Preparation method of STING agonist and MR1 antibody co-loaded MCC nano drug delivery system for enhancing tumor immunotherapy

Abstract

The invention relates to the technical field of pharmaceutical preparations, and particularly discloses a preparation method of an MCC nano drug delivery system co-carrying an STING agonist and an MR1 antibody and used for enhancing tumor immunotherapy. Carboxymethyl chitosan is used as a hydrophilic saccharide skeleton, and conjugated linoleic acid hydrophobically modifies the carboxymethyl chitosan through an EDC mediated reaction; the preparation method comprises the following steps: grafting D-mannose activated by acetic acid and a conjugated linoleic acid-carboxymethyl chitosan polymer to obtain an MCC nano-carrier; through ultrasonic emulsification, the MCC nano-carrier encapsulates the STING agonist and the MR1 antibody to obtain the MCC nano-drug release system. The nano drug release system is transdermally delivered into the body of a tumor patient, the MCC nano particles can reach the tumor part under the targeted guidance of mannose groups, the targeted immune cells release the STING agonist so as to positively activate the immune response reaction, and meanwhile, the MR1 antibody released by the MCC nano particles can inhibit the immune escape phenomenon of the tumor cells so as to activate the immune response reaction. And the immune response reaction is reversely activated, so that the bidirectional synergistic activation of tumor immunity is finally realized.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a method for preparing an MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy. Background Art

[0002] Interferon response gene (STING) protein agonists can enhance the body's inherent immune response and activate immune memory cells to exert the advantages of long-term immune memory effect. Using the body's own immune response to kill and inhibit tumor cells is one of the current ways of tumor treatment. Therefore, STING agonists are used as potential target drugs for the treatment of malignant tumor recurrence and metastasis, which can positively activate the tumor's own immune function.

[0003] MR1 is an immune molecule whose main function is to activate MAIT cells (regulatory T cells). The antigen peptide presented by MR1 binds to the T cell receptor (TCR) of MAIT cells, thereby activating these cells and triggering an immune response. The MR1 molecule on the surface of tumor cells is an inhibitory molecule of MAIT cells (regulatory T cells), which promotes the immune escape of tumor cells and reduces the killing effect on tumor cells. The MR1 antibody drug designed for this purpose can block the immune escape of tumor cells and reversely activate the tumor's own immune function.

[0004] However, STING agonists are not efficient in passing through the phospholipid bilayer and are difficult to directly bind to the STING protein on the endoplasmic reticulum membrane to exert their effects. Moreover, if they do not completely enter the tumor microenvironment, there is a risk of inducing systemic inflammation. When MR1 antibody drugs are directly injected, there are problems such as immunogenicity and pharmacokinetics. MR1 antibodies are not only easily identified as foreign substances and quickly cleared, but also have the problem of being off-target and unable to effectively reach target cells. Summary of the invention

[0005] The purpose of the present invention is to solve the above technical problems and provide a method for preparing an MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy. The stability of the drug and the efficiency of entering cells are improved by co-loading the STING agonist and the MR1 antibody drug.

[0006] In order to solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy, comprising the following steps:

[0007] S1: Carboxymethyl chitosan is used as the hydrophilic sugar backbone, and conjugated linoleic acid is used to hydrophobically modify carboxymethyl chitosan through an EDC-mediated reaction to obtain a conjugated linoleic acid-carboxymethyl chitosan polymer;

[0008] S2: using acetic acid activated D-mannose to graft with conjugated linoleic acid-carboxymethyl chitosan polymer to obtain MCC nanosuspension crude product, and then dialyzing and freeze-drying to obtain MCC nanocarrier;

[0009] S3: The STING agonist and MR1 antibody were encapsulated in MCC nanocarriers through ultrasonic emulsification, and then dialyzed and filtered to obtain the MCC nanodrug delivery system with bidirectional synergistic activation of tumor autoimmunity.

[0010] As a further optimization of the preparation method of the MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: the specific method of the EDC-mediated reaction in step S1 is: pouring methanol containing conjugated linoleic acid and methanol dissolved in EDC into an aqueous solution dissolved in carboxymethyl chitosan, and continuously stirring the reaction.

[0011] As a further optimization of the preparation method of the MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: the preparation method of acetic acid-activated D-mannose in step S2 is: stirring and dissolving mannose in acetic acid.

