figure 4IFN-γplatelet reverses hyperglycemia in diabetic NOD mice, where: A is a fluorescence spectrum image taken after injection of IFN-γplatelet via tail vein, showing its biodistribution in major organs and pancreas; B is a pancreatic slice of diabetic NOD mice given Cy5.5-IFN-γplatelet; C, D, E are blood glucose levels of diabetic NOD mice under different treatments, see the annotation for details (n=10); F is the average blood glucose level of diabetic NOD mice under different treatments, see the annotation for details (n=10); dark green line: unreversed diabetic NOD mice (n=2); light green line: reversed diabetic NOD mice (n=8). G is the incidence of NOD mice developing diabetes (n=10); H is the error bar for the concentration of C-Peptide in serum detected by ELISA after 60 days (n=10); I is the insulin in pancreatic islet slices + Representative confocal images of β cells; J is the detection of IFN-γ in serum by ELISA after 60 days (n=10); K is the detection of IL-10 in serum by ELISA after 60 days (n=10).

[0058] Figure 5 is the effect of IFN-γ on the activity of T cells infiltrating the pancreas, where: A is CD3 + The percentage of T cells in the total cells; B is CD4 + The percentage of T cells in the total cells; C is CD8 + The percentage of T cells in the total cells; D, E are analyzed by flow cytometry (using CD8 + T cell gate) to analyze pancreatic infiltration of INF-γ in different treatment groups + CD8 + Representative images (D) and quantification (E) of T cells (n=7); F, G are flow cytometry (using CD8 + T cell gate) to analyze pancreatic infiltration of GzmB in different treatment groups + CD8 + Representative images (F) and quantification (G) of T cells (n=7); H, I are flow cytometry analysis of pancreatic infiltration of INF-γ in different treatment groups (using CD4+ T cell gate). + CD4 + Representative images (H) and quantification (I) of T cells (n=7); J is immunohistochemical analysis of CD3 T cells in the pancreas of normal mice and after treatment; K is immunofluorescence analysis of CD8 T cells in the pancreas of normal mice and after treatment.

[0059] Figure 6 IFN-γ platelets enhance the infiltration of Treg cells and macrophages into the pancreas, where: A and B are different groups of CD25 + Foxp3+ Representative images (A) and quantification (B) of Treg cell infiltration into the pancreas (CD3 + CD4 + T cells were gated, n=7); C, D are flow cytometric analysis showing the percentage of macrophages in the entire pancreatic cells (gated as a percentage of total cells, n=7); E, F are flow cytometric analysis showing the percentage of M1 macrophages (F4 / 80 cells were gated, n=7); G, H are flow cytometric analysis showing the percentage of M2 macrophages (F4 / 80 cells were gated, n=7); I is immunofluorescence analysis of F4 / 80 and INOS cells in the pancreas of normal mice and after treatment; J is immunofluorescence analysis of F4 / 80 and CD206 cells in the pancreas of normal mice and after treatment.

[0060] Figure 7 IFN-γ promotes the proliferation of pancreatic β cells and inhibits their apoptosis. A is Insulin in the pancreas of normal mice and after treatment. + Ki67 + Cell immunofluorescence analysis B is Insulin in the pancreas of normal mice and after treatment + Casepase3 + Cell immunofluorescence analysis; C is the immunohistochemical analysis of CD31 cells in the pancreas of normal mice and after treatment; D is the immunofluorescence analysis of CD31 cells in the pancreas of normal mice and after treatment; E, F, G and H are blood routine examinations showing the concentrations of leukocytes, lymphocytes, neutrophils and monocytes at different time points after engineered platelet injection (n=3). DETAILED DESCRIPTION

[0061] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0062] Example 1 Preparation of engineered platelets

[0063] 1. Experimental Materials

[0064] L8057 cells: The L8057 cell line is derived from mouse bone marrow cells. These cells are cultured and treated specifically to maintain proliferation in vitro and differentiate into mature megakaryocytes through appropriate induction. These megakaryocytes can show the ability to divide and produce platelets in vitro, so the L8057 cell line has become a common model for studying megakaryocyte development and platelet production.

