CN113546045B

CN113546045B - Nanometer preparation for recovering tumor microenvironment inactivated dendritic cell function and application thereof

Info

Publication number: CN113546045B
Application number: CN202110950305.9A
Authority: CN
Inventors: 游剑; 陆益超; 李青坡; 姜新东
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2022-11-11
Anticipated expiration: 2041-08-18
Also published as: CN113546045A

Abstract

The invention provides a nano preparation for recovering the function of inactivated dendritic cells in a tumor microenvironment and application thereof, which can simultaneously inhibit the oxidative stress and endoplasmic reticulum stress of DCs in the tumor microenvironment to recover the activity of the DCs. The nanometer preparation loads the endoplasmic reticulum stress inhibitor into a carrier containing an ROS scavenger, and improves the three-high phenomenon (high endoplasmic reticulum and oxidative stress, high lipid accumulation) and the three-low current situation (low maturity, low activity and low infiltration) of DCs in a tumor microenvironment, thereby improving the quantity and the quality of the DCs and enhancing the tumor immunotherapy effect. The effect can be realized by (1) the body administration of direct nano preparation and (2) the adoptive administration of DCs pretreated by nano preparation. The nano preparation can be a drug-carrying system such as nano emulsion, liposome, micelle or lipid nanoparticle, and the like, can restore the anti-tumor immune blueprint of an organism, and has the characteristics of novel scheme, simple preparation, wide application range and suitability for personalized or universal treatment.

Description

Nanometer preparation for recovering tumor microenvironment inactivated dendritic cell function and application thereof

Technical Field

The invention belongs to the field of pharmacy, and relates to a nano preparation for recovering the function of inactivated dendritic cells in a tumor microenvironment and application thereof. The preparation is a nano preparation capable of restoring the function of dendritic cells in a tumor microenvironment in vivo and in vitro, can reactivate the dendritic cells in a patient body, ensures the function and distribution of an adoptive dendritic cell vaccine, and can be applied to the anti-tumor field.

Background

Tumors are one of the most serious malignant diseases threatening the health and quality of life of humans, and Dendritic Cells (DCs) are one of the most important immune cells in patients to fight this malignant disease. DCs are the most powerful professional Antigen Presenting Cells (APCs) in the body, and are essential for activating and maintaining innate and adaptive immunity, as well as promoting T cell diverse activation. DCs in vivo can sample "suspicious" molecules in the tumor microenvironment, present them on Major Histocompatibility Complex (MHC) I and MHC II molecules via antigen (crossover), and then transmit antigen information to APCs such as other DCs or B cells in lymph nodes, and play a role in stimulating T cell effect differentiation into Cytotoxic T Lymphocytes (CTLs). In addition, DCs distributed in the tumor region can also directly recruit a large number of T cells (including adoptive CAR-T and TCR-T) at the tumor site, play a role in-situ reactivation and T cell expansion, and are important for improving infiltration of tumor killer T cells.

Although tumor-infiltrating DCs play an irreplaceable role in tumor rejection or tumor regression, such DCs are often also affected by the immunosuppressive tumor microenvironment, resulting in a large increase in intracellular Reactive Oxygen Species (ROS) in DCs, with subsequent surges in the Endoplasmic Reticulum (ER) pressure in DCs. Subsequent activation of inositol-requiring kinase 1 α (IRE 1 α) -X-box binding protein 1 (XBP 1) by the ER stress sensor induces DCs to synthesize lipids far beyond normal and accumulate in large quantities in the form of lipid droplets, and these abnormally stored lipids can be peroxidized by intracellular redundant ROS, and the resulting peroxidized lipid by-products can further cause ER stress. Also, too intense ER stress and oxidative stress may be associated with apoptosis and decreased infiltration of DCs at the tumor site. Meanwhile, abnormally accumulated lipids cause increased levels of fatty acid oxidation and oxidative phosphorylation associated with immature DCs, affecting the initiation of glycolytic metabolic recombination necessary for the activation of DCs, resulting in failure of the maturation activation of DCs. In a word, the tumor part is a double-edged sword for DCs, which is full of opportunities and challenges, and DCs have the opportunity to capture tumor specific antigens and tumor associated antigens in a tumor area, so that the normal immune function of a patient is started; meanwhile, the complex tumor microenvironment can increase the oxidative stress and ER stress of the DCs through various factors, and the functions and the quantity of the DCs are seriously weakened.