[0012] As a further optimization of the preparation method of the MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: the concentration of mannose is 0.005-0.015 g / mL.

[0013] As a further optimization of the preparation method of the MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: the specific method of grafting D-mannose with conjugated linoleic acid-carboxymethyl chitosan polymer in step S2 is: under stirring, adding activated D-mannose acetic acid to conjugated linoleic acid-carboxymethyl chitosan polymer, and continuously stirring the reaction for 24-48 hours.

[0014] As a further optimization of the preparation method of the MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: in the step S2, the crude MCC nano suspension is dialyzed against anhydrous ethanol and distilled water in sequence to remove unreacted substances, cross-linking agents and by-products, and finally the dialyzate is freeze-dried to obtain the MCC nanocarrier.

[0015] As a further optimization of the preparation method of the MCC nano drug delivery system for co-loading STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: the step S3 is specifically: adding MCC nanocarriers into distilled water, and then sequentially adding STING agonist drugs and MR1 antibody drugs, completing the encapsulation of drugs by MCC nanocarriers through ultrasonic emulsification, and then sequentially dialyzing against anhydrous ethanol and distilled water to remove unencapsulated drugs and small molecule impurities; filtering through a filter membrane to remove macromolecular nanoparticles, and obtaining an MCC nano drug release system for co-loading STING agonist and MR1 antibody with bidirectional synergistic activation of tumor autoimmunity.

[0016] As a further optimization of the preparation method of the MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: MCC nanocarrier is added to distilled water at a concentration of 0.5-1.5 mg / mL, STING agonist drug is added to the mixed solution at a concentration of 0.02-0.08 mg / mL, and MR1 antibody drug is added to the mixed solution at a concentration of 0.15-0.35 μL / mL.

[0017] As a further optimization of the preparation method of the MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody for enhancing tumor immunotherapy of the present invention: the ultrasonic emulsification conditions are: ultrasonic emulsification three times, each time for 5-15 minutes, and a power of 300-400W.

[0018] The present invention also provides an MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy. The MCC nano drug delivery system is prepared by the above method.

[0019] The present invention has the following beneficial effects:

[0020] 1. The present invention integrates the dual mechanisms of STING immune enhancement and MR1 immune escape to design a bidirectional synergistic tumor immunotherapy combination strategy. By delivering the nano drug delivery system percutaneously to tumor patients, under the targeted guidance of the mannose group, the MCC nanoparticles will reach the tumor site and release the STING agonist in the targeted immune cells to positively activate the immune response. At the same time, the MR1 antibody released by the MCC nanoparticles will inhibit the immune escape of tumor cells to reversely activate the immune response, ultimately achieving bidirectional synergistic activation of tumor immunity.

[0021] Second, the present invention uses mannose receptor as the target group for nanosystem delivery of agonists. Mannose receptor, as an immune adhesion molecule, is mainly expressed on the surface of immature DC cells. D-mannose is used to modify the existing CC coupling compound and then prepare a multifunctional nanocarrier, which can target the delivery of agonists into DC cells, activate the STING signaling pathway, induce the expression of type I IFN, and enhance the anti-tumor effect of T cells. diABZI is a diaminobenzimidazole drug with a small molecular weight and strong hydrophobicity. It belongs to the small molecule STING agonist class of drugs. It is suitable for loading into the self-assembled MCC polysaccharide nanocarrier by hydrophobic force and covalent bonding to further activate the STING signaling pathway; while MR1 antibody, as a monoclonal antibody, has a large molecular weight, much larger than small molecule drugs, and a complex structure. It can be grafted onto the MCC polysaccharide skeleton by covalent bonding; finally, MCC nanocarrier, as an amphiphilic structure, can self-assemble into nanoparticles in the aqueous phase, realizing the co-loading of diABZI and MR1 antibody drugs and the release of drugs under the pH response of the tumor microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The infrared spectrum of MCC nanocarriers detected by FTIR infrared spectroscopy;

[0023] Figure 2 Transmission electron microscopy image and particle size analysis of MCC nanosuspension;

[0024] Figure 3 This is the particle size analysis data diagram of the MCC nano drug delivery system;

[0025] Figure 4 This is a fluorescent microscopic merged image of DC cells after the MCC nano drug delivery system labeled with FITC fluorescence at different concentrations acted on them;

[0026] Figure 5 The results of toxicity tests on DC cells after different concentrations of MCC nano drug delivery system acted on them;

