Engineered dendritic cell preparation as well as preparation method and application thereof

Abstract

The invention discloses an engineered dendritic cell preparation as well as a preparation method and application thereof. According to the engineering dendritic cell, a dendritic cell is modified through a genetic engineering method, so that the cell overexpresses a FasL receptor, and then, a click chemistry method is utilized to load an anti-inflammatory drug entrapped liposome on the surface of the dendritic cell, so that the engineering dendritic cell preparation is obtained. The engineered dendritic cell not only can induce apoptosis of an over-activated T cell at an inflammation part through a Fas / FasL mediated cell apoptosis effect, but also can induce apoptosis of the over-activated T cell at the inflammation part; in addition, migration and recruitment of inflammatory cells to inflammatory sites can be reduced by releasing RvE-1, and neutrophil apoptosis and polarization of anti-inflammatory macrophage phenotypes can be induced. Therefore, the engineered dendritic cell provided by the invention can efficiently, specifically and safely improve tissue damage and immune disorder caused by inflammation, and has a good application prospect in treatment of inflammatory bowel disease and enteritis related tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to an engineered dendritic cell preparation, a FasL-overexpressing dendritic cell, a liposome encapsulating an anti-inflammatory drug, and a preparation method thereof, as well as their use in preparing drugs for treating, improving and / or preventing inflammatory bowel disease and enteritis-related tumors. Background Art

[0002] The intestinal inflammatory microenvironment is closely related to the occurrence and development of many diseases, including inflammatory bowel disease (IBD) and intestinal tumors. A large number of studies have shown that it is mainly related to genetic susceptibility, changes in environmental factors, immune regulation, and intestinal microecological disorders. When inflammation occurs, there is a large amount of lymphocyte infiltration in the intestinal mucosal tissue. Among them, the increase in T lymphocytes plays a key role in inducing enteritis. Overactivation of T lymphocytes causes intestinal epithelial cells to be attacked by T cells and apoptosis. At the same time, overactivated T lymphocytes secrete excessive proinflammatory cytokines, leading to damage and chronic inflammation. Therefore, finding effective biomarkers and therapeutic targets is crucial to improving the intestinal inflammatory microenvironment and improving the treatment of various intestinal diseases.

[0003] Fas receptor is one of the most important members of the death receptor family. After interacting with its natural ligand Fas legend (FasL), it initiates the cell death signal. In the process of continuous activation of T cells caused by specific antigens of foreign pathogenic microorganisms, activated effector T cells, on the one hand, upregulate FasL to mediate the clearance of pathogenic microorganisms or diseased cells, and on the other hand, upregulate the expression level of Fas, enhance the sensitivity to FasL-induced apoptosis, and inhibit the excessive activation of T cells. Summary of the invention

[0004] Considering that excessive activation of T cells is one of the important inducing factors of the intestinal inflammatory microenvironment, the present invention analyzes the activation state of T lymphocytes and the expression of Fas and FasL in the intestinal tissues of patients with enteritis or colorectal cancer, and finds that a large number of T cells in the intestinal tissues of patients with enteritis are accumulated in the intestinal lamina propria, and the T cells are in an activated state, and the expression levels of surface Fas and FasL are increased compared with normal intestinal tissues. Based on this, the present invention develops a biological preparation that can inhibit excessive activation of T cells.

[0005] One object of the present invention is to provide an engineered dendritic cell preparation, which includes dendritic cells that overexpress FasL and liposomes that encapsulate anti-inflammatory drugs and are coupled to the cell surface. The engineered dendritic cell preparation has intestinal-specific targeting; its highly expressed FasL induces apoptosis of overactivated T cells; at the same time, the liposomes loaded with RvE-1 coupled to the cell surface inhibit the infiltration of neutrophils and M1 macrophages by releasing RvE-1, thereby achieving multi-pathway regulation of the inflammatory microenvironment.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned engineered dendritic cells.

[0007] Another object of the present invention is to provide a method for preparing FasL-overexpressing dendritic cells, and FasL-overexpressing dendritic cells prepared by the method.

[0008] Another object of the present invention is to provide a method for preparing liposomes encapsulating anti-inflammatory drugs, and the liposomes encapsulating anti-inflammatory drugs prepared by the method.

[0009] Another object of the present invention is to provide the use of the above-mentioned engineered dendritic cells, dendritic cells overexpressing FasL and / or liposomes encapsulating anti-inflammatory drugs in the preparation of drugs for treating, improving and / or preventing inflammatory bowel disease and enteritis-related tumors.

[0010] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0011] The first aspect of the present invention provides an engineered dendritic cell preparation, which includes dendritic cells overexpressing FasL and liposomes encapsulating anti-inflammatory drugs coupled to the cell surface.

[0012] In some embodiments, the surface of the liposomes encapsulating the anti-inflammatory drug contains an alkynyl group, and the dendritic cells overexpressing FasL are coupled to the alkynyl groups on the surface of the liposomes encapsulating the anti-inflammatory drug through an azido sugar analog (click chemistry). By coupling the liposomes to the cell surface, the intestinal targeting characteristics of dendritic cells after intraperitoneal injection can be utilized to increase the enrichment of liposomes in the intestine and increase the content of anti-inflammatory drugs in the intestinal inflammation site. Compared with simply mixing dendritic cells overexpressing FasL and liposomes encapsulating anti-inflammatory drugs, the coupling strategy has a more significant effect in relieving intestinal inflammation.

[0013] In some embodiments, the azido sugar analog is co-cultured with dendritic cells overexpressing FasL, introduced into glycoproteins through the intracellular polysaccharide biosynthesis pathway, and then coupled to the alkyne group in the liposomes encapsulating the anti-inflammatory drug through the azide group. In some embodiments, the azido sugar analog is selected from one or more of 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose (Ac4ManNAz), N-azidoacetylmannosamine (ManNAz), N-azidoacetyl glucosamine (GlcNAz), and N-azidoacetylgalactosamine (GalNAz); preferably 1,3,4,6-tetra-O-acetyl N-azidoacetylaminomannose (Ac4ManNAz) with better solubility.