Because DCs are in the central location of immune response and usually DCs in patients are eliminated at an early stage of tumor development, the anti-tumor "aggressiveness" of immune cells such as DCs, T cells and NK cells in vivo is often mobilized clinically by means of large-scale adoptive in vitro activated DCs vaccines to enhance the overall anti-tumor response of patients. Such DCs vaccines are often isolated or differentiated from autologous or allogeneic healthy volunteers, and reinfused after whole tumor lysate or multiple tumor-associated/specific antigen pulses, and adjuvant treatment. sipuleucel-T (trade name Provenge) is the DCs vaccine of milestone significance, the first vaccine-based immunotherapy currently approved for metastatic castration-resistant prostate cancer, and the only cell-based vaccine approved in the united states [ Nat Rev Drug Discov,2010,9 (7): 513-514 ]. There are also currently a number of phase I-III clinical trials involving DCs vaccines, of which renal cell carcinoma [ Hum VaccImmunother,2013,9 (6): 1217-1227], prostate Cancer [ J Immunother,2015,38 (2): 71-76], melanoma [ Clin Cancer Res,2016,22 (9): 2155-2166], and glioma [ Cancers,2018,10 (10): 372] are the more common therapeutic models. However, these DCs vaccines may still be affected by inhibitory microenvironment [ Cancer Res,2020,80 (10): 1942-1956 ], and still exhibit high ER stress and high oxidative stress, leading to the unexpected intratumoral and lymph node distribution [ Clin Cancer Res,2009,15 (7): 2531-2540] and function [ Chonnam Med J,2015,51 (1): 1-7], and further restricting the clinical efficacy. The existing DCs vaccines or DCs vaccine inducers do not consider that excessive stress in DCs can inhibit the initiation of their activation process and even induce direct apoptosis of DCs.

Therefore, the restoration of normal immune function of the tumor microenvironment DCs is crucial. The three-high phenomena of high endoplasmic reticulum stress, high oxidative stress, high lipid accumulation and the like expressed by the tumor microenvironment DCs can induce the three-low current situations of low infiltration, low activity and low maturity. Therefore, the simultaneous inhibition of the high endoplasmic reticulum stress and the high oxidative stress of the micro-environment DCs is expected to solve the 'three-high' phenomenon inhibiting the normal functions of the DCs, and further solve the 'three-low'.

Disclosure of Invention

The first purpose of the invention is to provide a nano preparation for restoring the function of inactivated dendritic cells in a tumor microenvironment, which consists of an oil phase and a water phase, wherein the oil phase is selected from medium chain triglyceride (5-70%, w/w), lecithin (5-70%, w/w), ROS scavenger tocopherol (0.1-50%, w/w), oil-soluble IRE1 alpha-XBP 1 targeted inhibitor 0.1-30%, w/w), and the water phase is selected from deionized water solution (0.1-30%, w/w) or deionized water of water-soluble inhibitor.

The oil-water ratio (oil phase: water phase) of the nano preparation is 0.01-70%, and w/w.

The nanometer preparation is oil-in-water (O/W) nanometer emulsion. But not limited to, nanoemulsions, and can also be extended to other nanopreparations such as liposomes, micelles, and lipid nanoparticles.

(1) In addition to functional ROS scavengers and IRE1 α -XBP1 targeted inhibitors, other oily components may be substituted or added with other lipid components that are highly biocompatible, poorly immunogenic, readily available and inexpensive to produce, including cephalins, phosphatidylserines, phosphatidylglycerols, cardiolipins, phosphatidylinositols, sphingomyelins, soybean oils, olive oils, other medium chain fatty acid esters, long chain fatty acid esters, glycerol stearates, sucrose fatty acid esters, squalene, cholesterol.

(2) The oil-soluble ROS scavenger tocopherol can be replaced by beta-carotene, N-acetyl-L-cysteine, N' -dimethylthiourea, polydopamine, fulvic acid, tea polyphenol, ceO2, mnO2-x, fe3O4, prussian blue, superoxide dismutase, catalase, glutathione peroxidase and other enzyme scavengers and agonists thereof.

(3) The oil soluble small molecule inhibitor can be KIRA6, KIRA7, KIRA8, sunitinib D10, MKC8866, 6-bromo-2-hydroxy-3-methoxybenzaldehyde, GSK2850163, NSC95682, STF083010, 4 μ 8C, toyocamycin (Toyocamycin). In addition, the water-soluble micromolecule inhibitor can be dissolved in the water phase, accounts for 0.1-30% of the total mass of the water phase, is combined on the surface of the nano preparation in a mode of ultra-detection or electrostatic adsorption and the like, and can be KIRA8 hydrochloride and 3,6-DMAD hydrochloride.