[0065] 2. Preparation method

[0066] 1) When the cell density was 70%, interferon-γ and phorbol 12-myristate 13-acetate were added. Phorbol 12-myristate 13-acetate at a working concentration of 100 nM and interferon-γ at a working concentration of 100 ng / mL were added to the culture medium and cultured in RPMI 1640 medium for 3 days to allow maturation.

[0067] 2) Mature L8057 cells will release platelets within the next 3 days. After 3 days, the culture medium was collected and centrifuged at 1200 rpm for 20 minutes. The precipitate was then discarded. The supernatant was centrifuged at 12,000 rpm for 20 minutes.

[0068] 3) Finally, carefully store the platelet pellet in PBS or Tyrode's buffer.

[0069] Example 2 Characterization of engineered platelets

[0070] 1. Verification of engineered platelet protein expression

[0071] 5× loading buffer was added to the cell lysate and engineered platelets, diluted to 1×, boiled at 100°C for 10 min. Bring to a boil and separate on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skim milk powder (Tris buffer solution Tween (TBST, Tween 20, 0.1%) buffer) at room temperature for 2 hours. The membrane was incubated with PD-L1, PD-L2, CD272, TGF-β, CD73, Nectin-2, SIRP-α, VTCN1, or IDO antibodies (1:1000) at 4°C overnight. Subsequently, the membrane was washed three times with TBST buffer for approximately 15 minutes each time and incubated with secondary antibodies for 1 hour at room temperature. After three 5-minute TBST washes, imaging was performed using enhanced chemiluminescence detection (ECL, FDbio-Pico).

[0072] 2. Nanoparticle size and Zeta analysis

[0073] The particle size and zeta potential of engineered platelets were measured using nanoparticle size and zeta analyzer.

[0074] The results are as follows Figure 1 As shown, J is the particle size distribution diagram of the engineered platelets, and K is the Zeta potential analysis result of the engineered platelets. The results show that the average diameter of the engineered platelets is about 1000nm, and the Zeta potential of the engineered platelets is about -12mV.

[0075] 3. Transmission electron microscopy observation

[0076] The morphology of negatively stained engineered platelets was characterized using transmission electron microscopy (TEM). The specific method is as follows: After clamping the copper mesh with tweezers, fix it with a dovetail clip to prevent the copper mesh from falling. Fix the tweezers on ice with tape, add 10μL of platelet solution, let it stand for 5 minutes, and use filter paper to absorb the liquid from the edge of the copper mesh. Repeat this six times, then add 10μL of 3% uranyl acetate to stain for 5 minutes, and use filter paper to absorb the liquid from the edge. Finally, dry the copper mesh at room temperature, and observe the morphology of platelets and take pictures using a 120kV transmission electron microscope. During the experiment, the gun tips and filter paper related to uranyl acetate should be handled in accordance with safety guidelines.

[0077] The results are as follows Figure 2 As shown in G and H, the engineered platelet structure is shown.

[0078] 4. Confocal laser scanning microscopy observation of engineered platelets

[0079] 10 μL of engineered platelets obtained by centrifugation of mature L8057 cells were dripped onto a glass slide, covered with a cover slip, sealed with nail polish around the slide, and dried at room temperature in the dark. The fluorescence of the engineered platelets was then observed under a laser confocal microscope.

[0080] The results are as follows Figure 2 As shown in I, it shows that the present invention successfully prepares engineered platelets.

[0081] 5. Determination of engineered platelet concentration

[0082] The concentration of engineered platelets was determined using the BCA protein concentration assay kit from Biyuntian. The 0.5 mg / mL BSA protein standard was gradiently diluted to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg / mL, and the engineered platelets were diluted 10 times with PBS2:18. Take 20 μL of each concentration standard and the sample to be tested, vortex and mix with 200 μL BCA working solution, then pipette 200 μL of the mixture to the ELISA plate, put on PE film gloves, and incubate in a 37°C incubator for 30 minutes. The standard and sample were read using the A562 absorbance of the ELISA reader. According to the test results, the concentration-reading standard curve was made using the reading of the standard, and the formula and R square value were calculated and substituted into the reading of the sample to be tested to calculate the concentration of the diluted sample. After diluting to the required concentration, save it for later use.

