A drug delivery system for improving tumor microenvironment and preparation method and application thereof

Abstract

The present application relates to a drug delivery system for improving tumor microenvironment and a preparation method and application thereof, the drug delivery system comprises liposomes, an anti-tumor drug loaded in the liposomes and a polymer particle wrapped in the core of the liposomes; the components of the polymer particle comprise PC7A polymer and Poly(I:C) polymer; the anti-tumor drug is selected from an RXR alpha agonist, an ITPR2 inhibitor or an MCU inhibitor; the RXR alpha agonist is selected from aurorex, SR11237, CD3257, GW0791 or an isomer of the foregoing drug, or a pharmaceutically acceptable salt of the foregoing drug. The drug delivery system can maintain stability for a long time under in-vivo and in-vitro physiological conditions, and quickly release the loaded anti-tumor drug molecules into the cytoplasm, help them to realize lysosome escape, and effectively enter the cytoplasm. When delivering the anti-tumor drug molecules, the delivery system exhibits excellent tumor microenvironment regulation ability, can effectively participate in inhibiting T cell exhaustion, and improves the efficiency of anti-tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug delivery system for improving the tumor microenvironment, its preparation method, and its application. Background Technology

[0002] The tumor microenvironment is the unique internal environment upon which tumor cells depend for survival and development. It plays a crucial role in tumor development and progression, and largely determines the efficacy of tumor immunotherapy. By shaping an inhibitory immune microenvironment, tumor cells hijack surrounding normal cells, significantly limiting the efficiency of tumor immunotherapy. Ultimately, this leads to T cell depletion in the tumor microenvironment, resulting in the loss of tumor-specific killing function and the development of immune tolerance.

[0003] Currently, methods to inhibit T cell exhaustion and actively regulate the tumor immunosuppressive microenvironment include activating immune cells using immune adjuvants or cytokines; developing and utilizing immune checkpoint inhibitors; and modulating classical metabolic pathways of various cells within the tumor. However, clinical data indicate that existing immunotherapy methods have low efficiency and poor therapeutic effects. Therefore, it is necessary to find more effective methods to improve the tumor immunosuppressive microenvironment to maintain the activity and function of immune cells.

[0004] Dendrobine is a small molecule compound with medicinal value extracted from Dendrobium Sw., a plant of the Dendrobium genus. It is readily soluble in organic solvents and possesses direct anti-tumor pharmacological properties. It not only significantly inhibits tumor cell invasion and angiogenesis, but is also a novel CRAF and MEK1 / 2 enzyme inhibitor, capable of dual-targeting the RAS-RAF-MEK-EPK signaling pathway to inhibit tumor cell proliferation and induce tumor cell apoptosis. Furthermore, it is a novel ferroptosis inducer, increasing intracellular calcium levels not only through mediating transferrin receptor 1 (TFR1) and targeting calmodulin (CaM). 2+ and Fe 2+ This level promotes the production and accumulation of ROS, increases lipid peroxidation, and thus induces ferroptosis in tumor cells.

[0005] Although dendriticin molecules can effectively inhibit the growth of different tumors, they require multiple administrations due to their poor water solubility and low bioavailability.

[0006] CN109602707A discloses a liposome composition containing dendritic lecithin, made from the following raw materials in parts by weight: 1-3 parts dendritic lecithin, 1.5-4 parts soybean lecithin, 0.1-0.3 parts polyethylene glycol modified with distearate phosphatidylethanolamine, 0.05-0.2 parts cholesterol, and 0.05-0.3 parts L-histidine. This liposome composition exhibits high encapsulation efficiency and good stability of dendritic lecithin; however, the dendritic lecithin liposomes have insufficient regulatory effect on the tumor microenvironment.

[0007] Therefore, developing a stable drug delivery system that allows dendritic lanolin to be combined with other adjuvant materials to improve the tumor microenvironment and enhance the anti-tumor immune response has become an urgent technical problem to be solved. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a drug delivery system for improving the tumor microenvironment, its preparation method, and its applications. This drug delivery system maintains stability for extended periods under in vitro and in vivo physiological conditions and rapidly releases the loaded antitumor drug molecules into the cytoplasm, facilitating lysosomal escape and effective entry into the cytoplasm. In delivering antitumor drug molecules, this delivery system exhibits excellent tumor microenvironment regulation capabilities, effectively participating in the inhibition of T cell exhaustion and improving the efficiency of antitumor immunotherapy.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a drug delivery system for improving the tumor microenvironment, the drug delivery system comprising liposomes and an antitumor drug loaded in the liposomes, and polymer particles encapsulated in the core of the liposomes;

[0011] The polymer particles consist of PC7A polymer and Poly(I:C) polymer;

[0012] The antitumor drug is selected from RXRα agonists, ITPR2 inhibitors, or MCU inhibitors;

[0013] The RXRα agonist is selected from roximate, SR11237, CD3257, GW0791, or isomers of the aforementioned drugs, or pharmaceutically acceptable salts of the aforementioned drugs.

[0014] This invention creatively discovers that dendritic molecule can effectively inhibit CD8 by activating the retinol receptor subtype RXRα in the T cell nucleus, thereby inhibiting the activity of the ITPR2-MCU pathway and suppressing the influx of calcium ions into the cells' mitochondria. + T cell depletion improves the tumor immunosuppressive microenvironment; simultaneously, RXRα pathway agonists, ITPR2 inhibitors, and MCU inhibitors involved in this process can exert effects similar to those of roximate, thereby inhibiting CD8. + T cell depletion improves the tumor immunosuppressive microenvironment.