[0027] Figure 6 This is a picture of cell apoptosis after DC cells were acted on by MCC nano drug delivery system with different concentrations;

[0028] Figure 7 The concentration of TNF-α released by DC cells treated with MCC nano drug delivery system at different concentrations

[0029] Figure 8 This is the OD value diagram of the effect of different concentrations of MCC nano drug delivery system on the secretion of TNF-α factor by DC cells;

[0030] Fig. 9 This is an OD value graph showing the effect of different concentrations of MCC nano-drug delivery system on the secretion of IFN-γ factor by DC cells. DETAILED DESCRIPTION

[0031] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with embodiments, but the content of the present invention is not limited to the following embodiments.

[0032] A method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy, comprising the following steps:

[0033] S1: Carboxymethyl chitosan is used as a hydrophilic sugar skeleton, and conjugated linoleic acid is hydrophobically modified to carboxymethyl chitosan through an EDC-mediated reaction to obtain a conjugated linoleic acid-carboxymethyl chitosan polymer.

[0034] The specific method of the EDC-mediated reaction is: pouring methanol containing conjugated linoleic acid and methanol dissolving EDC into an aqueous solution dissolving carboxymethyl chitosan, and continuously stirring the reaction.

[0035] S2: using acetic acid activated D-mannose to graft with conjugated linoleic acid-carboxymethyl chitosan polymer to obtain MCC nanosuspension crude product, and then dialysis and freeze-drying to obtain MCC nanocarrier.

[0036] The preparation method of acetic acid-activated D-mannose is as follows: mannose is dissolved in acetic acid by stirring (the concentration of mannose is 0.005-0.015 g / mL).

[0037] The specific method of grafting is: under stirring, adding activated D-mannose acetic acid to conjugated linoleic acid-carboxymethyl chitosan polymer, and continuously stirring and reacting for 24-48 hours.

[0038] The specific treatment method of the crude MCC nanosuspension is: perform dialysis against anhydrous ethanol and distilled water in sequence to remove unreacted substances, cross-linking agents and by-products, and finally freeze-dry the dialyzate to obtain the MCC nanocarrier.

[0039] The reaction formula for covalent coupling and mannosylation of carboxymethyl chitosan (CMC) and conjugated linoleic acid (CLA) is as follows:

[0040]

[0041] S3: The STING agonist and MR1 antibody were encapsulated in MCC nanocarriers through ultrasonic emulsification, and then dialyzed and filtered to obtain the MCC nanodrug delivery system with bidirectional synergistic activation of tumor autoimmunity.

[0042] Specifically, the MCC nanocarrier is added into distilled water, and then the STING agonist drug and the MR1 antibody drug are added in sequence. After ultrasonic emulsification, the drug is encapsulated by the MCC nanocarrier. Then, the drug is dialyzed against anhydrous ethanol and distilled water in sequence to remove the unencapsulated drug and small molecule impurities. The macromolecular nanoparticles are removed by filtration through a filter membrane to obtain the MCC nanodrug delivery system that co-loads the STING agonist and the MR1 antibody and has the function of bidirectionally synergistically activating tumor autoimmunity.

[0043] Among them, MCC nanocarriers are added to distilled water at a concentration of 0.5-1.5 mg / mL, STING agonist drugs are added to the mixed solution at a concentration of 0.02-0.08 mg / mL, and MR1 antibody drugs are added to the mixed solution at a concentration of 0.15-0.35 μL / mL.

[0044] The conditions for ultrasonic emulsification are: three times of ultrasonic emulsification, each time for 5-15 minutes, and a power of 300-400W.

[0045] During tumor immunotherapy, STING agonist therapy can be used to activate immune cells. However, STING agonists are poorly lipid-soluble and difficult to pass through cell membranes, and are easily off-target and easily degraded, resulting in poor immune response. By loading them onto nanocarriers with uniform size, stable chemical properties and targeting immune cells, the MCC nano drug delivery system can effectively overcome the above limitations and enhance the immune response.

[0046] During tumor immunotherapy, MR1 antibody therapy can be used to block the immune escape of tumor cells. However, when MR1 antibody drugs are directly injected, there are problems such as immunogenicity and pharmacokinetics. MR1 antibodies are not only easily identified as foreign substances and quickly cleared, but also have the problem of being off-target and unable to effectively reach target cells. By loading it onto a nanocarrier with uniform size, stable chemical properties and immune cell targeting, it is prepared into an MCC nano drug delivery system, which can effectively overcome the above limitations and enhance the immune response.