The second purpose of the invention is to provide the application of the nano preparation in preparing a medicine for restoring the function of dendritic cells in tumor microenvironment in vivo and in vitro.

The application of the medicine of the invention is realized by the following two main ways:

(1) The nanometer preparation can be used for direct in vivo delivery at a concentration of 0.1-100 mg/kg. By means of injection, oral administration, inhalation, cavity administration, transdermal administration and other administration modes, the preparation can directly relieve pressure and lipid accumulation of DCs inactivated by stress in a patient body, and high maturation, high activity and high tumor differentiation of endogenous DCs can be realized.

(2) The nano-preparation can be used for treating DCs in vitro and inducing the DCs to become a cell vaccine capable of being subjected to adoptive reinfusion, and the in vitro cell administration concentration is 1nM-40mM. Allogeneic or autologous initial DCs induced and differentiated in vitro or DCs activated by tumor antigens and tumor cell lysate are treated by the drug-loaded nano preparation for 1-48h to become therapeutic or preventive DCs vaccine for reinfusion.

The nano preparation or DCs treated by the preparation can be used together with one or more immune checkpoint inhibitors to improve the immunity of organisms, including PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, TIM3 antibodies and the like. DCs treated by the nano preparation can keep the activities of recruiting, stimulating, amplifying and activating T cells in a tumor area or a tumor drainage lymph node for a long time, and improve the overall immune condition of a patient, and an immune checkpoint inhibitor can maintain the killing function of host immune cells, so that the synergy is realized, and the systemic strong anti-tumor immune response is aroused.

The invention hopefully recovers the function of the DCs in the tumor microenvironment by double inhibition of the pressure of the DCs. First, ROS scavengers are effective in eliminating excess ROS intracellular in DCs, and the oxidative stress caused by these ROS is often associated with tumor zone infiltration of DCs and apoptosis and endoplasmic reticulum stress of adoptive DCs vaccines. Meanwhile, the endoplasmic reticulum stress inhibitor can effectively reduce the condition of massive synthesis and accumulation of DC intracellular lipid caused by the XBP1 protein as a transcription factor, weaken fatty acid oxidation and oxidative phosphorylation, ensure the necessary glycolytic metabolism reprogramming during DsC activation, reduce the oxidized truncated lipid source causing endoplasmic reticulum stress, and further relieve the pressure of DCs. DCs co-inhibited by oxidative stress and endoplasmic reticulum stress can maintain the activity of recruiting, stimulating, amplifying and activating T cells in tumor regions or tumor draining lymph nodes for a long time, and improve the overall immune status of patients, thereby arousing a systemic strong anti-tumor immune response. Therefore, the invention focuses on preparing a nano preparation with dual functions of oxidative stress and endoplasmic reticulum stress inhibition to recover the function of the tumor microenvironment DCs.

The invention is innovative in that the tumor microenvironment passive targeting preparation which is convenient to produce, low in cost, stable in property and dependent on the strong phagocytosis of DCs is prepared, and has good production and application prospects. According to the reason of DCs dysfunction in the tumor microenvironment, the method can be used for specifically and simultaneously relieving the overload ER stress and oxidative stress which are not beneficial to the survival and activation of the DCs, promoting the metabolism of the DCs to be reprogrammed in the direction beneficial to the activation, further recovering the functions of the DCs in the tumor area/tumor drainage lymph nodes, and recovering and regulating the whole body anti-tumor response capability.

Drawings

Figure 1 is the drug-loaded nanoemulsion particle size (prescription 5).

Fig. 2 is a transmission electron microscope morphology of drug-loaded nanoemulsion (formula 5).

Figure 3 is the seven day particle size stability of the drug loaded nanoemulsion (formula 5).

Figure 4 is drug-loaded nanoemulsion 24h cytotoxicity.

Figure 5 is a gradient inhibition of ID8 tumor infiltration DCs XBP1s expression with drug-loaded nanoemulsion.

Figure 6 is a drug-loaded nanoemulsion that reduces intracellular ROS and lipid accumulation levels in DCs in the ID8 tumor microenvironment.

Fig. 7 shows that the uptake level of the drug-loaded nanoemulsion by the DCs is much higher than that of the ID8 tumor cells.