[0015] The drug delivery system of this invention combines an antitumor drug with a pH-responsive adjuvant material, PC7A polymer, and a TLR3 agonist, Poly(I:C) polymer. This combination allows the drug to maintain stability for extended periods under in vitro and in vivo physiological conditions and rapidly releases the loaded antitumor drug molecules into the cytoplasm, facilitating lysosomal escape and effective cytoplasmic entry. In delivering the antitumor drug molecules, this delivery system exhibits excellent tumor microenvironment modulation capabilities, effectively participating in the inhibition of T cell exhaustion and improving the efficiency of antitumor immunotherapy.

[0016] Preferably, the ITPR2 inhibitor comprises 2-aminoethyl diphenylboronic acid ester (2-APB) or Xestospongin-C.

[0017] Preferably, the MCU inhibitor includes ruthenium red, Ru265, or Ru360.

[0018] Preferably, the raw materials for preparing the liposomes include phospholipids, cholesterol, and tumor cell membrane materials.

[0019] Preferably, the tumor cells include lung cancer cells.

[0020] Preferably, the tumor cell membrane material is a fragment of the tumor cell membrane.

[0021] The hydrophilic and highly targeted tumor cell membrane carries membrane-specific antigens that can be recognized by dendritic cells and promote the phagocytosis of drug delivery systems by dendritic cells.

[0022] Preferably, the mass ratio of the phospholipids, cholesterol and tumor cell membrane material is (10-200):(10-20):1, more preferably (50-100):(10-20):1.

[0023] Among them, the specific point values ​​in the range of 10 to 200 can be 10, 40, 70, 100, 130, 160, 200, etc.; the specific point values ​​in the range of 10 to 20 can be 10, 12, 14, 16, 18, 20, etc.; and the specific point values ​​in the range of 50 to 100 can be 50, 60, 70, 80, 90, 100, etc.

[0024] Preferably, the mass ratio of the PC7A polymer to the Poly(I:C) polymer is 1:(0.2-10), more preferably 1:(0.5-2).

[0025] Among them, the specific point values ​​in the range of 0.2 to 10 can be 0.2, 1, 3, 5, 7, 9, 10, etc., and the specific point values ​​in the range of 0.5 to 2 can be 0.5, 1, 1.5, 2, etc.

[0026] Preferably, the number average molecular weight of the PC7A polymer is 4000 to 7000, for example, it can be 4000, 5000, 6000, 7000, etc.

[0027] Preferably, the number-average molecular weight of the Poly(I:C) polymer is 500 to 800, for example, it can be 500, 600, 700, 800, etc.

[0028] Preferably, the mass ratio of the antitumor drug to the liposome is 1:(10-20).

[0029] Among them, the specific point values ​​in the range of 10 to 20 can be selected as 10, 12, 14, 16, 18, 20, etc.

[0030] Preferably, the mass ratio of the polymer particles to the liposomes is (1-10):(1-10).

[0031] Among them, the specific point values ​​from 1 to 10 can be selected as 1, 3, 5, 7, 9, 10, etc.

[0032] When the components meet the above ratio, a more stable and uniform drug delivery system can be formed, while improving the loading efficiency of anti-tumor drugs.

[0033] In a second aspect, the present invention provides a method for preparing a drug delivery system for improving the tumor microenvironment as described in the first aspect, the method comprising: preparing liposomes loaded with antitumor drugs; mixing polymer particles with liposomes loaded with antitumor drugs, extruding the mixture to obtain a drug delivery system.

[0034] Preferably, the preparation of liposomes loaded with antitumor drugs includes any one of the following two methods:

[0035] (a) The antitumor drug is mixed with phospholipids, cholesterol and tumor cell membrane materials and dissolved in an organic solvent. The solvent is removed by rotary evaporation to form a lipid film. The film is then hydrated to obtain liposomes loaded with the antitumor drug.

[0036] (b) The antitumor drug is mixed with phospholipids and cholesterol, then mixed with tumor cell membrane material and dissolved in an organic solvent. The solvent is removed by rotary evaporation to form a lipid film, which is then hydrated to obtain liposomes loaded with the antitumor drug.

[0037] When anti-tumor drugs are first mixed with phospholipids and cholesterol, and then mixed with tumor cell membrane materials, the function of tumor cell membrane fragments can be better maintained, and the regulation of the tumor microenvironment can be better achieved.

[0038] Preferably, the method for preparing the polymer particles includes the following steps:

[0039] PC7A polymer and Poly(I:C) polymer were mixed in water and self-assembled to obtain polymer particles.

[0040] Preferably, the mixing temperature is 20–40°C and the mixing time is 5–300 s.

[0041] Among them, the specific point values ​​in the range of 20 to 40℃ can be 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the specific point values ​​in the range of 5 to 300s can be 5s, 10s, 50s, 100s, 150s, 200s, 250s, 300s, etc.

[0042] Preferably, the polymer particles are mixed with the liposomes loaded with antitumor drugs at a temperature of 20–40°C for a time of 0.5–10 min.

[0043] Among them, the specific point values ​​in the 20~40℃ range can be 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the specific point values ​​in the 0.5~10min range can be 0.5min, 2min, 4min, 6min, 8min, 10min, etc.