[0047] Although the MCC nano drug delivery system alone carries a STING agonist, it can enhance the immune response. However, the MR1 molecule on the surface of tumor cells is an inhibitory molecule of MAIT cells (regulatory T cells), which will promote the immune escape of tumor cells and reduce the killing effect on tumor cells. Antibody drugs targeting MR1 can significantly inhibit the immune escape of tumors. On the one hand, STING agonists activate immune responses; on the other hand, MR1 antibodies inhibit the immune escape of tumors. By integrating agonist therapy and immune checkpoint blockade therapy, MCC nanocarriers are co-loaded with STING agonists and MR1 antibodies to prepare an MCC nano drug delivery system. During tumor immunotherapy, it can effectively target target cells and achieve bidirectional synergistic activation of tumor autoimmunity.

[0048] <Example 1>

[0049] Weigh 1.0g of natural polysaccharide carboxymethyl chitosan (CMCS) and completely dissolve it in 100ml of distilled water, measure 1ml (conjugated linoleic acid) CLA and add it to a 100ml beaker, then add 85ml of methanol, shake well and add it to the CMCS solution, stir evenly. Take 0.5g EDC and add it to 15ml of methanol, stir evenly and add it to the mixed solution, stir magnetically for 24h, and obtain the conjugated linoleic acid-carboxymethyl chitosan polymer (CC) reaction solution.

[0050] Weigh 0.1 g of D-mannose and add it to 10 ml of acetic acid solution. After it is completely dissolved, stir and add it to conjugated linoleic acid-carboxymethyl chitosan polymer (CC). Continue stirring and activating for 36 hours to complete the D-mannose modification, and obtain the crude MCC nanosuspension.

[0051] The crude MCC nanosuspension was dialyzed with anhydrous ethanol for 24 hours. Then, it was dialyzed with distilled water for 48 hours, during which the distilled water was changed several times to remove unreacted substances, cross-linking agents and by-products. The obtained dialysate was freeze-dried to finally obtain the freeze-dried product of the MCC nano drug delivery system.

[0052] The lyophilized product of MCC nanocarrier was placed in distilled water at a concentration of 1 mg / ml, and STING agonist drugs and MR1 antibody drugs were added in sequence at concentrations of 0.05 mg / mL and 0.25 μL / mL, respectively. Ultrasonic emulsification (350W, 10 min each time, three times in total) was performed under ice-water bath conditions to achieve drug encapsulation by MCC nanocarrier. The drug was then dialyzed against anhydrous ethanol and distilled water to remove unencapsulated drugs and small molecule impurities; the drug was filtered through a filter membrane (0.22 μm) to remove nanoparticles with larger molecules. Finally, an MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody was prepared.

[0053] like Figure 1As shown: NEXUS470 infrared spectrometer was used to detect the MCC nano drug delivery system by infrared spectrum: According to the literature review, the peak of the ester group is at 1700cm -1 After testing, the prepared MCC nanocarrier has a wavelength of 1700 cm -1 The vibration peak begins to appear around 1738.07cm -1 The peak value was reached, proving that D-mannose had been covalently bound to the CC coupler through an ester bond, completing the mannosylation modification of the CC coupler.

[0054] like Figure 2 , 3 As shown: The size of MCC nanocarriers is uniform, the Z-Average average particle size is about 235.9nm, and the polydispersity is 27%. The nanoparticles under the electron microscope are approximately round and have a complete shape, proving that they present a uniform spherical structure. The surface of the nanoparticles is smooth without obvious protrusions. The distribution on the substrate is relatively uniform, without obvious aggregation.

[0055] The MCC nano drug delivery system was fluorescently labeled with FITC fluorescent dye. DC cells were treated with different concentrations of MCC nano drug delivery system for 8 hours, and the effect of MCC nanoparticles on DC cells was analyzed based on the fluorescence photos. After 8 hours, the wells of the cell culture dish were rinsed with PBS, and randomly selected areas were excited under 488nm wavelength blue light and fluorescence photos were taken.