[0083] Example 3 Characterization of in vitro biological activity of engineered platelets

[0084] 1. Experiment on the interaction between engineered platelets and CI.LY 1+2- / 9 cells

[0085] In order to study whether the engineered platelets can bind to the PD-1 receptor, Tim-3 receptor or HVEM receptor, this example uses co-immunoprecipitation (CO-IP) and cell binding experiments to study the binding of IFN-γplatelet and Free platelet to CI.LY 1 + 2 - / 9 cells T cells molecular interaction. The specific method is as follows: CI.LY 1 + 2 - / 9 cells were resuspended in a culture dish dedicated to laser confocal microscopy with 1640 culture medium to a density of about 40-50%, and 100 μg of engineered platelets (prepared in Example 1) were added to 2 ml of culture medium and mixed horizontally. After culturing in a cell culture incubator for 6 hours, the culture dish was removed, and then DAPI dye solution was added to stain the cell nucleus. Under a laser confocal microscope, the co-localization of engineered platelets and cells was observed.

[0086] The results are as follows Figure 3 As shown in A and B, it is shown that the engineered platelets can be combined with CI.LY 1 + 2 - In addition, CO-IP experiments showed that PD-1 and PD-L1 can be bound together by PD-L1 antibodies and precipitated, BTLA and HVEM can be bound together by BTLA antibodies and precipitated, and Tim-3 and Galectin-9 can be bound together by Galectin-9 antibodies and precipitated, indicating that IFN-γplatelet can bind to PD-1 receptors, HVEM receptors and Tim-3 receptors on the surface of T cells (such as Figure 3 C, D and E in FIG.

[0087] Furthermore, IFN-γplatelet was combined with CI.LY 1 + 2 - After co-incubation with 100 μg / mL / 9 cells for 24 h, the inhibitory effect of platelets on T cells and T cell apoptosis were observed.

[0088] like Figure 3 As shown in F and G, after co-incubation with IFN-γ platelet, CI.LY 1 + 2 - The proportion of apoptosis in / 9 / 9 cells increased significantly, presumably due to the binding of PD-L1 ligand on platelets to PD-1 receptor on T cells, BTLA ligand to HVEM receptor, and Galectin-9 ligand to Tim-3 receptor, thus activating negative immune checkpoints.

[0089] 2. Experiment on the interaction between engineered platelets and raw264.7 macrophages

[0090] In subsequent experiments, the engineered platelets were co-cultured with macrophages and undifferentiated RAW264.7 cells to study the effect of their interaction on macrophage polarization.

[0091] The results showed that Free platelet promoted the polarization of M0 macrophages to M2 macrophages ( Figure 3 H, I). In contrast, IFN-γplatelet resulted in a higher proportion of M0 macrophages polarizing to M2 macrophages ( Figure 3 H, I).

[0092] Example 4 Distribution experiment of engineered platelets in mouse organs

[0093] The engineered platelets were labeled with Cy 5.5-NHS dye and injected into tumor-bearing mice via the tail vein. The mice were killed and dissected 24 hours after administration, and their hearts, livers, spleens, lungs, kidneys, and pancreases were obtained for fluorescence imaging to observe the in vivo distribution of extracellular platelets and the platelet content levels in various organs.

[0094] The results are as follows Figure 4 As shown in A, 24 hours after administration, a large number of engineered platelets infiltrated into the liver and kidneys, and engineered platelets were detected to infiltrate into the pancreatic tissue.