[0044] Preferably, the preparation method of the PC7A polymer includes the following steps:

[0045] (1) Dissolve cuprous bromide and monomer C7A in tetrahydrofuran, remove oxygen, and react by bubbling at 50-90℃ for 10-20 min to obtain a reaction mixture;

[0046] (2) Dissolve ethyl 2-bromoisobutyrate and pentamethyldiethylenetriamine in tetrahydrofuran, mix with the reaction mixture obtained in step (1) and react for 10-30 min, then react at 40-70 °C for 10-20 h;

[0047] (3) Dissolve the product obtained in step (2) with dilute hydrochloric acid, dry, and dialyze to obtain PC7A polymer.

[0048] Specifically, the specific point values ​​for 10–20 min can be 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc.; the specific point values ​​for 10–30 min can be 10 min, 15 min, 20 min, 25 min, 30 min, etc.; the specific point values ​​for 10–20 h can be 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, etc.; the specific point values ​​for 50–90 °C can be 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, etc.; and the specific point values ​​for 40–70 °C can be 40 °C, 50 °C, 60 °C, 70 °C, etc.

[0049] Preferably, the method for preparing the monomer C7A includes the following steps:

[0050] (1) Mix 2-(hexamethyleneimino)-ethyl methacrylate (C7), triethylamine, and methacrylamide chloride in a solvent and react at 0-10°C for 1-3 h, and then react at 20-40°C for 4-8 h.

[0051] (2) Mix the mixture obtained in step (1) with Na2CO3 aqueous solution, separate the oil phase and repeat 2 to 6 times;

[0052] (3) The oil phase collected in step (2) is separated and purified to obtain monomer C7A.

[0053] Preferably, the solvent in step (1) includes tetrahydrofuran or chloroform.

[0054] Among them, the specific point values ​​in 0~10℃ can be 0℃, 2℃, 4℃, 6℃, 8℃, 10℃, etc.; the specific point values ​​in 20~40℃ can be 20℃, 25℃, 30℃, 35℃, 40℃, etc.; the specific point values ​​in 1~3h can be 1h, 1.5h, 2h, 2.5h, 3h, etc.; the specific point values ​​in 4~8h can be 4h, 5h, 6h, 7h, 8h, etc.; and the specific point values ​​in 2~6 times can be 2 times, 3 times, 4 times, 5 times, 6 times.

[0055] Thirdly, the present invention provides the application of a drug delivery system for improving the tumor microenvironment as described in the first aspect in the preparation of antitumor drugs.

[0056] Fourthly, the present invention provides a method for treating tumors by combining radiotherapy and chemotherapy with a drug delivery system for improving the tumor microenvironment as described in the first aspect, the method comprising concurrent administration of radiotherapy and a drug delivery system for improving the tumor microenvironment as described in the first aspect.

[0057] Radiation therapy combined with drug delivery systems that improve the tumor microenvironment can be used to maintain the activity and function of immune cells. A schematic diagram illustrating the intratumoral antitumor immune response process of drug delivery systems that improve the tumor microenvironment is shown below. Figure 1As shown: Radiotherapy kills some tumor cells, inducing immunogenic death and releasing some tumor neoantigens. Intratumoral injection of a drug delivery system that improves the tumor microenvironment induces ferroptosis in some tumor cells using the tumor drugs it carries. Tumor cell membranes in the drug delivery system carry membrane-specific antigens and some tumor neoantigens, which can be recognized by dendritic cells and promote phagocytosis of the drug delivery system by dendritic cells. In the endosomes of dendritic cells, under acidic conditions, the release of Poly(I:C) polymers activates TLR3; while the PC7A polymer adjuvant helps tumor antigens escape from lysosomes to the cytoplasm and activates the STING pathway, inducing immune cell maturation and the release of pro-inflammatory cytokines. Tumor antigens are cleaved into short peptides by proteasomes in the cytoplasm, and then presented to CD8 via MHC I. + Lymphocytes (and MHC II can present antigens to CD4) + Lymphocytes). Antitumor drugs loaded in drug delivery systems can not only induce ferroptosis in some tumor cells, but also further regulate the immunosuppressive microenvironment within the tumor during the accumulation of chemotactic lymphocytes into the tumor, thereby maintaining the activity and function of immune cells.

[0058] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] This invention creatively discovers that dendritic molecule can effectively inhibit CD8 by activating the retinol receptor subtype RXRα in the T cell nucleus, thereby inhibiting the activity of the ITPR2-MCU pathway and suppressing the influx of calcium ions into the cells' mitochondria. + T cell depletion improves the tumor immunosuppressive microenvironment; simultaneously, RXRa pathway agonists, ITPR2 inhibitors, and MCU inhibitors involved in this process can exert effects similar to those of roximate, thereby inhibiting CD8. + T cell depletion improves the tumor immunosuppressive microenvironment;

[0061] The drug delivery system of this invention combines an antitumor drug with a pH-responsive adjuvant material, PC7A polymer, and a TLR3 agonist, Poly(I:C) polymer. This combination allows the drug to maintain stability for extended periods under in vitro and in vivo physiological conditions and rapidly releases the loaded antitumor drug molecules into the cytoplasm, facilitating lysosomal escape and effective cytoplasmic entry. In delivering the antitumor drug molecules, this delivery system exhibits excellent tumor microenvironment modulation capabilities, effectively participating in the inhibition of T cell exhaustion and improving the efficiency of antitumor immunotherapy.