Figure 8 is the drug-loaded nanoemulsion improved internalization levels for tumor microenvironment DCs antigen uptake.

Figure 9 is that the drug-loaded nanoemulsion increases the degree of maturation activation of the ID8 and B16 tumor microenvironment DCs.

FIG. 10 drug-loaded nanoemulsion KTNE improves the secretion of DCs factor in ascites of ID8 ovarian cancer.

FIG. 11 drug-loaded nanoemulsion KTNE increases T cell number and factor secretion in ascites of ID8 ovarian cancer.

Figure 12 drug-loaded nanoemulsion KTNE delays the progression of ID8 ovarian cancer.

Figure 13 drug loaded nanoemulsion KTNE reduced ID8 ovarian cancer ascites accumulation.

Figure 14 drug loaded nanoemulsion KTNE reduced peritoneal metastasis of ID8 ovarian cancer.

FIG. 15 drug-loaded nanoemulsion KTNE and PD-1mAb administered in combination to restore maturity of lymph node DCs with ID8 ovarian cancer and DC-T interaction.

Figure 16 adoptive loading of nanoemulsion KTNE treated lysate activated DCs slowed tumor progression.

Figure 17 adoptively loaded nanoemulsion KTNE treated lysate activated DCs stimulated cell expansion.

Figure 18DCs taken up both oxidative stress and endoplasmic reticulum stress dual inhibitory nano-formulations.

Figure 19 particle size of long-circulating drug-loaded liposomes.

Detailed Description

The invention is further illustrated by the accompanying drawings and examples.

Example 1 prescription screening, antigen Loading and cellular uptake of nanoemulsions

(1) Prescription screening and physicochemical property characterization of drug-loaded nanoemulsion

Table one: prescription composition of nano-emulsion

Firstly, preparing a drug-loaded nano-emulsion by an emulsification ultrasonic method, and screening a prescription of the drug-loaded nano-emulsion. The formulation 5, which has high stability, i.e., when PL100M is used as a membrane material (PL 100M: α -T: MCT =0.98%:0.17%:0.81%, w/w), was screened for subsequent studies by changing the types of phosphatidylcholine (PL 100M and E-80), the ratio of the lipid components, and the content of the aqueous phase. The nanoemulsion particle size under the prescription is about 150nm (detected by a dynamic light scattering method) (see figure 1); typical emulsion morphology under Transmission Electron Microscopy (TEM) (see fig. 2); high stability and easy storage, and no obvious change of particle size after storage for one month at 4 ℃ (see figure 3).

(2) Inhibitor dosing concentration screening

KIRA6 is an IRE1 α kinase and RNase inhibitor, and is effective in attenuating downstream XBP1 mRNA cleavage and the associated lipid synthesis in large quantities. In order to obtain a proper administration concentration of the inhibitor, namely the drug-loaded nanoemulsion can effectively inhibit XBP1 protein expression and weaken intracellular lipid accumulation and has certain biological safety, gradients of 0 mu M, 1 mu M, 2 mu M, 4 mu M, 6 mu M, 10 mu M, 15 mu M and 20 mu M are set according to the concentration of the inhibitor. The preparation is found to have better biological safety, under the treatment of higher administration dosage for 24h, the preparation still has no obvious cytotoxicity to terminal differentiated cells such as DCs (named BMDCs, obtained by taking mouse tibia and femur bone marrow cells and inducing and differentiating the cells in vitro by GM-CSF and IL-4 for 5-6 days) and the like, but has certain inhibition effect on cells with vigorous growth and division such as ID8 ovarian cancer cells and the like under higher dosage (see figure 4). And XBP1 protein expression was significantly down-regulated at 6 μ M (see FIG. 5), intracellular ROS levels and lipid accumulation were relieved (see FIG. 6).

(3) Cellular uptake of drug-loaded nanoemulsion

According to the selected administration concentration, the nanoemulsion is subjected to fluorescent labeling by DID, 24h uptake of the drug-loaded nanoemulsion is investigated by taking DCs and ID8 tumor cells as model cells, and the imaging result of a fluorescence inverted microscope shows that DCs can take the nanoemulsion (shown in figure 7) far more than ID8 in 24h due to the strong phagocytic property of the DCs, so that the drug-loaded nanoemulsion can be regarded as a DCs passive targeting preparation of a tumor microenvironment.