[0056] like Figure 4 As shown: the blank control group did not excite green light, while in the experimental groups with MCC concentrations of 0.2 and 0.4, almost all active DC cells excited green fluorescence. This indicates that MCC nanoparticles have a certain degree of DC cell targeting, so that they can enter the cells; it also shows that DC cells have a good uptake ability for MCC nanoparticles.

[0057] DC cells were evenly inoculated in a 96-well plate, and a control group and five gradient experimental groups were set up, with 10 replicates in each group. Subsequently, the control group and experimental group cells were treated with different concentrations of MCC nano-drug delivery system. After 8 hours, the old culture medium and MCC nano-drug delivery system were removed and rinsed with PBS. Finally, CCK-8 solution was added and incubated in a 37°C constant temperature incubator for another 2 hours. After 2 hours, the OD value was detected at a wavelength of 450nm.

[0058] like Figure 5As shown: Taking the blank control group (MCC nano drug delivery system concentration = 0) as the benchmark, the results show that the OD values ​​of the five experimental groups with different gradients are all higher than the control group, which indicates that the MCC nano drug delivery system has no toxicity or no significant toxicity to DC cells. In the concentration range of 0-0.2, as the MCC concentration increases, the OD value gradually increases; in the concentration range of 0.2-0.5, as the MCC concentration increases, the OD value gradually decreases, but it is always higher than the control group. In contrast to the toxic effect, this indicates that a certain concentration of MCC nano drug delivery system may promote the proliferation of DC cells, and the specific conclusions and mechanisms need to be verified.

[0059] DC cells were evenly inoculated in a 6-well plate, and a control group and 5 gradient experimental groups were set up. Then, the control group and experimental group cells were treated with different concentrations of MCC nano-drug delivery system. After 8 hours, the old culture medium and MCC nano-drug delivery system were removed and rinsed with PBS. Finally, AOEB double staining reagent was added and incubated in a 37°C constant temperature incubator for 20 minutes. Cells were stimulated with green light and blue light sources to detect cell apoptosis. Red fluorescence represents apoptotic cells, and green fluorescence represents cells in an active state.

[0060] like Figure 6 As shown in the figure, only a very small number of cells in both the control group and the experimental group were apoptotic cells, while the vast majority of DC cells were in a normal active state, indicating that the MCC nano drug delivery system had no effect on cell apoptosis.

[0061] TNF-α is a cytokine secreted by DC cells, which has multiple functions in immune response, such as promoting inflammatory response and regulating the proliferation of immune cells. The cGAS-STING signaling pathway is a natural immune signaling pathway present in cells. The activation of the pathway can cause cells to produce immune responses and then secrete a variety of cytokines, including TNF-α and INF-γ. STING agonists can effectively enhance immune responses.

[0062] First, the standard curve was drawn using the standard substance in the ELISA kit for detecting TNF-α cytokines. 2 =1, the curve is available. Then, DC cells were evenly inoculated in the microplate of the ELISA kit, and a control group and 5 gradient experimental groups were set up, with 2 replicates in each group. The control group and experimental group cells were treated with different concentrations of MCC nano drug delivery system carrying STING agonist. After 8 hours, the cell culture supernatant was taken to detect the secretion of TNF-α factor by DC cells under different MCC nano concentrations.

[0063] like Figure 7As shown: With the increase of MCC concentration, MCC concentration is positively correlated with the secretion of TNF-α. Substituting the OD values ​​of the control group and the experimental group into the standard curve, it can be calculated that after the DC cells were treated with MCC nanoparticles at a concentration of 0.5, the concentration of TNF-α factor in the cell culture supernatant was about 243.9ng / L. Compared with the control group (133.5ng / L), the secretion of TNF-α increased by about 83%, proving that MCC nanoparticles carrying STING agonists have a very significant effect on enhancing the immune response of DC cells.

[0064] The effect of different MCC nano concentrations on the secretion of TNF-α by DC cells was determined by OD values. Using the ELISA kit for detecting TNF-α, at a specific wavelength, the higher the OD value of the microwell, the higher the concentration of TNF-α cytokine. The OD value can be substituted into the TNF-α standard curve to obtain the specific TNF-α concentration, see Figure 8 .