[0062] The drug delivery system of the present invention improves the solubility of antitumor drugs, increases their bioavailability, and enhances the cell entry efficiency of antitumor drugs. Attached Figure Description

[0063] Figure 1 This is a schematic diagram of the intratumoral antitumor immune response process of a drug delivery system that improves the tumor microenvironment;

[0064] Figure 2 This is a synthetic route diagram for the PC7A polymer;

[0065] Figure 3 This is a schematic diagram illustrating the fabrication of a drug delivery system to improve the tumor microenvironment;

[0066] Figure 4 This is the 1H NMR spectrum of the C7A monomer;

[0067] Figure 5 This is a comparison diagram of the expression of different membrane proteins in Example 2;

[0068] Figure 6 This is a characterization result diagram of a drug delivery system that improves the tumor microenvironment;

[0069] Figure 7 This is a schematic diagram of the treatment process for each experimental group in Test Example 1;

[0070] Figure 8 This is a comparison chart of PD-1 / TIM-3 expression levels in each experimental group of Test Example 1;

[0071] Figure 9 This is a comparison chart of the concentrations of inflammatory factors secreted by T cells in each experimental group of Test Example 1;

[0072] Figure 10 This is a comparison chart of mitochondrial oxidative stress and glycolytic metabolism levels in T cells of different experimental groups in Test Example 1;

[0073] Figure 11 These are comparative images of mitochondrial membrane potentials of T cells in different experimental groups under a laser confocal microscope.

[0074] Figure 12 This is a graph showing the toxicity assessment results of the liposome nanoparticles of romaine to T cells in Test Example 2;

[0075] Figure 13 The graphs show the regulatory effects of the RXRα agonists SR11237 and CD3254 on T cell exhaustion in Test Example 3, as well as the regulatory effects of the ITPR2 inhibitor 2-APB and the MCU inhibitor ruthenium red on T cell exhaustion. Figure 14 This is the flowchart for constructing subcutaneous tumors in test case 4 mice;

[0076] Figure 15 This is a comparison chart of the therapeutic effects of different experimental groups on subcutaneous tumors in mice in Test Example 4;

[0077] Figure 16 This is a test image showing the therapeutic effect of a drug delivery system combined with radiotherapy on subcutaneous tumors in mice (Example 4).

[0078] Figure 17 This is a test image showing the recurrence of tumors in mice after a second tumor graft in test case 4;

[0079] Figure 18 This is a graph showing the changes in the expression levels of central memory T cells and effector memory T cells in mice after secondary tumor grafting in test case 4.

[0080] Figure 19 This is a comparison chart of the proportions of DC cells and CD3 / CD8 T cells in the tumor microenvironment of mice in each experimental group in Test Example 5;

[0081] Figure 20 This is a comparison chart of the secretion levels of pro-inflammatory and anti-inflammatory cytokines in mice in each experimental group of test example 5;

[0082] Figure 21 This is a comparison of cell apoptosis levels in tumor tissue sections from different experimental groups of mice in Test Example 5;

[0083] Figure 22 These are cross-section images of various organs from mice in each experimental group of test case 6;

[0084] Figure 23 This is a graph showing the levels of liver and kidney function-related indicators in mice of each experimental group in Test Example 6. Detailed Implementation

[0085] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0086] Example 1

[0087] This embodiment provides a drug delivery system for improving the tumor microenvironment, which is prepared using the following method:

[0088] (1) Preparation of C7A monomer:

[0089] ① Weigh 5g of C7 and add it to a 250ml round-bottom flask. Place the flask in an ice-water bath in a fume hood and slowly add 100ml of tetrahydrofuran to dissolve it. Then add 6.3ml of triethylamine (TEA) to the flask.

[0090] ② Mix 4 ml of methacryloyl chloride and 30 ml of chloroform, and add dropwise to the round-bottom flask of step ①. Stir and react at 4°C for 2 h, then react at 25°C for 6 h.

[0091] ③ At 4℃, add 2M Na2CO3 aqueous solution dropwise to the mixture obtained in step ②, and measure the pH value of the mixture until the reaction solution is alkaline;

[0092] ④ Add the mixture obtained in step ③ to a separatory funnel for separation. The upper layer is an aqueous phase and the lower layer is an oil phase. Collect the oil phase into a beaker, then pour the oil phase into the separatory funnel. Add 2M Na2CO3 aqueous solution and repeat 3 times. On the third time, add 10ml of 2M Na2CO3 aqueous solution and 70ml of NaCl aqueous solution, mix thoroughly, separate the liquid, and collect the oil phase.

[0093] ⑤ Take the oil phase collected in step ④ using a capillary tube and drop it onto a silica gel plate. Place the silica gel plate in the expander (V(ethyl acetate):V(petroleum ether) = 1:2) and observe the liquid until it reaches 1 / 2 to 2 / 3 of the silica gel plate. Perform column chromatography on the obtained oil phase (mobile phase is V(ethyl acetate):V(petroleum ether) = 1:3, with 1% triethylamine added). Drop the liquid that drips off the column onto the silica gel plate and collect all the liquid from the appearance of the red dot on the silica gel plate until it disappears.