Example 2 verification of restoration of tumor microenvironment DCs function by drug-loaded nanoemulsion

In the example of prescription 5 of example 1, DCs were co-incubated with tumor cells for 24h, followed by treatment with drug-loaded nanoemulsion for 24h. The treated DCs were pulsed with FITC-OVA and the location of their nuclei was marked with DAPI and their antigen uptake capacity was recorded by fluorescent inverted microscope observation. The ID8 tumor associated DCs treated with the nanoemulsion were found to have better antigen uptake and accumulation capacity within 24h (see FIG. 8). The activation of DCs in the micro-environment of tumors such as ID8 and B16 after being treated with nanoemulsion for 24h is verified by flow cytometry, the labeled DCs are stained with appropriate amounts of fluorescein labeled DC11c, CD80 and MHC I flow antibodies according to the use amount of the specification, incubated at 37 ℃ for 45 minutes, washed 2-3 times with fresh 4 ℃ PBS, resuspended in 500 muL PBS every 100 ten thousand, and the expression representing the double positive of mature activated MHC I molecules and CD80 molecules in the DCs subgroup of CD11c + is observed and analyzed by a multi-color flow meter. The results show that the DCs phenotype after the drug-loaded nanoemulsion treatment is mature (see figure 9), namely the DCs functional activity in various tumor microenvironments can be recovered.

Example 3 drug-loaded nanoemulsion restoration of antitumor Activity of tumor microenvironment DCs

The formula of the nanoemulsion comprises:

yolk lecithin PL100M 25mg

Triglyceride 20mg

2-5mg of alpha-tocopherol

KIRA62 mg

1mL of water.

After an ID8-LUC high serous ovarian cancer model of a C57BL/6 mouse is constructed, the drug-loaded nanoemulsion (KTNE) is injected into the abdominal cavity to improve the vitality damage phenotype of high-stress high-lipid accumulation of tumor microenvironment DCs and restore the antitumor activity of the tumor microenvironment DCs. High serous ovarian cancer is a tumor model rich in DCs, but due to its inhibitory tumor microenvironment, these DCs antigen presentation and T cell activation etc. hypoimmunity infiltrated in malignant ascites and tumor draining lymph nodes are caused, and no immune checkpoint Inhibitor (ICB) is currently approved for clinical ovarian cancer treatment [ Nat Rev Immunol,2018,18 (3): 153-167], which is believed to be possibly associated with severely dysfunctional DCs in its microenvironment. The KTNE nanoemulsion can effectively improve the secretion of functional factors (IL-12 p70, IFN alpha) of DCs in a tumor microenvironment (see figure 10) and the number and the function of infiltrating T cells (see figure 11), and finally delay the development of ovarian cancer (see figure 12), reduce ascites accumulation (see figure 13) and inhibit peritoneal metastasis of tumors (see figure 14). Compared with the therapy using the PD-1 monoclonal antibody alone, the anti-tumor effect (see figures 12-14) and the microenvironment immune property (see figures 10-11) of the KTNE nanoemulsion are greatly improved after the KTNE nanoemulsion is used in combination, and the DCs maturation and DC-T cell interaction (see figure 15) in the tumor draining lymph nodes are remarkably improved.

Example 4 adoptive reinfusion of tumor cell lysate activated and nanoemulsion processed DCs vaccine

The formula of the nano-emulsion comprises:

egg yolk lecithin E80 mg

Cholesterol 2-10mg

Triglyceride 6mg

2mg of alpha-tocopherol

Sunitinib D10.6-5 mg

1mL of water.

The tumor cell lysate accounts for 0.1-70% (w/w) of the normal DCs culture medium.

Adjuvant: lipopolysaccharide 0.1-10mg/mL or polyinosinic acid-polycytidylic acid 0.1-10mg/mL.

After 24h of pulse treatment of DCs with a certain proportion of tumor cell lysate and adjuvant, DCs were obtained that captured and presented tumor antigens to some extent, and such DCs generally exhibited a state of incapacity of high stress, high lipid accumulation. These DCs were treated with KIRA 6-loaded nanoemulsion 24h before their adoptive reinfusion back into ID8 tumor-bearing hosts and the tumor lysate was either autologous or syngeneic/homogeneous tumor of the patient. Compared with the DCs vaccine processed by the tumor lysate, the DCs vaccine can better exert the anti-tumor effect to a greater extent and weaken the tumor growth (see figure 16) because the activated preparation process inhibits the ER stress and the oxidative stress of the DCs vaccine doubly and the DCs vaccine is not influenced by inhibitory components in the lysate and inhibitory microenvironment in vivo, and the DCs vaccine with higher activity in vitro is also beneficial to better distribute to the tumor and/or lymph node area in vivo.