[0095] Example 5 Use of engineered platelets in reversing newly diagnosed type 1 diabetes

[0096] In order to study whether engineered platelets can reverse newly-onset type 1 diabetes, this example used 12-week-old female non-obese diabetic mice NOD / Lt (NOD), and randomly divided the NOD mice into 3 groups, 7 mice in each group:

[0097] ①Untreated group (untreated group);

[0098] ②Free platele treatment group (control group);

[0099] ③IFN-γplatelet treatment group;

[0100] Starting from 12 weeks of age, the blood glucose of mice was tested every 2 days. Healthy mice maintained normal blood glucose, with a blood glucose range of 80-130 mg dL -1 Once the blood glucose level of NOD mice exceeded 250 mg dL for two consecutive days -1, the mice were considered to have new-onset diabetes. Subsequently, 250 μg of engineered platelets were injected intravenously every 2 days until the end of the experiment (treatment period of 60 days).

[0101] 1. Blood sugar change detection

[0102] Blood sugar test results Figure 4 Figures C, D, E, and F show that when NOD mice with newly developed T1D were not treated, blood glucose gradually increased, eventually reaching severe hyperglycemia with blood glucose levels >600 mg dL -1 In contrast, in newly developed T1D mice treated with IFN-γplatelet, the progression of T1D was significantly suppressed and hyperglycemia was reversed to normoglycemia, with 75% of mice successfully reversing from hyperglycemia to normoglycemia levels.

[0103] 2. C-peptide and IL-10 detection

[0104] C-peptide is a secretory product of pancreatic β cells. The detection of C-peptide can reveal the function of pancreatic cells and is of great significance for the diagnosis and treatment of diabetes. The C-peptide level in mouse serum was detected to detect the level of insulin secretion. After the above-mentioned Freeplatele and IFN-γplatelet treatment, blood was collected from the eyeballs of mice in each treatment group, and the blood was collected in a 1.5mL EP tube without RNAse. The blood was allowed to stand for 1-2h, and the blood was centrifuged at 200g and 20min to collect serum. The collected serum was used to detect the C-peptide level in the blood according to the method of the ELISA kit (Cat. No. E-EL-M0354c).

[0105] The results of C-peptide detection are shown in H in 4. After Free platelet treatment, the C-peptide levels of mice in the IFN-γplatelet treatment group were significantly higher than those in the untreated group, close to the level of normal mice without the disease. At the same time, after IFN-γplatelet treatment, the IL-10 content in the blood of diabetic mice significantly returned to normal levels.

[0106] 3. Immunofluorescence detection of insulin and glucagon levels in mouse pancreas

[0107] To further investigate the damage to insulin-producing β cells, pancreata from NOD mice in different treatment groups were collected and analyzed by immunofluorescence. The specific process was as follows: After the mice were given the drug, they were killed by cervical dislocation, the pancreas was dissected, washed twice with PBS, dried with filter paper, embedded with OCT, and placed in a -20°C refrigerator until OCT solidified. The cryostat was precooled, the slice thickness was adjusted to 7 μm, and the slices were sliced. The slices were placed at room temperature for 1-2 hours before downstream operations to prevent detachment. The slices that returned to room temperature were soaked in PBS for 15 minutes to remove OCT. Most of the water was absorbed with filter paper, and a circle was drawn with an immunohistochemistry pen away from the tumor tissue to avoid edge effects. 100 μL of 0.2% Trixton-x100 prepared with 3wt% BSA was added and incubated at room temperature for 30 minutes. After the liquid was absorbed, the mixed primary antibodies of insulin and glucagon were added, and the slices were placed in a wet box at 4°C overnight. On the second day, recover the primary antibody, soak it in PBS, and wash it three times on a shaker for 10 minutes each time. After absorbing the liquid with filter paper, add the fluorescent secondary antibody mixture and incubate it at room temperature in the dark for 1 hour. After absorbing the secondary antibody, soak it in PBS and wash it three times on a shaker for 10 minutes each time. After absorbing most of the water with filter paper, add DAPI dye solution with a final concentration of 1μg / mL and incubate it at room temperature in the dark for 15 minutes. After absorbing the dye solution, soak it in PBS and wash it three times on a shaker for 10 minutes each time. After absorbing the water, add anti-fluorescence quencher to seal the slide, cover it with a coverslip, and fix the coverslip with nail polish. Observe and film it as soon as possible with a laser confocal microscope.