[0014] In some embodiments, the dendritic cells overexpressing FasL can be obtained by genetic engineering methods. For example, the dendritic cells can be made to contain a FasL gene overexpression plasmid. The FasL gene overexpression plasmid can be obtained by genetic engineering methods. For example, the FasL gene fragment and the linearized vector are connected according to homologous recombination or T4 connection methods to obtain a FasL gene overexpression plasmid. Subsequently, the purified FasL gene overexpression plasmid is obtained by transfecting competent cells, screening positive clones, gene sequencing, plasmid extraction, etc. Alternatively, the FasL gene overexpression plasmid can be obtained by introducing the FasL gene (GeneID: 14013) into an expression vector (for example, a commercial expression vector (for example, pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO plasmid vector)). In some embodiments, the FasL gene overexpression plasmid structure can be pHBLV-FASLG-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO obtained by introducing the FasL gene into the pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO plasmid vector.

[0015] In some embodiments, the liposomes encapsulating anti-inflammatory drugs contain hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and distearoylphosphatidylethanolamine-polyethylene glycol-dibenzocyclooctyne (DSPE-PEG-DBCO), and the mass ratio thereof is HSPC: Cholesterol: DSPE-PEG: DSPE-PEG-DBCO = (5-10): (1-5): (1-5): (0.5-1); in some embodiments, the anti-inflammatory drugs encapsulated in the liposomes encapsulating anti-inflammatory drugs may refer to resolvin, acetaminophen, salicylic acid, 6-mercaptopurine, etc., and examples thereof may be one or more selected from Resolvin E1, Resolvin D1, Resolvin D2, and Resolvin D3, preferably Resolvin E1; In some embodiments, the mass ratio of the anti-inflammatory drug to hydrogenated soybean phosphatidylcholine (HSPC) is 1:(300-500).

[0016] In some embodiments, in the engineered dendritic cell preparation, the ratio of liposomes to dendritic cells is 50-300 μg liposomes / 1 million cells, preferably 50-150 μg liposomes / 1 million cells.

[0017] The second aspect of the present invention provides a method for preparing FasL-overexpressing dendritic cells, which are obtained by transfecting dendritic cells with a lentiviral vector, adenoviral vector or cationic lipid vector loaded with a FasL gene overexpression plasmid.

[0018] In some embodiments, the method for preparing FasL-overexpressing dendritic cells comprises the following steps:

[0019] (1) constructing a FasL gene overexpression plasmid and packaging it using a lentiviral vector, adenoviral vector or cationic lipid vector;

[0020] (2) Inducing the production of dendritic cells from monocytes from blood or bone marrow using granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4;

[0021] (3) Transfecting the FasL gene overexpression plasmid packaged by the lentiviral vector, adenoviral vector or cationic lipid vector in (1) into the dendritic cells induced in step (2) to obtain FasL overexpressing dendritic cells.

[0022] In some embodiments, in step (1), the construction of the FasL gene overexpression plasmid can be obtained by genetic engineering methods. For example, the FasL gene fragment and the linearized vector are connected by homologous recombination or T4 connection to obtain the FasL gene overexpression plasmid. Subsequently, the purified FasL gene overexpression plasmid is obtained by transfecting competent cells, screening positive clones, gene sequencing, plasmid extraction and other steps. Alternatively, the FasL gene overexpression plasmid can be pHBLV-FASLG-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO obtained by introducing the FasL gene into the pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO plasmid vector.

[0023] In some embodiments, in step (2), the monocytes from blood or bone marrow can be monocytes from the patient's own blood or bone marrow. In some embodiments, in step (2), the concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 used to induce monocytes to produce dendritic cells are 10-100 ng / mL, respectively, preferably 20 ng / mL of granulocyte-macrophage colony stimulating factor (GM-CSF) and 10 ng / mL of IL-4.

[0024] In some embodiments, the specific steps of step (2) inducing monocytes to generate dendritic cells include:

[0025] The mononuclear cells were dispersed into 1 × 10 cells using complete medium containing 10% serum. 6 -1×10 7 / mL, adherent culture for 8-24 hours; then aspirate the suspended cells and supernatant culture medium, add complete cell culture medium containing GM-CSF and IL-4 at concentrations of 10-100 ng / mL respectively for induction culture, the induction culture conditions are 37°C, 5% carbon dioxide; wherein, half volume of the medium is replaced every 2 days and the culture is continued.

[0026] In some embodiments, the specific step of transfecting the packaged FasL gene overexpression plasmid into the dendritic cells induced in step (2) in step (3) comprises:

[0027] On the 5th day of monocyte induction culture, the suspended cells are counted, and a lentiviral vector, adenoviral vector or cationic lipid vector loaded with a FasL gene overexpression plasmid with a multiplicity of infection MOI = 15-40, preferably MOI = 20-30 is added to the culture medium, and a transfection-promoting reagent Polybrene is added, and the culture is continued for 2 days; on the 7th day, the suspended dendritic cells are aspirated, centrifuged at 100-500g for 3-30 minutes at 4°C-10°C, and redispersed in physiological saline to a cell suspension of 1 million-10 million / mL, thereby obtaining FasL-overexpressing dendritic cells.

[0028] In some embodiments, mononuclear cells from blood can be isolated from blood by using a lymphocyte separation solution and gradient density centrifugation.

[0029] In some embodiments, mononuclear cells from bone marrow can be obtained by isolating and culturing primary bone marrow cells.

[0030] The third aspect of the present invention provides FasL-overexpressing dendritic cells prepared by the above-mentioned method for preparing FasL-overexpressing dendritic cells.

[0031] A fourth aspect of the present invention provides a method for preparing a liposome encapsulating an anti-inflammatory drug, comprising the following steps:

[0032] (i) taking liposome materials hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and distearoylphosphatidylethanolamine-polyethylene glycol-dibenzocyclooctyne (DSPE-PEG-DBCO), wherein the mass ratio is HSPC: Cholesterol: DSPE-PEG: DSPE-PEG-DBCO = (5-10): (1-5): (1-5): (0.5-1), and dissolving them in an organic phase; uniformly mixing the ethanol solution of the anti-inflammatory drug with the organic phase in which the liposome material is dissolved to form a mixed solution;

[0033] (ii) subjecting the mixed solution to reduced pressure rotary evaporation to remove the organic solvent to form a film; adding an aqueous phase to the film-forming lipids and hydrating them at 30° C.-90° C.; and then treating them under high pressure or high shear conditions to obtain the liposomes encapsulating the anti-inflammatory drug.