Example 5 nanoemulsion-treated DCs vaccine under adoptive reinfusion tumor Co-incubation System

The formula of the nanoemulsion comprises:

soybean lecithin S10030mg

Squalene 5-30mg

N, N' -dimethylthiourea 4mg

KIRA80.07-25mg

1mL of water.

The tumor cells and the DCs in a certain proportion are mixed and incubated for 24h, and a certain adjuvant is added for stimulation to simulate the DCs in a tumor microenvironment, the DCs have the opportunity to capture, process and present various tumor antigens, but the DCs often process the state that the DCs can not complete activation due to the existence of various immunosuppressive factors/molecules. Subsequently, these tumor-associated DCs were treated with KIRA 8-loaded nanoemulsion for 24h and transferred to adoptive tumor-loaded patients. The DCs vaccine thus treated can present more types of tumor antigens and can remain viable in the host, thus expanding and activating more TCR-specific T cells (see fig. 17), improving anti-tumor immunity.

Example 6 adoptive reinfusion of one/more tumor specific antigen/neoantigen, tumor associated antigen activated and nanoemulsion processed DCs vaccine

The formula of the nano-emulsion comprises:

phosphatidylserine 15mg

Sucrose fatty acid ester 8mg

Beta-carotene 2mg

MKC88660.5-12mg

1mL of water.

Tumor specific antigen/neoantigen: carrying out personalized sequencing on a patient, and detecting and screening to obtain several types of antigens which are specific, highly expressed and only presented on MHC I on the surface of the tumor; tumor associated antigens: screening to obtain the antigen highly expressed in the tumor of the patient. The resulting fusion protein of the tumor antigen or a mixture thereof can be used to activate DCs.

DCs are pulsed in vitro with a fusion protein of a tumor antigen or a mixture thereof and stimulated with a suitable adjuvant to give a mature DCs vaccine. The DCs vaccine can be activated and matured normally in vitro, shows mature phenotype and metabolic characteristics, and can recognize multiple types of tumor antigens, but the DCs vaccine is still likely to be inhibited by immune microenvironment after entering the body, so that the DCs vaccine presents a high-stress and high-lipid accumulation state, and finally has impaired vitality and even apoptosis. Therefore, the DCs are treated by the nanoemulsion for inhibiting the excessive oxidative stress and the ER stress and then are returned, so that the DCs can be possibly protected from being influenced by tumors and can be continuously kept in a high-activity state.

Example 7 direct injection of double inhibitory nanoemulsion of ER stress and oxidative stress

The formula of the nano-emulsion comprises:

yolk lecithin E8036mg

Stearic acid glyceride 24mg

Alpha-tocopherol of 15mg

Toyocamycin 0.1-4mg

1mL of water.

For various ascites tumor or heat tumor models with more DCs infiltrated, such as ovarian cancer, cervical cancer, colon cancer, liver cancer and the like, administration modes such as injection and the like can be selected directly to directly regulate high-stress high-lipid DCs in bodies or tumor areas of patients, improve the functional activity of infiltrated DCs, and be beneficial to recruiting and stimulating more T cells and other immune cells and improving the tumor microenvironment. Serous tumors often have the characteristics of high malignancy, difficult healing, easy metastasis, poor prognosis and the like, and the expected survival period and the life quality of patients are poor, but the tumors often have more DCs and T cells due to the tumor microenvironment of ascites, so the quality of the DCs in the tumors is very important. In the case of serous ovarian cancer, DCs in the ascites often exhibit an immunosuppressive phenotype and are not very sensitive to a variety of immune checkpoint Inhibitors (ICBs), and thus no ICB is currently clinically approved for the treatment of serous ovarian cancer. After the double stress-inhibited nanoemulsion is directly injected, the quality of DCs in a patient body phagocytose a nano preparation in a large amount (figure 18) and is shown as an immune activation phenotype, so that peritoneal metastasis and the whole cancer process of serous ovarian cancer can be effectively inhibited when the double stress-inhibited nanoemulsion is used in combination with ICB (intensive care therapy) such as PD1 monoclonal antibody.