[0065] IFN-γ cytokine is another cytokine secreted by DC cells in the immune response, and it also plays a key role in the stimulation and regulation of the immune system. Therefore, the concentration of IFN-γ secreted by DC cells can also be used as one of the indicators to determine whether the immune response of DC cells is enhanced. Using an ELISA kit for detecting IFN-γ, at a specific wavelength, the higher the OD value of the microwell, the higher the concentration of IFN-γ cytokine. DC cells were evenly inoculated in the microplate of the ELISA kit, and a control group and two gradient experimental groups were set up, with 2 replicates in each group. The control group and experimental group cells were treated with different concentrations of MCC nano drug delivery system carrying STING agonists. After 8 hours, the cell culture supernatant was taken to detect the secretion of IFN-γ factor by DC cells under different MCC nano concentrations.

[0066] like Fig. 9 As shown: With the increase of MCC concentration, the OD value gradually increased, and the MCC concentration was positively correlated with the secretion of IFN-γ. It also proved that MCC nanoparticles carrying STING agonists have a significant effect on enhancing the immune response of DC cells.

[0067] <Example 2>

[0068] Weigh 1.0g of natural polysaccharide carboxymethyl chitosan (CMCS) and completely dissolve it in 100ml of distilled water, measure 1ml (conjugated linoleic acid) CLA and add it to a 100ml beaker, then add 85ml of methanol, shake well and add it to the CMCS solution, stir evenly. Take 0.5g EDC and add it to 15ml of methanol, stir evenly and add it to the mixed solution, stir magnetically for 24h, and obtain the conjugated linoleic acid-carboxymethyl chitosan polymer (CC) reaction solution.

[0069] Weigh 0.05 g of D-mannose and add it to 10 ml of acetic acid solution. After it is completely dissolved, stir and add it to conjugated linoleic acid-carboxymethyl chitosan polymer (CC). Continue stirring and activating for 24 hours to complete the D-mannose modification, and obtain the crude MCC nanosuspension.

[0070] The crude MCC nanosuspension was dialyzed with anhydrous ethanol for 24 hours. Then, it was dialyzed with distilled water for 48 hours, during which the distilled water was changed several times to remove unreacted substances, cross-linking agents and by-products. The obtained dialysate was freeze-dried to finally obtain the freeze-dried product of the MCC nano drug delivery system.

[0071] The lyophilized product of MCC nanocarrier was placed in distilled water at a concentration of 0.5 mg / ml, and STING agonist drugs and MR1 antibody drugs were added in sequence at concentrations of 0.08 mg / mL and 0.15 μL / mL, and ultrasonic emulsification (400W, 5 min each time, three times in total) was performed under ice-water bath conditions to achieve drug encapsulation by MCC nanocarrier. Then, it was dialyzed against anhydrous ethanol and distilled water in turn to remove unencapsulated drugs and small molecule impurities; it was filtered through a filter membrane (0.22 μm) to remove nanoparticles with larger molecules. Finally, an MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody was prepared.

[0072] <Example 3>

[0073] Weigh 1.0g of natural polysaccharide carboxymethyl chitosan (CMCS) and completely dissolve it in 100ml of distilled water, measure 1ml (conjugated linoleic acid) CLA and add it to a 100ml beaker, then add 85ml of methanol, shake well and add it to the CMCS solution, stir evenly. Take 0.5g EDC and add it to 15ml of methanol, stir evenly and add it to the mixed solution, stir magnetically for 24h, and obtain the conjugated linoleic acid-carboxymethyl chitosan polymer (CC) reaction solution.

[0074] Weigh 0.15 g of D-mannose and add it to 10 ml of acetic acid solution. After it is completely dissolved, stir and add it to conjugated linoleic acid-carboxymethyl chitosan polymer (CC). Continue stirring and activating for 48 hours to complete the D-mannose modification, and obtain the crude MCC nanosuspension.

[0075] The crude MCC nanosuspension was dialyzed with anhydrous ethanol for 24 hours. Then, it was dialyzed with distilled water for 48 hours, during which the distilled water was changed several times to remove unreacted substances, cross-linking agents and by-products. The obtained dialysate was freeze-dried to finally obtain the freeze-dried product of the MCC nano drug delivery system.

[0076] The lyophilized product of MCC nanocarrier was placed in distilled water at a concentration of 1.5 mg / ml, and STING agonist drugs and MR1 antibody drugs were added in sequence at concentrations of 0.02 mg / mL and 0.35 μL / mL, and ultrasonic emulsification (300W, 15 min each time, three times in total) was performed under ice-water bath conditions to achieve drug encapsulation by MCC nanocarrier. Then, it was dialyzed against anhydrous ethanol and distilled water in turn to remove unencapsulated drugs and small molecule impurities; it was filtered through a filter membrane (0.22 μm) to remove nanoparticles with larger molecules. Finally, an MCC nano drug delivery system co-loaded with STING agonist and MR1 antibody was prepared.