[0034] In some embodiments, in step (i), the organic phase is selected from one or more of ethanol, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, dichloromethane, chloroform, and acetone, preferably dichloromethane. In some embodiments, the amount ratio of hydrogenated soybean phosphatidylcholine (HSPC) to the organic phase is (2-8) mg: 3 mL.

[0035] In some embodiments, in step (i), the anti-inflammatory drug is selected from resolvin, acetaminophen, salicylic acid, 6-mercaptopurine, etc., preferably one or more of Resolvin E1, Resolvin D1, Resolvin D2, Resolvin D3, and more preferably Resolvin E1; the concentration of the anti-inflammatory drug in the ethanol solution of the anti-inflammatory drug is 40-60 μg / mL.

[0036] In some embodiments, in step (i), the mass ratio of the anti-inflammatory drug to hydrogenated soybean phosphatidylcholine (HSPC) is 1: (300-500).

[0037] In some embodiments, in step (ii), the temperature of the reduced pressure rotary evaporation is in the range of 35 to 45° C., and the time is 0.5 to 3 hours.

[0038] In some embodiments, in step (ii), the ratio of hydrogenated soybean phosphatidylcholine (HSPC) to the added aqueous phase is (0.5-2) mg: 1 mL.

[0039] In some embodiments, in step (ii), the hydration temperature is 40-60° C., and the hydration time is 0.5-2 h, preferably 1 h.

[0040] In some embodiments, in step (ii), the high pressure or high shear force condition is: homogenization is performed under instrument conditions such as a high pressure homogenizer, an ultrasonic disruptor, a high shear mixer or a high shear agitator.

[0041] In some embodiments, in step (ii), the particle size of the liposome encapsulating the anti-inflammatory drug is 100 to 200 nm, preferably 140 to 169 nm.

[0042] A fifth aspect of the present invention provides liposomes encapsulating anti-inflammatory drugs prepared by the above-mentioned method for preparing liposomes encapsulating anti-inflammatory drugs.

[0043] A sixth aspect of the present invention provides a method for preparing the engineered dendritic cell preparation according to the first aspect, comprising the following steps:

[0044] S1: Dendritic cells overexpressing FasL were co-cultured with azidosugar analogs;

[0045] S2: After co-culture, the suspended dendritic cells are aspirated, the dendritic cells are separated by centrifugation at 4°C-10°C, and the dendritic cells are redispersed in physiological saline to form a cell suspension; then, the liposomes encapsulating the anti-inflammatory drug are added to the cell suspension pre-cooled at 4°C, and incubated at 4°C for coupling; after the incubation is completed, the cells are separated by centrifugation at 4°C-10°C, and redispersed in physiological saline to obtain an engineered dendritic cell preparation.

[0046] In some embodiments, in step S1, during the co-culture, the concentration of the azido sugar analog is 30-60 μM; in some embodiments, the azido sugar analog is selected from one or more of 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose (Ac4ManNAz), N-azidoacetylmannosamine (ManNAz), N-azidoacetylglucosamine (GlcNAz), and N-azidoacetylgalactosamine (GalNAz); preferably 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose (Ac4ManNAz).

[0047] In some embodiments, in step S1, the co-culture conditions are: 37° C., 5% carbon dioxide concentration; and the co-culture time is 3 days.

[0048] In some embodiments, in step S1, part of the suspended FasL-overexpressing dendritic cells after co-culture can be aspirated, and the expression of the azide group N3 on the cell surface can be detected by flow cytometry or immunofluorescence imaging technology. When more than 90% of the cells express the azide group N3, subsequent experiments can be performed.

[0049] In some embodiments, in step S2, the centrifugation conditions at 4°C-10°C are: 100-500g, 3-30 minutes.

[0050] In some embodiments, in step S2, the cells are redispersed in physiological saline to form a cell suspension of 0.5 million to 10 million cells / mL, preferably 0.5 million to 2 million cells / mL.

[0051] In some embodiments, in step S2, the cell suspension is precooled in a 4° C. refrigerator for 20-30 min.

[0052] In some embodiments, in step S2, the liposomes encapsulating the anti-inflammatory drug are added at a ratio of 50-300 μg liposomes / 1 million cells, preferably 50-150 μg liposomes / 1 million cells.

[0053] In some embodiments, in step S2, liposomes encapsulating anti-inflammatory drugs are added to the pre-cooled cell suspension, and then incubated in a 4° C. refrigerator for 20-30 min.

[0054] In some embodiments, in step S2, the cell concentration in the engineered dendritic cell preparation is 0.5-10 million cells / mL, preferably 0.5-2 million cells / mL.

[0055] The seventh aspect of the present invention provides the use of the above-mentioned engineered dendritic cells, dendritic cells overexpressing FasL and / or liposomes encapsulating anti-inflammatory drugs, or the engineered dendritic cells, dendritic cells overexpressing FasL and / or liposomes encapsulating anti-inflammatory drugs prepared by the above-mentioned preparation method in the preparation of drugs for treating, improving and / or preventing inflammatory bowel disease and enteritis-related tumors.

[0056] In particular, the engineered dendritic cell preparation of the present invention can induce apoptosis of overactivated T lymphocytes at the inflammatory site, inhibit the infiltration of inflammatory cells, and increase the proportion of anti-inflammatory immune cells, thereby regulating the intestinal inflammatory microenvironment. The engineered dendritic cell preparation has intestinal targeting, can specifically regulate the intestinal inflammatory microenvironment, and has a significant improvement effect on a variety of intestinal inflammations. In some embodiments, the inflammatory bowel disease includes: ulcerative colitis, Crohn's disease, viral enteritis, bacterial enteritis and indeterminate colitis.

[0057] In particular, the engineered dendritic cell preparation of the present invention can also alleviate adenomatous polyps and tumor-like lesions induced by chronic colitis. After administration, by regulating the intestinal inflammatory microenvironment, it can effectively inhibit the transformation of colitis to intestinal cancer and significantly reduce the volume and number of tumors. In some embodiments, the enteritis-related tumors include: adenomatous polyps and tumor-like lesions induced by chronic colitis.