[0108] Test results such as Figure 4 As shown, the pancreatic islet morphology of mice treated with IFN-γplatelet was relatively complete, indicating that they were able to produce insulin normally.

[0109] 4. Immunofluorescence staining and flow cytometric analysis of pancreatic infiltrating T cells

[0110] In order to further compare the therapeutic effects of different treatments on type 1 diabetic mice, immunofluorescence staining and flow cytometry were used to analyze the number of pancreatic infiltrating T cells in mice with different treatments, including:

[0111] The immunofluorescence staining method is as follows: After the mice are given the drug, the pancreas is removed, washed twice with PBS, dried with filter paper, embedded with OCT, and placed in a -20℃ refrigerator until OCT solidifies. Precool the cryostat, adjust the slice thickness to 7μm, and slice. Place the sliced ​​slices at room temperature for 1-2h before performing downstream operations to prevent detachment. Soak the slices that have returned to room temperature in PBS for 15min to remove OCT. Use filter paper to absorb most of the water, and use an immunohistochemistry pen to draw a circle away from the tumor tissue to avoid edge effects. Add 100μL of 0.2v / v% Trixton-x100 prepared with 3wt% BSA and incubate at room temperature for 30min. After absorbing the liquid, add the mixed primary antibody of CD4 and CD8, place in a wet box, and place in a humidified box at 4℃ overnight. The next day, recover the primary antibody, soak it in PBS, and wash it three times on a shaker, 10min each time. After absorbing the liquid with filter paper, add the fluorescent secondary antibody mixture and incubate at room temperature for 1h in the dark. After absorbing the secondary antibody, soak it in PBS and wash it three times on a shaker for 10 minutes each time. After absorbing most of the water with filter paper, add DAPI dye solution with a final concentration of 1μg / mL and incubate it at room temperature in the dark for 15 minutes. After absorbing the dye solution, soak it in PBS and wash it three times on a shaker for 10 minutes each time. After absorbing the water, add anti-fluorescence quenching agent to seal the slide, cover it with a coverslip, and fix the coverslip with nail polish. Observe and shoot the slide as soon as possible with a laser confocal microscope.

[0112] The pancreatic cell flow cytometry method is as follows: After the mice are given the drug, they are killed by cervical dislocation, the mouse pancreas is dissected, and washed twice with PBS to remove blood. The pancreas is placed on a 70μm cell sieve, and a 50mL centrifuge tube is placed under the sieve. The whole process is carried out on ice. After adding PBS containing 2v / v% FBS to moisten, gently grind, and continuously add PBS containing 2v / v% FBS to rinse to obtain a single cell suspension. The single cell suspension is placed at 4°C, centrifuged at 350g for 5min, the supernatant is removed, and then PBS containing 2v / v% FBS is added to wash twice, and resuspended with 500μL 2v / v% FBS PBS, and divided into 1.5mL EP tubes for downstream experiments. The flow cytometry steps are as follows.

[0113] (1) Cell surface molecule detection: Resuspend each tube of cells in PBS containing 2 v / v% FBS to a volume of 30-50 μL, add flow cytometry antibodies, and incubate at 4°C for 30 min. After staining, wash twice with PBS containing 2% FBS, resuspend the cells in 500 μL PBS containing 2 v / v% FBS, and detect them as soon as possible.