[0077] Drugs and reagents used in the preparation of nano drug delivery system: Carboxymethylchitosan (low molecular weight, Sigma); conjugated linoleic acid (Qingdao Aohai Biological Co., Ltd.); 1-ethyl-3-(3-dimethylpropyl)-carbodiimide (EDC); D-mannose (Chengdu Mansite Biotechnology Co., Ltd.); acetic acid (Zibo Lishuo Chemical Co., Ltd.); dialysis bag (molecular weight cutoff is 3000Da); STING agonist diABZI, MR1 antibody, and others such as anhydrous methanol, anhydrous ethanol and hydrochloric acid (Tianjin Yongda Chemical Reagent Co., Ltd.).

[0078] The test equipment used is detailed as follows:

[0079] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy, characterized in that: The following steps are involved: S1: Carboxymethyl chitosan is used as the hydrophilic sugar backbone, and conjugated linoleic acid is used to hydrophobically modify carboxymethyl chitosan through an EDC-mediated reaction to obtain a conjugated linoleic acid-carboxymethyl chitosan polymer; S2: using acetic acid activated D-mannose to graft with conjugated linoleic acid-carboxymethyl chitosan polymer to obtain MCC nanosuspension crude product, and then dialyzing and freeze-drying to obtain MCC nanocarrier; S3: The STING agonist and MR1 antibody were encapsulated in MCC nanocarriers through ultrasonic emulsification, and then dialyzed and filtered to obtain the MCC nanodrug delivery system with bidirectional synergistic activation of tumor autoimmunity.

2. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy according to claim 1, characterized in that: The specific method of the EDC-mediated reaction in step S1 is: pouring methanol containing conjugated linoleic acid and methanol dissolving EDC into an aqueous solution dissolving carboxymethyl chitosan, and continuously stirring the reaction.

3. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy according to claim 1, characterized in that: The method for preparing acetic acid-activated D-mannose in step S2 is: stirring and dissolving mannose in acetic acid.

4. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy as claimed in claim 3, characterized in that: The concentration of mannose is 0.005-0.015 g / mL.

5. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy according to claim 1, characterized in that: The specific method for grafting D-mannose with conjugated linoleic acid-carboxymethyl chitosan polymer in step S2 is: adding activated D-mannose acetic acid to conjugated linoleic acid-carboxymethyl chitosan polymer under stirring, and continuously stirring and reacting for 24-48 hours.

6. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy according to claim 1, characterized in that: In step S2, the crude MCC nano suspension is dialyzed against anhydrous ethanol and distilled water in sequence to remove unreacted substances, cross-linking agents and by-products, and finally the dialyzate is freeze-dried to obtain the MCC nanocarrier.

7. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy according to claim 1, characterized in that: The step S3 specifically comprises: adding the MCC nanocarrier to distilled water, and then sequentially adding the STING agonist drug and the MR1 antibody drug, performing ultrasonic emulsification to complete the encapsulation of the drug by the MCC nanocarrier, and then performing dialysis against anhydrous ethanol and distilled water in sequence to remove the unencapsulated drug and small molecule impurities; After filtration with a membrane, the macromolecular nanoparticles were removed to obtain the MCC nano-drug delivery system that co-loaded the STING agonist and the MR1 antibody and had the function of bidirectionally synergistically activating tumor autoimmunity.

8. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy according to claim 7, characterized in that: The MCC nanocarrier was added to distilled water at a concentration of 0.5-1.5 mg / mL, the STING agonist drug was added to the mixed solution at a concentration of 0.02-0.08 mg / mL, and the MR1 antibody drug was added to the mixed solution at a concentration of 0.15-0.35 μL / mL.

9. The method for preparing a MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy according to claim 7, characterized in that: The conditions of ultrasonic emulsification were as follows: three times of ultrasonic emulsification, each time for 5-15 minutes, and a power of 300-400W.

10. A MCC nano drug delivery system co-loaded with a STING agonist and an MR1 antibody for enhancing tumor immunotherapy, characterized in that: Prepared by the method described in any one of claims 1 to 9.

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