[0058] Beneficial Effects

[0059] The engineered dendritic cell preparation of the present invention has intestinal-specific targeting. After intraperitoneal injection, it can migrate to the intestine and related lymphoid tissues, such as mesenteric lymph nodes and Peyer's patches, through transvascular action and blood circulation, thereby achieving intestinal-specific targeting. At the same time, the intestinal migration of dendritic cells is mainly induced by inflammatory chemokines such as CCL2, CCL3, and CCL5 in the intestinal inflammatory microenvironment. Compared with normal intestinal tissue, the enrichment of dendritic cells in inflammatory intestinal tissue is significantly increased.

[0060] The dendritic cell preparation of the present invention can overcome the shortcomings of existing intestinal inflammation treatment strategies and is effectively used for the treatment of intestinal inflammation. Using genetic engineering and bioorthogonal engineering methods, FasL-overexpressing dendritic cells are prepared, and liposomes loaded with anti-inflammatory drugs are coupled to the cell surface. The intestinal tropism of dendritic cells after intraperitoneal injection is utilized to target the intestine and regulate the intestinal inflammatory microenvironment by inducing apoptosis of overactivated T cells and inhibiting inflammatory cell infiltration, etc., and the effect is significant in the treatment of various types of intestinal inflammation and enteritis-related tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is a schematic diagram of the research idea of ​​the engineered dendritic cells of the present invention.

[0062] Figure 2 It is a transmission electron micrograph of the liposomes encapsulating the anti-inflammatory drug in Example 1, in which A and B are transmission electron micrographs of empty liposomes and drug-loaded liposomes, respectively.

[0063] Figure 3 This is a particle size characterization diagram of the drug-loaded liposomes in Example 1.

[0064] Figure 4 The particle size of the drug-loaded liposomes in Example 1 changes with time.

[0065] Figure 5 This is the expression of N3 on the cell surface after DCs were co-cultured with Ac4ManNAz for 3 days in Example 2; the dendritic cells were labeled with DBCO-Cy5.

[0066] Figure 6 This is an immunofluorescence staining picture of FasL expression in engineered dendritic cells in Example 2; FasL was labeled with AlexaFlour488, and DC was used as a blank control.

[0067] Figure 7 This is a photo of immunofluorescence staining of the engineered dendritic cell liposome coupling in Example 2; the liposome encapsulated fluorescent probe DiR as an imaging molecule, and FasL-DC not co-cultured with Ac4ManNAz was used as a blank control.

[0068] Figure 8 This is the distribution of the engineered dendritic cells in Example 3 in the mouse intestine 24 hours after intraperitoneal injection; the liposome-encapsulated fluorescent probe DiR is used as an imaging molecule.

[0069] Fig. 9 This is the experimental result of Example 4 that the engineered dendritic cells induced a decrease in activated T cells at the site of intestinal inflammation in a mouse model.

[0070] Fig.10This is the experimental result of Example 4 that the engineered dendritic cells induced neutropenia in the intestinal inflammation site in the mouse model.

[0071] Fig.11 This is the pharmacodynamic evaluation of the engineered dendritic cells in Example 5 in the DSS-induced acute enteritis model in mice, wherein A and B are the weight change curve of mice and the DAI score, respectively.

[0072] Fig.12 This is the pharmacodynamic evaluation of the engineered dendritic cells in Example 6 in the TNBS-induced acute enteritis model in mice, wherein A and B are the weight change curves of mice and the DIA scores, respectively.

[0073] Fig.13 This is the pharmacodynamic evaluation of the engineered dendritic cells in Example 7 in the mouse inflammation-induced intestinal cancer model, wherein A is a picture of colon tumors in each group, B is the statistical result of the number of colon tumors in each group, and C is the statistical result of the tumor size in each group. DETAILED DESCRIPTION

[0074] In order to enable persons with ordinary knowledge in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and the scope of the patent application. Unless otherwise specified, all technical and scientific words used in the text have the usual meanings understood by those skilled in the art for the present invention. When there is a conflict, the definition of this specification shall prevail. In this article, the terms "comprise", "include", "have", "contain" or any other similar terms are open-ended transitional phrases, which are intended to cover non-exclusive inclusions. For example, a composition or product containing multiple elements is not limited to the elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent to the composition or product. In addition, unless otherwise explicitly stated, the term "or" refers to an inclusive "or" rather than an exclusive "or". For example, any of the following situations satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist). In addition, in this article, the interpretation of the terms "comprising", "including", "having", and "containing" should be deemed to have been specifically disclosed and simultaneously cover closed or semi-closed conjunctions such as "consisting of" and "consisting essentially of". In this article, all features or conditions defined in the form of numerical ranges or percentage ranges are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have covered and specifically disclosed all possible secondary ranges and individual values ​​within the range, especially integer values. For example, the range description of "1 to 8" should be deemed to have specifically disclosed all secondary ranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., especially secondary ranges defined by all integer values, and should be deemed to have specifically disclosed individual values ​​within the range such as 1, 2, 3, 4, 5, 6, 7, 8, etc. Unless otherwise specified, the above explanation method applies to all contents of the present invention, whether the scope is broad or not. If the quantity or other numerical value or parameter is expressed as a range, a preferred range or a series of upper and lower limits, it should be understood that all ranges consisting of any upper limit or preferred value of the range and the lower limit or preferred value of the range have been specifically disclosed herein, regardless of whether these ranges are disclosed separately. In addition, if a numerical range is mentioned herein, unless otherwise specified, the range should include its endpoints and all integers and fractions within the range.

[0075] The following detailed description is merely illustrative in nature and is not intended to limit the present invention and its use. In addition, this document is not limited by any theory described in the above prior art or invention summary or the following detailed description or examples.