[0114] (2) Detection of intracellular cytokines: Add the pancreatic cell suspension to a 48-well plate containing 500 μL of 1640 culture medium, add PMA at a final concentration of 50 ng / mL and 1 μg / mL ionomycin, and culture in a cell culture incubator for 1 hour. Then add brefeldin A at a final concentration of 1-50 μM and continue to culture for 4-6 hours. After removing the culture medium, wash twice with PBS. Resuspend each tube of cells in PBS containing 2 v / v% FBS to a volume of 30-50 μL, add surface molecule flow cytometry antibodies, and incubate at 4°C for 30 minutes. After staining, wash twice with PBS containing 2 v / v% FBS, and fix with 4wt% paraformaldehyde at room temperature in the dark for 20 minutes. Centrifuge at 4°C, 350g for 5 minutes, remove the supernatant, and wash twice with PBS containing 2 v / v% FBS. Add 0.2 v / v% Trixton-x100 to break the membrane of the fixed cells, incubate at room temperature for 20 minutes, and centrifuge at 350g for 5 minutes. Resuspend each tube of cells with 30-50μL 0.01v / v% Trixton-x100, add intracellular flow cytometry antibodies, and incubate at 4℃ for 30min. Centrifuge at 4℃, 350g for 5min, remove the supernatant, and wash twice with 2% FBS PBS. Resuspend the fixed and stained cells with 500μL 2v / v% FBS PBS and detect them as soon as possible.

[0115] Results of pancreatic infiltrating T cell activity Figure 5 The results of immunofluorescence staining are shown in Figure 4 As shown in J and K, CD8 + T cells and CD3 + T cell infiltration Figure 5 As shown in J and K, a small number of CD8 + However, a large number of T cells appeared in the pancreatic margins and islets of hyperglycemic NOD mice. After treatment with IFN-γ platelet, pancreatic infiltrating CD8 + The number of CD8 T cells infiltrating the pancreas of normal non-lesional mice was significantly reduced. + T cell numbers were similar.

[0116] Activated CD8 cytotoxic T cells secrete cytokines such as IFN-γ and TNF-α to kill β cells. Therefore, this example further detected the infiltrating CD8 + The proportion of T cells in the pancreas was shown to be increased after treatment with IFN-γ platelet compared with untreated mice. + CD8 + The proportion of T cells was significantly reduced (eg Figure 5Compared with the control group, the expression of TNF-α in the pancreas of mice treated with IFN-γplatelet was significantly higher than that in the control group. + CD8 + The proportion of T cells also decreased significantly (such as Figure 5 ). Flow cytometry results showed that the proportion of Treg cells in the pancreatic islets of untreated hyperglycemic NOD mice was significantly reduced, while the proportion of Treg cells in the pancreatic islets of NOD mice treated with IFN-γplatelet was significantly increased, indicating that there are still sufficient numbers of Treg cells in the pancreatic islets to maintain immune tolerance ( Figure 6 Middle A). IFN-γ platelet can effectively inhibit pancreatic infiltration of CD8 + T cell activity and increased Foxp3 + The proportion of Tregs was increased, thereby reversing hyperglycemia in new-onset type 1 diabetes in NOD mice.

[0117] In addition, this example evaluates the ratio of macrophages and M1 and M2 macrophages in the pancreas. Flow cytometry results also showed that IFN-γplatelet can significantly inhibit the infiltration of macrophages into the pancreas, reduce the proportion of M1 macrophages, and promote the transformation of M1 macrophages to M2 macrophages. In the pancreas of normal mice, the ratio of M1 macrophages to M2 macrophages is low. At the same time, in the pancreatic margins and islets of NOD mice with hyperglycemia, the infiltration ratio of M1 macrophages is high, and the ratio of M2 macrophages is low. Through treatment, the proportion of M1 macrophages infiltrating the pancreas was significantly reduced, and the number of M2 macrophages infiltrating the pancreas was significantly increased. In contrast, free platelets have limited effects on preventing M1 macrophage infiltration and promoting M2 macrophage infiltration ( Figure 6 (C, D, E, FG, H). Previous studies have shown that in the pancreatic islet microenvironment, synergy between macrophages and endothelial and islet cells promotes β-cell regeneration. Studies have shown that macrophages play a key role in the regeneration of β-cells after loss. Endothelial cell proliferation may indirectly affect the β-cell regeneration process by regulating the infiltration and phenotype of macrophages. In addition, the recruitment of macrophages is essential for the recovery of β-cells, and the phenotypic shift may contribute to the regeneration and regression of β-cells.