[0094] ⑥ Perform rotary evaporation on the oil phase collected in step ④. After the rotary evaporation is completed, check the consistency of the product before and after rotary evaporation by applying silica gel to the plate again.

[0095] ⑦ After rotary evaporation, the liquid in the flask is dried under vacuum to obtain the C7A monomer.

[0096] (2) Preparation of PC7A polymer:

[0097] ① Weigh 0.0142g of cuprous bromide and 1.5g of monomer C7A and dissolve them in tetrahydrofuran. Seal the reaction apparatus, remove water and oxygen, and fill it with nitrogen. React at 70℃ using the bubbling method for 18min.

[0098] ② Dissolve 19.688 μl of ethyl 2-bromoisobutyrate and 34.166 μl of pentamethyldiethylenetriamine in tetrahydrofuran and slowly add it to the apparatus of step ①. React at 70 °C for 15 min and then at 50 °C for 12 h.

[0099] ③ After the reaction in step ② is completed, tetrahydrofuran is added to dilute the product, and the diluted product is transferred to a round-bottom flask for rotary evaporation. After rotary evaporation, dilute hydrochloric acid is added to dissolve the product, freeze-drying and dialysis are performed to obtain the PC7A polymer.

[0100] The synthetic route diagram of PC7A polymer is as follows: Figure 2As shown.

[0101] (3) Extraction of tumor cell membrane fragments:

[0102] ① Passage LL2 lung cancer cells, discard the old culture medium, and wash twice with pre-chilled PBS. Gently scrape cells off with a cell scraper, centrifuge at 4°C, 1000 rpm for 5 min, and collect the cell pellet. Resuspend the cell pellet in pre-chilled PBS, centrifuge at 4°C, 600 g for 5 min, and discard the supernatant; then centrifuge at 4°C, 600 g for 1 min, and discard the supernatant again. Use a cell membrane protein extraction kit, add protease inhibitor to membrane protein extraction reagent A at a volume ratio of 1:100, then add 1 ml of reagent A with added protease inhibitor to the cell pellet, resuspend, and incubate on ice for 15 min.

[0103] ② Transfer the cells to a pre-cooled glass homogenizer and homogenize 35 times. Take 2.5 μl of the cell homogenate and drop it onto a coverslip. Observe it under a microscope. 70-80% of the cells have no perinuclear halo and no intact cell morphology, indicating that the cells have been broken.

[0104] ③ After homogenization, transfer the liquid to a centrifuge tube, centrifuge at 4℃ and 700g for 10 minutes, carefully collect the supernatant into a new centrifuge tube, and then centrifuge at 4℃ and 14000g for 30 minutes to obtain the precipitate, which is the LL2 tumor cell membrane fragment.

[0105] (4) Preparation of linolenic acid liposome nanoparticles (abbreviated as linolenic acid-LIP):

[0106] 0.012g of soybean lecithin and 0.003g of cholesterol were dissolved in chloroform to obtain a lipid solution; 0.001g of dendritic molecule was fully dissolved in chloroform, and dendritic molecule, soybean lecithin and cholesterol were mixed in a mass ratio of 1:15. The mixture was transferred to a round bottom flask and the chloroform was removed by rotary evaporation. After full hydration, dendritic liposome nanoparticles were obtained.

[0107] (5) Preparation of a drug delivery system (DDS) to improve the tumor microenvironment:

[0108] The liposome nanoparticles of romaine were mixed with LL2 tumor cell membrane fragments at a mass ratio of 10:1 to form romaine complex liposome nanoparticles.

[0109] 0.01 g of PC7A polymer and 0.01 g of Poly(I:C) polymer were dissolved in water to obtain 40 mg / ml mother liquor PC7A and mother liquor poly(I:C), which were then diluted to 800 μg / ml aqueous solutions of PC7A and poly(I:C). The 800 μg / ml PC7A and poly(I:C) aqueous solutions were mixed in equal volumes at 25 °C for 30 s, resulting in self-assembly of polymer particles.

[0110] The above polymer particles were mixed with lanolin-based liposome nanoparticles at a volume ratio of 1:1 at 25°C, sonicated for 20 seconds, and then extruded back and forth 20 times using an extrusion film technique to obtain a drug delivery system. A schematic diagram of its preparation is shown below. Figure 3 As shown.

[0111] Examples 2-5

[0112] Examples 2-5 each provide a liposome nanoparticle, which are respectively SR11237 liposome nanoparticle (abbreviated as SR11237-LIP), CD3254 liposome nanoparticle (abbreviated as CD3254-LIP), 2-APB liposome nanoparticle (abbreviated as 2-APB-LIP), and ruthenium red liposome nanoparticle (abbreviated as ruthenium red-LIP). The difference between them and steps (1) to (4) of Example 1 is that 0.001g of lanolin molecules in step (4) of Example 1 are replaced with equal masses of SR11237 molecules, CD3254 molecules, 2-APB molecules, and ruthenium red molecules, respectively, to obtain SR11237 liposome nanoparticles, CD3254 liposome nanoparticles, 2-APB liposome nanoparticles, and ruthenium red liposome nanoparticles.