[0076] Example

[0077] The present disclosure will be further described in detail below through examples. The sources of raw materials and instruments used in the examples of the present disclosure are as follows:

[0078] Hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and distearoylphosphatidylethanolamine-polyethylene glycol-dibenzocyclooctyne (DSPE-PEG-DBCO) were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.; dimethylformamide solution and dichloromethane were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai); Resolvin E1 (abbreviated as RvE-1, CAS: 552830-51-0) was purchased from Cayman Company, USA; the lentivirus loaded with FasL gene overexpression plasmid was commissioned to Hanheng Biotechnology (Shanghai) Co., Ltd., which was obtained by introducing FasL gene into pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO plasmid vector (purchased from Hanheng Biotechnology Co., Ltd.) to obtain pHBLV-FASLG-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO; 1,3,4,6-tetra-O-acetyl N-azidoacetyl aminomannose (Ac4ManNAz) was purchased from Aladdin Technology (China) Co., Ltd.; 1640 culture Culture medium and fetal bovine serum (FBS) were purchased from Gibco, Canada; granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and lipopolysaccharide (LPS) were purchased from Peprotech, USA; phosphate buffered saline (PBS), 2,4,6-trinitrobenzenesulfonic acid (TNBS), and azomethane (AOM) were purchased from Dalian Meilun Biotechnology Co., Ltd.; dextran sulfate sodium (DSS) was purchased from Yishen Biotechnology Co., Ltd.; Anti-CD11b-FITC, Anti-Ly-6G-PerCp-Cy5.5, Anti-CD3-PerCp-Cy5.5, and Anti-CD8-PE were purchased from eBioscience, UK.

[0079] C57BL / 6 mice: 4-6 weeks old, 18-22 g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The breeding conditions and experimental procedures were strictly in accordance with the relevant requirements and standards of the Ethics Committee for the Management and Use of Laboratory Animals of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0080] Rotary evaporator (Heidlph, Germany); high pressure homogenizer (Microfluidizer, USA); transmission electron microscope (JEOL JEM-1230, Japan); laser particle size analyzer (ZEN3690, Malven, USA); desktop centrifuge (Eppendorf); carbon dioxide incubator (Thermo, USA); flow cytometer (BD, Calibur, USA);

[0081] Cell clean bench (BJ-1CD, Shanghai Boxun Industrial Co., Ltd.).

[0082] Example 1: Preparation of liposomes loaded with RvE-1

[0083] Accurately weigh 5 mg of hydrogenated soybean phosphatidylcholine (HSPC), 2.5 mg of cholesterol (Chol), 2.5 mg of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and 0.5 mg of distearoylphosphatidylethanolamine-polyethylene glycol-dibenzocyclooctyne (DSPE-PEG-DBCO) (the mass ratio of which is HSPC: Chol: DSPE-PEG: DSPE-PEG-DBCO=10:5:5:1), and dissolve them in 3 mL of dichloromethane; accurately weigh RvE-1 ethanol solution, the solution concentration is 50 μg / mL, the mass ratio of RvE-1 to HSPC is 1:400, and the ethanol solution of the anti-inflammatory drug RvE-1 and the lipid organic phase are evenly stirred. The organic solvent was removed by rotary evaporation under reduced pressure at 40°C for 1 hour to form a film; 5 mL of aqueous phase was added to the film-forming lipids and hydrated at 50°C for 1 hour; the obtained liposomes were then treated with a high-pressure homogenizer to obtain the final liposome preparation loaded with RvE-1.

[0084] The preparation of unloaded liposomes was carried out according to the same steps as above, except that the ethanol solution did not contain RvE-1.

[0085] Figure 2 The unloaded liposomes ( Figure 2 A) and liposomes loaded with RvE-1 (( Figure 1 (B) Transmission electron microscope photograph obtained by observation. Figure 3 This is a characterization diagram of the drug-loaded liposome particle size obtained using a laser particle size analyzer. Figure 2-3 It can be seen that the particle size of the unloaded liposomes is about 145nm, and the PDI value is 0.216; the particle size of the liposomes loaded with RvE-1 is 151.3±4.7nm, the particle size distribution is relatively uniform, and the PDI value is 0.258.

[0086] In addition, the prepared liposomes were placed at 4°C for one week and their particle size changes were observed daily. Figure 4, showing that the particle size of drug-loaded liposomes remained stable within one week.

[0087] Example 2: Construction and characterization of engineered dendritic cells

[0088] The FasL gene fragment (Gene ID: 14013) and the linearized vector were connected by homologous recombination or T4 ligation to obtain the FasL gene overexpression plasmid pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO. Subsequently, the purified FasL gene overexpression plasmid was obtained by transfecting competent cells, screening positive clones, gene sequencing, plasmid extraction and other steps. The plasmid structure is pHBLV-FASLG-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO.

[0089] Mononuclear cells were isolated from the tibia and humerus of C57BL / 6 mice (4-6 weeks old). The mononuclear cells were dispersed into 2×10 6 1 mL / well was added to a 12-well cell culture plate for 12 hours; the suspended cells and supernatant culture medium in each well were then aspirated and discarded, and 1640 culture medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 at concentrations of 20 ng / mL and 10 ng / mL, respectively, was added and cultured for 2-3 days in a 37°C cell culture incubator with a carbon dioxide concentration of 5%; the culture medium in the well was then replaced by half volume and cultured for another 2 days; on the 5th day, the suspended cells in the well were counted, and a lentivirus loaded with a FasL gene overexpression plasmid or a lentivirus without a plasmid was added to the culture medium at a multiplicity of infection (MOI) of 25, and 2 μL Polybrene was added (2 mg / mL), and continue to culture for 2 days; on the 7th day, the suspended dendritic cells were aspirated, centrifuged at 300g for 5 minutes at 4°C, and redispersed in physiological saline to form a cell suspension of 1 million / mL, thereby obtaining FasL-overexpressing dendritic cells (FasL-DC) and blank control dendritic cells (DC).

[0090] Dendritic cells overexpressing FasL (FasL-DC) were cultured, and 1640 medium with a final concentration of Ac4ManNAz of 50 μM was added to the culture medium for 3 days in a 37°C cell culture incubator with a carbon dioxide concentration of 5%; then, part of the suspended cells were aspirated, and the expression of N3 or FasL on the cell surface was detected by flow cytometry or immunofluorescence imaging. Figure 5Confocal fluorescence microscopy (CLSM) was used to observe the N3 group on the surface of engineered dendritic cells (FasL-DC), where the dendritic cells were labeled with DBCO-Cy5. The results showed that almost all cells expressed the N3 group. Figure 6 This is an immunofluorescence staining picture of FasL expression in engineered dendritic cells (FasL-DC), where FasL is labeled by Alexa Flour488 and DC is a blank control. The results show that after transfection with the FasL overexpression plasmid, the engineered dendritic cells can express FasL.