[0118] In order to evaluate the situation of pancreatic infiltration of macrophages, the pancreas of NOD mice in different treatment groups was collected in this example. The results of immunofluorescence staining showed that the infiltration of M1 macrophages and M2 macrophages in the pancreas of NOD mice with normal blood sugar levels was less, while the islets of NOD mice with hyperglycemia had a large number of M1 macrophage infiltrations. After IFN-γplatelet treatment, the infiltration of M1 macrophages in the pancreas was significantly reduced, and the infiltration of M2 macrophages was significantly increased. In the pancreas of normal mice, there are a small number of M1 macrophages and M2 macrophages. At the same time, in the pancreatic margins and islets of NOD mice with hyperglycemia, the infiltration of M1 macrophages is more intensive. Through treatment, the number of M1 macrophages infiltrating the pancreas was significantly reduced, and the number of M2 macrophages infiltrating the pancreas was significantly increased. In contrast, free platelets have limited effect on preventing M1 macrophage infiltration ( Figure 6 I, Figure 6 J). Previous studies have shown that M2 macrophages promote β-cell proliferation by releasing TGFβ1 and EGF. In addition, upregulation of SMAD7 in β-cells can also promote β-cell proliferation. Therefore, the growth factors released by M2 macrophages and the action of SMAD7 are factors that promote β-cell proliferation.

[0119] This example continues to study whether the therapeutic effect of platelets on diabetic mice is attributed to platelet-mediated proliferation and apoptosis of pancreatic islet cells. Figure 7 As shown in A and B, Ki67 in a single or multiple islets + Insulin + Immunofluorescence staining of cells showed that IFN-γplatele treatment promoted the proliferation of pancreatic β cells and enhanced insulin expression. + Staining of Insulin+ cells showed that apoptosis of insular and pancreatic cells was significantly inhibited after IFN-γplatelet treatment. These results indicate that IFN-γplatelet treatment has a great effect in promoting the proliferation of pancreatic β cells and inhibiting apoptosis to repair the damaged pancreas in T1D mice. These in vivo experimental results indicate that IFN-γplatelet treatment has a stronger ability to improve T1D than Free platelet treatment. In addition, PD-L1 on platelets plays an important role in the protection of pancreatic islets and β cells.

[0120] The vascular wall, especially the vascular endothelium, is the ultimate barrier to prevent autoimmune T cells from destroying pancreatic islets. CD31, as a specific marker for endothelial cells, is often used to assess the status of damage-related angiogenesis. Figure 7 As shown in C and D, immunohistochemistry and immunofluorescence staining analysis showed that compared with other groups, the CD31 +The number of cells increased significantly, suggesting that IFN-γ platelet treatment enhanced angiogenesis in the pancreas of diabetic mice. Previous studies have shown that blood vessels in the pancreatic islet microenvironment produce VEGF-A signals, which work in concert with intravascular endothelial cells (ECs) in the islets to promote β-cell regeneration. VEGF-A signals regulate the activation of pancreatic vascular endothelial cells through VEGFR2, leading to β-cell loss, thereby maintaining pancreatic vascular homeostasis and β-cell mass. In addition, VEGF-A induction also recruits CD45 + CD11b + Gr1 + Macrophages, which persist in islet remnants after VEGF-A stimulation is withdrawn, promote β-cell proliferation and regeneration by producing effector molecules, independent of islet location and systemic factors.

[0121] However, in order to accurately assess the systemic toxicity of IFN-γplatelet treatment, routine blood tests are necessary. After 60 days of treatment, blood immune cell counts were tested. The results showed that leukocytes, lymphocytes, neutrophils and monocytes. It is worth noting that IFN-γplatele significantly reduced the number of leukocytes, lymphocytes, neutrophils and monocytes in the blood in vivo. However, the number of leukocytes, lymphocytes, neutrophils and monocytes in the blood of hyperglycemic mice increased significantly, showing signs of an overactive immune system. At the same time, it was found that the number of leukocytes, lymphocytes, neutrophils and monocytes in the blood of IFN-γplatelet-treated mice was significantly reduced, which helps maintain immune tolerance. In contrast, free platelets have limited effect on reducing the number of leukocytes, lymphocytes, neutrophils and monocytes in the blood ( Figure 7 Middle E, Figure 7 Middle F, Figure 7 Middle G, Figure 7 Middle H).