[0113] Comparative Example 1

[0114] This comparative example provides nanoparticles (abbreviated as UDDS) without antitumor drugs. The only difference between UDDS and Example 1 is that no dendritic molecules are added in step (4). Instead, 0.012g of soybean lecithin and 0.003g of cholesterol are dissolved in chloroform to obtain a lipid solution. The lipid solution is transferred to a round-bottom flask and the chloroform is removed by rotary evaporation. After thorough hydration, blank liposome nanoparticles are obtained, which are then mixed with LL2 tumor cell membrane fragments at a mass ratio of 10:1 to form composite liposome nanoparticles. Polymer particles are prepared using the same method as in Example 1. The polymer particles and composite liposome nanoparticles are mixed at a volume ratio of 1:1 and extruded back and forth 20 times using an extrusion film technique to obtain nanoparticles without antitumor drugs.

[0115] Verification Example 1

[0116] The C7A monomer obtained in step (1) of Example 1 was subjected to proton nuclear magnetic resonance spectroscopy for analysis and comparison. 1The 1H NMR spectrum, by comparing the number of hydrogen atoms on the carbon atoms in the C7A structural formula, determines the purity and correctness of the synthesized C7A product. The 1H NMR spectrum is shown below. Figure 4 As shown, the results indicate that the synthesized product is a high-purity C7A monomer.

[0117] Verification Example 2

[0118] The consistency of expression in LL2 tumor cells (TCL), LL2 tumor cell membranes (CM), and LL2 tumor cell membrane fragments (DDS-CM) prepared in Example 1 was detected using vertical slab gel electrophoresis. Coomassie brilliant blue staining results are shown below. Figure 5 As shown, the proteins of different molecular weights expressed in LL2 tumor cells, LL2 tumor cell membranes, and the tumor cell membranes added to the drug delivery system are consistent, proving that the extracted cell membrane fragments have been successfully added to the drug delivery system.

[0119] Verification Example 3

[0120] The particle size and potential of the drug delivery system prepared in Example 1 were verified, and the results are as follows: Figure 6 As shown, the prepared delivery system has uniform particle size and stable structure, with a particle size of approximately 161.5 nm and an action potential of -5.18 mV.

[0121] Verification Example 4

[0122] The loading rate of the drug delivery system prepared in Example 1 was verified by using high performance liquid chromatography (HPLC) to detect and quantify the loading rate of dandelion in the drug delivery system.

[0123] Experimental Methods: Different concentrations of free dendritic molecules were used as standards. The mobile phases were acetonitrile and 0.1% phosphoric acid solution, respectively. The standard curve for dendritic molecules was determined as y = 934.07x + 3.0324. Subsequently, 0.001 g (mass denoted as m1) of dendritic molecules was weighed in Example 1 to prepare a drug delivery system (DDS). The prepared DDS was demulsified using methanol, and the concentration of dendritic molecules in the DDS was detected by HPLC. The dendritic content in the DDS was calculated using the standard curve and denoted as m2. The loading rate of dendritic molecules = m2 / m1. The calculated loading rate of dendritic molecules in the DDS prepared in Example 1 was 75%.

[0124] Test Example 1

[0125] Evaluation of the effect of rolanin liposome nanoparticles (rolanin-LIP) on inhibiting T cell exhaustion

[0126] (1) Obtain high-purity mouse spleen CD8 + T cells:

[0127] Seven-week-old female C57 mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were sacrificed, soaked in 75% ethanol for 5 minutes, and their spleens were removed. The spleens were crushed, filtered through a 70-mesh sterile filter, and centrifuged at 400g for 10 minutes. The supernatant was discarded, and erythrocytes were lysed in erythrocyte lysis buffer for 3 minutes. The lysis process was terminated with RPMI 1640 serum-free medium, filtered through a 40-mesh sterile filter, and centrifuged at 400g for 10 minutes. The supernatant was discarded, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 8 / ml, according to CD8 + Following the instructions of the magnetic bead sorting kit, high-purity mouse spleen CD8 cells were obtained through sorting. + T cells.

[0128] (2) Trial grouping and intervention methods:

[0129] T cells were seeded at a rate of 250,000 per well into 48-well plates pre-coated with CD3 / CD28 (coating concentration of CD3 and CD28 was 2 μg / ml, coating volume of 250 μl per well) 24 h in 21% O2 for activation. After 24 h, T cells from the same mouse were randomly divided into three groups, and each group was further treated as follows (see schematic diagram of the treatment process). Figure 7 As shown:

[0130] Non-exhaustion group: T cells were cultured in a medium with CD3 / CD28 stimulation removed and IL-7 added at 21% O2.

[0131] Exhaustion group: T cells were cultured in a 1.5% O2 environment without removing CD3 / CD28 stimulation;

[0132] The linalool group: without removing CD3 / CD28 stimulation, T cells were cultured in a 1.5% O2 environment, and linalool liposome nanoparticles (linalool-LIP) prepared in step (4) of Example 1 were added and co-cultured with T cells.

[0133] After culturing for another 4 days, T cells were collected, stained with live and dead dyes on ice in the dark for 15 min, washed 3 times with PBS, blocked with CD16 / 32 on ice for 10 min, washed 3 times with PBS, and centrifuged. Flow cytometry antibodies CD8-FITC, PD-1APC, and TIM-3PE were incubated on ice in the dark for 30 min. After incubation, the cells were washed 3 times with PBS, centrifuged, and the results were analyzed by flow cytometry.