Example 8 preparation and application of Long-circulating liposomes for double inhibition of microenvironment DCs stress

Composition of double-inhibition long-acting sequential liposomes:

dioleoyl phosphatidylethanolamine (DOPE) 3mg,

N-distearoylphosphatidylethanol-PEG (DSPE-PEG) ₂₀₀₀ )0.2mg，

0.1-4mg of tea polyphenol,

GSK2850163 0.2-3mg，

1mL of water.

The invention prepares the liposome for inhibiting ER stress and oxidative stress by a rotary evaporation-probe ultrasonic method. Dissolving the lipid and inhibitor in chloroform solution, ultrasonic dispersing, and removing organic solvent by rotary evaporation under reduced pressure in water bath at 45 deg.C to obtain uniform and complete lipid film. The lipid film was then thoroughly hydrated with ultrapure water at 45 ℃ and sonicated with a probe in an ice water bath to give stable drug-loaded liposomes of uniform particle size (around 100nm, FIG. 19). The liposome can eliminate excessive ROS by using tea polyphenol, and meanwhile IRE1 alpha-XBP 1s inhibitor GSK2850163 can further weaken ER stress related to lipid byproducts on the basis of reducing lipid accumulation, and recover the function of tumor resident DCs.

Example 9 preparation and application of manganese-iron nanoparticles for co-inhibiting ER stress and oxidative stress of microenvironment DCs

The drug-loaded manganese-iron nanoparticles comprise the following components:

MnCl ₂ ·4H ₂ O 2g

FeCl ₂ ·4H ₂ O 1.5g

FeCl ₃ ·6H ₂ O 1g

MnSO ₄ proper amount of solution

4μ8C 20-1000mg。

4 mu 8C-loaded Fe-containing ₃ O ₄ The magnetic core manganese (II) nanoparticles of (1) are prepared by a coprecipitation method. FeCl is added ₃ ·6H ₂ Dissolving O in ultrapure water, heating to 60 deg.C, adding appropriate amount of 4 μ 8C and MnCl ₂ ·4H ₂ O and FeCl ₂ ·4H ₂ O dissolved in FeCl ₃ In the solution, a total molar ratio manganese (II)/Fe of 0.3 was obtained. Mixing 5% polyethylene glycol ₂₀₀₀ (PEG ₂₀₀₀ ) Solution addition to metalsChloride solution and mixed by ultrasonic agitation. Dropping 2.5mol/L KOH solution into the mixed solution at 60 ℃, and stirring for coprecipitation. The precipitate is aged in mother liquor at 60 ℃, separated, washed and dried. 1.0g of the sample was dispersed in PEG by ultrasonic agitation ₂₀₀₀ To the solution, 100mL of 0.04mol/L MnSO was then added to the mixture ₄ The solution was heated to 60 ℃ and 0.045mol/L KMnO was added ₄ 85mL of the solution was stirred to form 4. Mu.8C-containing manganese-Fe/MnO ₂ The particles were magnetically separated, washed with ultrapure water and dried for 24 hours. Wherein the manganese-Fe/MnO ₂ The composition can be used for scavenging ROS, and 4 μ 8C can be used as inhibitor for relieving intracellular lipid accumulation of DCs.

Example 10 preparation and application of self-assembled micelle co-inhibiting ER stress and oxidative stress of microenvironment DCs

The drug-loaded self-assembled micelle comprises the following components:

25mg of poly-dopamine (polydopamine),

the content of fulvic acid is 75mg,

NSC95682 3-40mg。

the amine group of polydopamine is conjugated with the carboxyl group of fulvic acid. Briefly, fulvic acid was dissolved in THF, and then N, N' -Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were added and allowed to activate at 25 ℃ for 8 hours. Ice-cold n-hexane was added to the mixture to precipitate the activated fulvic acid, followed by drying at 40 ℃. Incubation of polydopamine and activated fulvic acid in dichloromethane for 15 hours resulted in formation of a fulvic acid-polydopamine conjugate, and the conjugate was dried using a rotary evaporator. The conjugate was dissolved using dilute hydrochloric acid and subsequently precipitated with ice-cold acetone. Thereafter, the conjugate was mixed with ultrapure water, followed by filtration and freeze-drying to obtain a fulvic acid-polydopamine conjugate. NSC95682 (3 mg) was dispersed into a mixture of triethylamine (TEA, 0.1 mL) and anhydrous DMSO (1 mL) and activated with equal amounts of NHS and EDC under anhydrous nitrogen for 2 hours at room temperature. Fulvic acid-polydopamine (3,100mg) was added to 25mL of ultrapure water, followed by dilution with 25mL of methanol and stirring until an optically clear solution was obtained, and then activated NSC95682 was added dropwise. The mixture was stirred at room temperature under nitrogen for 24 hours to bind NSC95682 to the polydopamine molecules. The mixture after termination was first dialyzed against Phosphate Buffered Saline (PBS) pH 7 for 3 days. 4 to eliminate excess unreacted substrate, and dialyzed with ultrapure water for 3 days. The polymer was isolated by lyophilization. In the self-assembled micelle, polydopamine and fulvic acid can be used as ROS scavengers, NSC95682 can be used as an IRE1 alpha-XBP 1 pathway inhibitor, the pressure and lipid accumulation of DCs related to tumors with impaired vitality can be effectively relieved, and the activation process is restarted.