[0122] The above results show that the number of specific T cells in the pancreatic islets of mice treated with IFN-γplatelet is smaller, so the β cells are protected, the pancreatic islet morphology is more intact, and type 1 diabetes is effectively reversed.

[0123] In summary, the present invention provides an engineered platelet and a preparation method and application thereof. The engineered platelet of the present invention is loaded with PD-L1 protein, PD-L2 protein, BTLA protein and Galectin-9 protein, and is prepared by stimulating megakaryocytes with the cytokine IFN-γ. The present invention has found through experiments that engineered platelets overexpressing PD-L1, PD-L2, BTLA and Galectin-9 molecules can accumulate in inflamed pancreatic tissue and inhibit the activity of pancreatic autoreactive T cells, thereby protecting insulin-producing β-cells from attack, and can rescue β-cells and restore insulin secretion by intervening in the immune system, thereby maintaining the blood sugar of NOD mice at a normal level, achieving the treatment of newly diagnosed type 1 diabetes, and applying it to the treatment of diabetes has a very good therapeutic effect.

Claims

1. An engineered platelet, characterized in that: The engineered platelets overexpress immunosuppressive molecules; The immunosuppressive molecules include PD-L1 protein, PD-L2 protein, BTLA protein and Galectin-9 protein.

2. The engineered platelet according to claim 1, characterized in that: The immunosuppressive molecule is increased relative to a reference level; The reference level is the wild-type level.

3. The engineered platelet according to claim 1, characterized in that: The particle size of the engineered platelets is 500 to 5000 nm; Preferably, the particle size of the engineered platelets is 800-1200 nm.

4. The engineered platelet according to claim 1, characterized in that: The zeta potential of the engineered platelets is -11 to -15 mV.

5. The method for preparing engineered platelets according to any one of claims 1 to 4, characterized in that: The following steps are involved: Megakaryocytes were treated with interferon-γ and phorbol 12-myristate 13-acetate, and platelets were collected.

6. The preparation method according to claim 5, characterized in that: The megakaryocytes are derived from at least one of mice and humans; Preferably, the mouse-derived megakaryocytes include at least one of L8057 (CMK cell line), MK-1 cell line, MKS-2 cell line, D1 cell line, and Mega 1 cell line. Preferably, the human-derived megakaryocytes include at least one of the Meg-01 cell line and the HEL cell line.

7. The preparation method according to claim 5, characterized in that: The concentrations of interferon-γ and phorbol 12-myristate 13-acetate are 0.4-0.6 nM and 80-120 ng / mL respectively.

8. The preparation method according to claim 5, characterized in that: The treatment time is 2 to 4 days.

9. Use of the engineered platelets according to any one of claims 1 to 4 in the preparation of products for treating and / or preventing diabetes.

10. The use according to claim 9, characterized in that: The use of the product includes at least one of A) to P): A) Lowering blood sugar levels; B) Increase serum C-peptide levels; C) Increase the level of IL-10 in the blood; D) reduce the amount of IFN-γ in the blood; E) Increase insulin or decrease glucagon levels; F) Reduction of pancreatic infiltration of CD8 + T cell content; G) Reduce IFN-γ in the pancreas + CD8 + The proportion of T cells; H) Reduce GzmB in the pancreas + CD8 + The proportion of T cells; I) Reduction of GzmB in the pancreas + CD4 + The proportion of T cells; J) Increase CD25 in the pancreas + Foxp3 + The proportion of Tregs; K) Reduce F4 / 80 in the pancreas + CD11B + The proportion of macrophages; L) Reduction of CD86 in the pancreas + F4 / 80 + The proportion of macrophages; M) Increase CD206 in the pancreas + F4 / 80 + The proportion of macrophages; N) increases the proliferation of beta cells in the pancreas; O) reduce apoptosis of β cells in the pancreas; P) Increases the proliferation of blood vessels in the pancreas.

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