[0134] (3) Analysis of T cell exhaustion level:

[0135] Flow cytometry detection of CD8 +The expression levels of the inhibitory receptors PD-1 / TIM-3 on the surface of T cells were analyzed, and the levels of late T cell exhaustion, early exhaustion, and total cell exhaustion were statistically analyzed. The results are as follows: Figure 8 As shown in the figure, compared with the non-depleted group, the depleted group showed a significant increase in T cell depletion level, indicating that the T cell depletion model was successfully established in vitro; the T cell depletion level in the linalool group showed a significant decrease, indicating that linalool can effectively inhibit T cell depletion.

[0136] The overall levels of inflammatory factors secreted by T cells during the induced T cell exhaustion period were detected and analyzed using ELISA in each experimental group. The results are as follows: Figure 9 As shown, the cyclophosphamide group maintained the function of T cells in secreting inflammatory factors compared with the non-depleted group and the depleted group.

[0137] The mitochondrial oxidative stress (OCR) and glycolytic metabolism (glyco-PER) levels of T cells in each experimental group were detected and statistically analyzed using a mitochondrial oxidative stress kit and a mitochondrial glycolysis metabolism kit, respectively. The results are as follows: Figure 10 As shown, the mitochondrial metabolic level of T cells in the depleted group was significantly reduced, while the mitochondrial energy metabolism in the dermal leucine group was maintained.

[0138] Changes in the mitochondrial membrane potential JC-1 of T cells in each experimental group were observed under a laser confocal microscope. The results are as follows: Figure 11 As shown, the depleted group showed enhanced green fluorescence, indicating a significant decrease in mitochondrial membrane potential, while the dendritic group showed weakened green fluorescence, indicating that the mitochondrial membrane potential remained normal.

[0139] Test Example 2

[0140] Evaluation of the toxicity of romaine liposome nanoparticles (romaine-LIP) to T cells

[0141] Under normoxic conditions, T cells were continuously stimulated with 15 nM liposome nanoparticles for 4 days. The effects of liposome nanoparticles on T cell viability and apoptosis levels were detected using a cell viability assay kit (CCK8) and an apoptosis assay kit (Annexin-V / PI), respectively. The results are as follows: Figure 12 As shown, 15 nM dendriticil has no significant effect on cell viability and can be used as a safe and effective concentration.

[0142] Test Example 3

[0143] (I) Evaluation of the effects of RXRα agonists SR11237 and CD3254, ITPR2 inhibitor 2-APB, and MCU inhibitor ruthenium red on inhibiting T cell exhaustion

[0144] (1) Obtain high-purity mouse spleen CD8 by referring to the method in step (1) of Test Example 1. + T cells;

[0145] (2) Trial grouping and intervention methods:

[0146] T cells were seeded at a rate of 250,000 per well into 48-well plates pre-coated with CD3 / CD28 (coating concentration of CD3 and CD28 was 2 μg / ml, coating volume of 250 μl per well) 24 h in 21% O2 for activation. After 24 h, T cells from the same mouse were randomly divided into 4 groups, and each experimental group was further treated as follows:

[0147] Non-exhaustion group: T cells were destimulated from CD3 / CD28 and cultured in a medium supplemented with IL-7 (10 ng / ml) at 21% O2.

[0148] Exhaustion group: T cells were cultured in a 1.5% O2 environment without removing CD3 / CD28 stimulation;

[0149] SR11237 group: T cells were cultured in a 1.5% O2 environment without removing CD3 / CD28 stimulation, and SR11237 liposome nanoparticles (SR11237-LIP) prepared in Example 2 were added to the T cells for co-culture at a final concentration of 15 nM.

[0150] CD3254 group: Without removing CD3 / CD28 stimulation, T cells were cultured in a 1.5% O2 environment, and CD3254 liposome nanoparticles (CD3254-LIP) prepared in Example 3 were added to the T cells at a final concentration of 15 nM and co-cultured. After culturing for 4 days, T cells were collected, stained with live and dead dyes on ice in the dark for 15 min, washed 3 times with PBS, blocked with CD16 / 32 on ice for 10 min, washed 3 times with PBS, and centrifuged. Flow cytometry antibodies CD8-FITC, PD-1APC, and TIM-3PE were incubated on ice in the dark for 30 min. After incubation, the cells were washed 3 times with PBS, centrifuged, and the results were detected and analyzed by flow cytometry. (3) Analysis of T cell exhaustion level:

[0151] Flow cytometry detection of CD8 + The expression levels of the inhibitory receptors PD-1 / TIM-3 on the surface of T cells were analyzed, and the levels of late exhaustion, early exhaustion, and total exhaustion of T cells were statistically analyzed. The results are as follows: Figure 13 As shown, the RXRα agonists SR11237 and CD3254 can also significantly inhibit T cell exhaustion.

[0152] (II) Evaluation of the efficacy of ITPR2 inhibitor 2-APB and MCU inhibitor ruthenium red in inhibiting T cell exhaustion

[0153] (1) Obtain high-purity mouse spleen CD8 by referring to the method in step (1) of Test Example 1. + T cells;

[0154] (2) Trial grouping and intervention methods:

[0155] T cells were seeded at a rate of 250,000 per well into 48-well plates pre-coated with CD3 / CD28 (coating concentration of CD3 and CD28 was 2 μg / ml, coating volume of 250 μl per well) 24 h in 21% O2 for activation. After 24 h, T cells from the same mouse were randomly divided into 4 groups, and each experimental group was further treated as follows:

[0156] Non-exhaustion group: T cells were destimulated from CD3 / CD28 and cultured in a medium supplemented with IL-7 (10 ng / ml) at 21% O2.