[0091] Another part of the suspended dendritic cells (FasL-DC) after co-culture with Ac4ManNAz for 3 days and the suspended dendritic cells (FasL-DC) not co-cultured with Ac4ManNAz were taken, centrifuged at 300g for 5 minutes at 4°C, and re-dispersed in physiological saline to a cell suspension of 1 million / mL; the cell suspension was placed in a 4°C refrigerator for pre-cooling for 30 minutes; then, liposomes loaded with RvE-1 (fluorescent probe DiR was encapsulated in the liposomes as an imaging molecule) were added to the cell suspension, wherein the liposomes were added at a ratio of 100 μg liposomes / 1 million cells, incubated in a 4°C refrigerator for 20 minutes, centrifuged at 300g for 5 minutes at 4°C, the supernatant was discarded, and the cell suspension was re-dispersed in physiological saline to a cell suspension of 1 million / mL, thus obtaining a liposome-coupled engineered dendritic cell preparation and a liposome-coupled blank control dendritic cell preparation. Immunofluorescence imaging technology was used to detect the coupling of dendritic cells to liposomes, and the results are shown in Figure 7 , in which FasL-DC without co-culture with Ac4ManNAz was used as blank control. The results showed that a large amount of liposomes were coupled to the surface of engineered dendritic cells, while almost no liposomes were coupled to the surface of blank control dendritic cells.

[0092] Example 3: Evaluation of the distribution of engineered dendritic cells in the mouse intestine

[0093] Using DiR as a fluorescent molecule, the fluorescent probe DiR was encapsulated in blank liposomes, and the liposomes were coupled to the surface of engineered dendritic cells according to the method described in Example 2. The liposomes and engineered dendritic cell preparations were given to mice by intraperitoneal injection, respectively. After 24 hours, the mice were imaged and analyzed using an in vivo optical imaging system (IVIS), and the distribution of the liposomes in the isolated colon was detected using a confocal fluorescence microscope (CLSM). The results are shown in Figure 2. Figure 8 As shown, a large amount of liposomes were distributed in the isolated colon of mice administered with the engineered dendritic cell preparation, while almost no liposomes were distributed in the isolated colon of mice administered with the liposome preparation.

[0094] Example 4: Study on the regulation of T cell and neutrophil infiltration in the intestinal inflammatory microenvironment of mice by engineered dendritic cells

[0095] C57BL / 6 mice were divided into 6 groups, 5 mice in each group. The healthy group was given drinking water for 7 days, and PBS was injected intraperitoneally on the 2nd, 4th and 6th days; the model group was given drinking water containing 3% (wt / vol) dextran sulfate sodium (DSS) for 7 days, and PBS, RvE-1 liposomes (Lipo), dendritic cells coupled to drug-loaded liposomes (DC-Lipo), FasL-overexpressing dendritic cells (FasL-DC) and engineered dendritic cells (FasL-DC-Lipo) were injected intraperitoneally on the 2nd, 4th and 6th days. 6 On the 8th day, the colon tissues of mice were collected, and the colon tissue lamina propria cells were separated using digestive fluid and lamina propria cell separation fluid. Anti-CD11b-FITC, Anti-Ly-6G-PerCp-Cy5.5, Anti-CD3-PerCp-Cy5.5, and Anti-CD8-PE were used for staining. The proportion of T cells and neutrophils in the colon tissues of mice was detected by flow cytometry. The results are shown in Fig. 9 As shown in 10, engineered dendritic cells can significantly reduce the proportion of T cells and neutrophils in the colon tissue of enteritis mice, and the proportion is reduced to a level similar to that of the healthy group.

[0096] Example 5: Pharmacodynamic evaluation of engineered dendritic cells in a DSS-induced acute enteritis model in mice

[0097] C57BL / 6 mice were divided into 6 groups, 5 mice in each group. The healthy group was given drinking water for 10 days, and PBS was injected intraperitoneally on the 2nd, 4th and 6th days respectively; the model group was given drinking water containing 3% (wt / vol) dextran sulfate sodium (DSS) for 7 days, and then changed to normal drinking water. On the 2nd, 4th and 6th days, PBS, drug-loaded liposomes (Lipo), dendritic cells coupled to drug-loaded liposomes (DC-Lipo), FasL-overexpressing dendritic cells (FasL-DC) and engineered dendritic cells (FasL-DC-Lipo) were injected intraperitoneally into the mice (liposome dosage was 100 μg / mouse, cell dosage was 2.5×10 6 The weight changes, fecal status, and fecal bleeding of mice were recorded every day, and the disease activity index (DAI) of each group was calculated according to the scoring table 1. Fig.11As shown, mice treated with engineered dendritic cells experienced less weight loss and decreased DAI scores, and compared with other groups, engineered dendritic cells were able to significantly alleviate the progression of DSS-induced colitis in mice.

[0098] Table 1

[0099]

[0100]

[0101] Example 6: Pharmacodynamic evaluation of engineered dendritic cells in the TNBS-induced acute enteritis model in mice

[0102] C57BL / 6 mice were divided into 6 groups, 5 mice in each group. The hair on the right back of C57BL / 6 mice was removed to expose about 1cm×1cm of skin. The healthy group was not treated; the model group used a pipette to slowly drip 150μL of 1% (wt / vol) 2,4,6-trinitrobenzenesulfonic acid (TNBS) aqueous solution on the back skin and gently spread it evenly. After 7 days, 150μL of 2.5% (wt / vol) TNBS aqueous solution was injected into the rectum of the mice, which was recorded as day 0. On days 2, 4, and 6, the model group used PBS, drug-loaded liposomes (Lipo), dendritic cells coupled to drug-loaded liposomes (DC-Lipo), FasL-overexpressing dendritic cells (FasL-DC), and engineered dendritic cells (FasL-DC-Lipo) to administer drugs to mice by intraperitoneal injection (liposome dosage was 100μg / mouse, cell dosage was 2.5×10 6 The weight changes, fecal status, and fecal bleeding of mice were recorded every day, and the disease activity index (DAI) of each group was calculated according to the scoring table. Fig.12 As shown, mice treated with engineered dendritic cells experienced less weight loss and decreased DAI scores, and compared with other groups, engineered dendritic cells were able to significantly alleviate the progression of TNBS-induced colitis in mice.