Example 11 preparation and application of solid lipid nanoparticles for co-inhibiting ER stress and oxidative stress of microenvironment DCs

The drug-loaded solid lipid nanoparticle comprises the following components:

0.1-15mg of glycerin monostearate,

PEG ₂₀₀₀ 15-100mg，

0.1-10mg of alpha-tocopherol,

KIRA8 0.1-15mg。

the solid lipid nanoparticles for inhibiting both ER stress and oxidative stress can be prepared by a solvent diffusion method. Dissolving KIRA8 and alpha-tocopherol in glyceryl monostearate and PEG ₂₀₀₀ Solid lipid solution of the mixture. The resulting organic solution was rapidly dispersed into poloxamer 188 solution (0.1%, w/v) and stirred in a water bath at 70 ℃ for 5 minutes at 400rpm with mechanical stirring. After the pre-emulsion (molten lipid drops) is cooled to room temperature, a dialysis membrane (MWCO: 3.5 KDa) and 10% polyvinylpyrrolidone K30 solution are dialyzed for 48 hours, and then the solid lipid nanoparticles carrying KIRA8 can be obtained by concentration.

Example 12 preparation and application of PEG-PLA micelles for co-inhibiting ER stress and oxidative stress of microenvironment DCs

The drug-loaded PEG-PLA micelle comprises the following components:

polyethylene glycol (PEG) -polylactic acid (PLA) 20-350mg,

0.1-10mg of tea polyphenol,

KIRA8 0.01-5mg。

DMSO containing KIRA8 and tea polyphenols was added to a commercially available DMSO solution of PEG-PLA conjugate, and the mixed solution was added dropwise to a pH 7.4 phosphate buffer solution and stirred for 24 hours. Dialyzing with ultrapure water for 24 hours to obtain the PEG-PLA micelle loaded with KIRA8 and tea polyphenol. ROS scavenger tea polyphenols and IRE1 alpha-XBP 1 inhibitor KIRA8 can be used as active ingredients to play a role in spatio-temporally co-inhibiting ER stress and oxidative stress of DCs in tumor microenvironment.

Claims

1. The application of the nano preparation in preparing the medicine capable of restoring the dendritic cell function in-vivo and in-vitro tumor microenvironment is characterized in that the nano preparation consists of an oil phase and a water phase, wherein the oil phase is selected from 5-70% of medium chain triglyceride, w/w, 5-70% of lecithin, w/w, 0.1-50% of ROS reducing agent tocopherol, w/w and 0.1-30% of oil-soluble IRE1 alpha-XBP 1 targeted inhibitor, and the water phase is selected from deionized water; oil phase of the nano-formulation: the water phase is 0.01-70% w/w.

2. The use according to claim 1, wherein said use is achieved by two ways:

(1) The nanometer preparation is prepared into medicine for direct in vivo delivery, the administration concentration is 0.1mg/kg-100mg/kg, and the administration route is injection, oral administration, inhalation, cavity administration and transdermal;

(2) The nanometer preparation is treated with DCs in vitro to induce the DCs to become cell vaccine capable of being delivered again, the in vitro cell administration concentration is 1nM-40mM, and the DCs which are induced and differentiated in vitro and are allogeneic or autologous initial DCs or DCs activated by tumor antigens and tumor cell lysate are treated with the drug-carrying nanometer preparation for 1-48h to become the preventive and therapeutic DCs vaccine.

3. The use according to claim 1, wherein the nano-formulation is an oil-in-water type nano-emulsion, or a liposome, a micelle, a lipid nanoparticle.