[0103] Example 7: Pharmacodynamic evaluation of engineered dendritic cells in a mouse inflammation-induced intestinal cancer model

[0104] C57BL / 6 mice were divided into 6 groups, 5 mice in each group. The healthy group was fed with normal drinking water. In the model group, C57BL / 6 mice were intraperitoneally injected with 100 μL of 10 mg / mL azomethane (AOM) aqueous solution, fed with normal drinking water for one week, and then fed with drinking water containing 2% (wt / vol) dextran sulfate sodium (DSS) for one week, and then fed with normal drinking water for 2 weeks; the above-mentioned normal drinking water and DSS drinking water replacement feeding steps were repeated 3 times; during the DSS drinking water feeding period, PBS, drug-loaded liposomes (Lipo), dendritic cells coupled to drug-loaded liposomes (DC-Lipo), FasL-overexpressing dendritic cells (FasL-DC) and engineered dendritic cells (FasL-DC-Lipo) were intraperitoneally injected into mice every two days (liposome dosage was 100 μg / mouse, cell dosage was 2.5×10 6 After the cycle, the colon tissue of the mice was taken to observe the number and size of tumor-like polyps on the inner wall of the colon. Fig.13 As shown, mice treated with engineered dendritic cells had a reduced number of colon tumors and a smaller tumor size, and engineered dendritic cells were able to significantly prevent the growth of intestinal inflammation-induced tumors compared to other groups.

Claims

1. An engineered dendritic cell preparation, comprising dendritic cells overexpressing FasL and liposomes encapsulating anti-inflammatory drugs coupled to the cell surface.

2. The engineered dendritic cell preparation according to claim 1, characterized in that: The surface of the liposomes encapsulating the anti-inflammatory drug comprises an alkynyl group, and the dendritic cells overexpressing FasL are coupled to the alkynyl groups on the surface of the liposomes encapsulating the anti-inflammatory drug via an azido sugar analog; Preferably, the azido sugar analog is introduced into the glycoprotein through the intracellular glycan biosynthesis pathway by co-culturing with dendritic cells overexpressing FasL, and then coupled with the alkyne group in the liposomes encapsulating the anti-inflammatory drug through the azide group; Preferably, the azido sugar analog is selected from one or more of 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose, N-azidoacetylmannosamine, N-azidoacetylglucosamine, and N-azidoacetylgalactosamine (GalNAz); more preferably, 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose; and / or The FasL-overexpressing dendritic cells are obtained by genetic engineering; Preferably, the genetic engineering method is to obtain dendritic cells containing a FasL gene overexpression plasmid; preferably, the FasL gene overexpression plasmid structure is pHBLV-FASLG-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO obtained by introducing the FasL gene into a pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO plasmid vector; and / or The liposomes encapsulating the anti-inflammatory drug contain hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and distearoylphosphatidylethanolamine-polyethylene glycol-dibenzocyclooctyne (DSPE-PEG-DBCO), and the mass ratio thereof is HSPC:Cholesterol:DSPE-PEG:DSPE-PEG-DBCO=(5-10):(1-5):(1-5):(0.5-1); Preferably, the anti-inflammatory drug encapsulated in the liposome encapsulating the anti-inflammatory drug is selected from resolvin, acetaminophen, salicylic acid, 6-mercaptopurine, preferably, the anti-inflammatory drug is selected from one or more of Resolvin E1, Resolvin D1, Resolvin D2, Resolvin D3, preferably Resolvin E1; Preferably, the mass ratio of the anti-inflammatory drug to hydrogenated soybean phosphatidylcholine (HSPC) is 1:(300-500); and / or In the engineered dendritic cell preparation, the ratio of liposomes to dendritic cells is 50-300 μg liposomes / 1 million cells, preferably 50-150 μg liposomes / 1 million cells.

3. A method for preparing FasL-overexpressing dendritic cells, which are obtained by transfecting dendritic cells with a lentiviral vector, an adenoviral vector or a cationic lipid vector loaded with a FasL gene overexpression plasmid; Preferably, the method for preparing FasL-overexpressing dendritic cells comprises the following steps: (1) constructing a FasL gene overexpression plasmid and packaging it using a lentiviral vector, adenoviral vector or cationic lipid vector; (2) Inducing the production of dendritic cells from monocytes from blood or bone marrow using granulocyte-macrophage colony-stimulating factor and IL-4; (3) Transfecting the FasL gene overexpression plasmid packaged by the lentiviral vector, adenoviral vector or cationic lipid vector in (1) into the dendritic cells induced in step (2) to obtain FasL overexpressing dendritic cells.

4. The method for preparing FasL-overexpressing dendritic cells according to claim 3, characterized in that: In step (1): The FasL gene overexpression plasmid is constructed by genetic engineering; preferably, the FasL gene fragment and the linearized vector are connected by homologous recombination or T4 connection to obtain the FasL gene overexpression plasmid; then, the purified FasL gene overexpression plasmid is obtained by transfecting competent cells, screening positive clones, gene sequencing, and plasmid extraction steps; or, the FasL gene overexpression plasmid is pHBLV-FASLG-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO obtained by introducing the FasL gene into the pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO plasmid vector; and / or In step (2): The mononuclear cells from blood or bone marrow are mononuclear cells from the patient's own blood or bone marrow; and / or The concentrations of granulocyte-macrophage colony stimulating factor and IL-4 used to induce monocytes to produce dendritic cells are 10-100 ng / mL, preferably 20 ng / mL for granulocyte-macrophage colony stimulating factor and 10 ng / mL for IL-4; Preferably, the specific steps of inducing monocytes to generate dendritic cells include: The mononuclear cells were dispersed into 1 × 10 cells using complete medium containing 10% serum. 6 -1×10 7 / mL, adherent culture for 8-24 hours; then aspirate the suspended cells and supernatant culture medium, add complete cell culture medium containing GM-CSF and IL-4 at concentrations of 10-100 ng / mL respectively for induction culture, the induction culture conditions are 37°C, 5% carbon dioxide; wherein, half volume of the medium is replaced every 2 days and culture is continued; and / or In step (3): The specific steps of transfecting the packaged FasL gene overexpression plasmid into the dendritic cells induced in step (2) include: On the 5th day of monocyte induction culture, the suspended cells are counted, and a lentiviral vector, adenoviral vector or cationic lipid vector loaded with a FasL gene overexpression plasmid with a multiplicity of infection MOI = 15-40, preferably MOI = 20-30 is added to the culture medium, and a transfection-promoting reagent Polybrene is added, and the culture is continued for 2 days; on the 7th day, the suspended dendritic cells are aspirated, centrifuged at 100-500g for 3-30 minutes at 4°C-10°C, and redispersed in physiological saline to a cell suspension of 1 million-10 million / mL, thereby obtaining FasL-overexpressing dendritic cells. 5 . The FasL-overexpressing dendritic cells prepared by the method for preparing FasL-overexpressing dendritic cells according to claim 3 or 4 .

6. A method for preparing liposomes encapsulating anti-inflammatory drugs, comprising the following steps: (i) taking liposome materials hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and distearoylphosphatidylethanolamine-polyethylene glycol-dibenzocyclooctyne (DSPE-PEG-DBCO) in a mass ratio of HSPC:Cholesterol:DSPE-PEG:DSPE-PEG-DBCO=(5-10):(1-5):(1-5):(0.5-1), and dissolving them in an organic phase; uniformly mixing the ethanol solution of the anti-inflammatory drug with the organic phase in which the liposome material is dissolved to form a mixed solution; (ii) subjecting the mixed solution to reduced pressure rotary evaporation to remove the organic solvent to form a film; adding an aqueous phase to the film-forming lipids and hydrating them at 30° C. to 90° C.; and then treating them under high pressure or high shear conditions to obtain the liposomes encapsulating the anti-inflammatory drug; Preferably, in step (i): The organic phase is selected from one or more of ethanol, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, dichloromethane, chloroform, and acetone, preferably dichloromethane; and / or, The dosage ratio of the hydrogenated soybean phosphatidylcholine (HSPC) to the organic phase is (2-8) mg: 3 mL; The anti-inflammatory drug is selected from resolvin, acetaminophen, salicylic acid, 6-mercaptopurine, etc., preferably one or more of Resolvin E1, Resolvin D1, Resolvin D2, Resolvin D3, and more preferably Resolvin E1; preferably, the concentration of the anti-inflammatory drug in the ethanol solution of the anti-inflammatory drug is 40-60 μg / mL; and / or The mass ratio of the anti-inflammatory drug to hydrogenated soybean phosphatidylcholine (HSPC) is 1:(300-500); Preferably, in step (ii): The temperature of the reduced pressure rotary evaporation is in the range of 35 to 45° C. and the time is 0.5 to 3 hours; and / or The ratio of hydrogenated soybean phosphatidylcholine (HSPC) to the added aqueous phase is (0.5-2) mg: 1 mL; and / or The hydration temperature is 40-60°C, the hydration time is 0.5-2h, preferably 1h; and / or The high pressure or high shear force conditions are: homogenization under the conditions of a high pressure homogenizer, an ultrasonic crusher, a high shear mixer or a high shear agitator; and / or The particle size of the liposome encapsulating the anti-inflammatory drug is 100 to 200 nm, preferably 140 to 169 nm.

7. The liposomes encapsulating anti-inflammatory drugs prepared by the method for preparing liposomes encapsulating anti-inflammatory drugs according to claim 6.

8. The method for preparing the engineered dendritic cell preparation according to claim 1 or 2, comprising the following steps: S1: Dendritic cells overexpressing FasL were co-cultured with azidosugar analogs; S2: After co-culture, the suspended dendritic cells are aspirated, the dendritic cells are separated by centrifugation at 4°C-10°C, and the dendritic cells are redispersed in physiological saline to form a cell suspension; then, the liposomes encapsulating the anti-inflammatory drug are added to the cell suspension pre-cooled at 4°C, and incubated at 4°C for coupling; after the incubation is completed, the cells are separated by centrifugation at 4°C-10°C, and redispersed in physiological saline to obtain an engineered dendritic cell preparation.

9. The method for preparing the engineered dendritic cell preparation according to claim 8, characterized in that: In step S1: In the co-culture, the concentration of the azido sugar analog is 30-60 μM; preferably, the azido sugar analog is selected from one or more of 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose, N-azidoacetylmannosamine, N-azidoacetylglucosamine, and N-azidoacetylgalactosamine; preferably 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose; and / or The co-culture conditions are: 37°C, 5% carbon dioxide concentration; the co-culture time is 3 days; and / or In step S2: Centrifugation at 4°C-10°C at 100-500 g for 3-30 minutes; and / or Redispersing in physiological saline to form a cell suspension of 0.5-10 million cells / mL, preferably 0.5-2 million cells / mL; and / or Precooling the cell suspension: placing it in a 4°C refrigerator for 20-30 minutes; and / or The liposomes encapsulating the anti-inflammatory drug are added at a ratio of 50-300 μg liposomes / 1 million cells, preferably 50-150 μg liposomes / 1 million cells; and / or After adding the liposomes encapsulating the anti-inflammatory drug to the pre-cooled cell suspension, incubate in a 4°C refrigerator for 20-30 minutes; and / or the cell concentration in the engineered dendritic cell preparation is 500,000-10 million cells / mL, preferably 500,000-2 million cells / mL.

10. Use of the engineered dendritic cells according to claim 1 or 2, the FasL-overexpressing dendritic cells according to claim 5, and / or the anti-inflammatory drug-encapsulated liposomes according to claim 7 in the preparation of drugs for treating, improving and / or preventing inflammatory bowel disease and enteritis-related tumors; Preferably, the inflammatory bowel disease is: ulcerative colitis, Crohn's disease, viral enteritis, bacterial enteritis or indeterminate colitis; Preferably, the enteritis-related tumor is: adenomatous polyps or tumor-like lesions induced by chronic colitis.

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