[0157] Exhaustion group: T cells were cultured in a 1.5% O2 environment without removing CD3 / CD28 stimulation;

[0158] 2-APB group: T cells were cultured in a 1.5% O2 environment without removing CD3 / CD28 stimulation, and 2-APB liposome nanoparticles (2-APB-LIP) prepared in Example 4 were added to the T cells for co-culture at a final concentration of 15 nM.

[0159] Ruthenium Red Group: T cells were cultured in a 1.5% O2 environment without removing CD3 / CD28 stimulation, and ruthenium red liposome nanoparticles (ruthenium red-LIP) prepared in Example 5 were added to the T cells at a final concentration of 15 nM and co-cultured.

[0160] After culturing for another 4 days, T cells were collected, stained with live and dead dyes on ice in the dark for 15 min, washed 3 times with PBS, blocked with CD16 / 32 on ice for 10 min, washed 3 times with PBS, and centrifuged. Flow cytometry antibodies CD8-FITC, PD-1APC, and TIM-3PE were incubated on ice in the dark for 30 min. After incubation, the cells were washed 3 times with PBS, centrifuged, and the results were analyzed using a flow cytometer. (3) Analysis of T cell exhaustion level:

[0161] Flow cytometry detection of CD8 + The expression levels of the inhibitory receptors PD-1 / TIM-3 on the surface of T cells were analyzed, and the late-stage exhaustion level of T cells was also determined. The results are as follows: Figure 13 As shown, the results indicate that the ITPR2 inhibitor 2-APB and the MCU inhibitor ruthenium red can also significantly inhibit T cell exhaustion.

[0162] Test Example 4

[0163] The antitumor effect of the drug delivery system prepared in Example 1 was evaluated.

[0164] The process of constructing subcutaneous tumors in mice is as follows: Figure 14 As shown.

[0165] (1) First tumor inoculation - Construction of LL2 subcutaneous tumor mouse model: 80 7-week-old female C57 mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were selected, and each mouse was subcutaneously inoculated with 1×10 6 One LL2 tumor cell was collected and recorded as day 0. On day 8, the length (L) and width (W) of the tumor were measured using calipers, and the result was calculated using the formula V = 0.5 × L × W. 2 The tumor volume in the mouse was calculated to be approximately 100 mm. 3 ;

[0166] (2) Experimental grouping and intervention method: On day 8, the mice were randomly divided into 8 groups of 10 mice each. Each experimental group was treated as follows:

[0167] The group of romaine-LIP: Starting from day 8, the romaine liposome nanoparticles (romaine-LIP) prepared in step (4) of Example 1 were injected into the tumor (injection dose of 30 mg / kg) once every 2 days for 3 consecutive injections.

[0168] UDDS group: Starting from day 8, nanoparticles (UDDS) without antitumor drugs prepared in Comparative Example 1 were injected into the tumor (injection dose of 30 mg / kg) once every 2 days for 3 consecutive times.

[0169] DDS group: Starting from day 8, the drug delivery system (DDS) prepared in Example 1 was injected intratumorally (injection dose of 30 mg / kg) once every 2 days for 3 consecutive times;

[0170] RT group: On day 8, mice were treated with X-rays at a dose of 8 Gy.

[0171] RT+Maolansu-LIP group: On day 8, the mice were treated with X-rays and the dose of the radiotherapy was 8 Gy; and on day 8, the maolansu liposome nanoparticles (maolansu-LIP) prepared in step (4) of Example 1 were injected into the tumor (injection dose was 30 mg / kg) once every 2 days for 3 consecutive injections.

[0172] RT+UDDS group: On day 8, mice were treated with X-rays at a dose of 8 Gy. Starting from day 8, the tumor was injected with nanoparticles (UDDS) without antitumor drugs prepared in Comparative Example 1 (injection dose of 30 mg / kg) every 2 days for 3 consecutive injections.

[0173] RT+DDS group: On day 8, mice were irradiated with X-rays at a dose of 8 Gy; and starting from day 8, the drug delivery system (DDS) prepared in Example 1 was injected into the tumor (injection dose of 30 mg / kg) once every 2 days for 3 consecutive injections.

[0174] Control group: Starting from day 8, 30 mg / kg of PBS was injected into the tumor once every 2 days for a total of 3 injections.

[0175] (3) Monitoring of tumor size and growth status in mice:

[0176] Continuously monitor the size and growth of mouse tumors until the tumor volume exceeds 2500 mm. 3 At that time, the mouse was determined to have died due to excessive tumor size. The tumor growth curve and survival curve of the mouse were analyzed and plotted based on the monitoring records.

[0177] The therapeutic effects of different components of the drug delivery system on subcutaneous tumors in mice were compared, and the results are as follows: Figure 15 As shown, the tumor growth rate from highest to lowest was the control group, the linolenic acid-LIP group, the UDDS group, and the DDS group. The duration of 100% mouse survival from highest to lowest was the DDS group, the UDDS group, the linolenic acid-LIP group, and the control group. This indicates that the drug delivery system can significantly inhibit tumor growth and improve mouse survival.

[0178] The therapeutic effect of a drug delivery system combined with radiotherapy on subcutaneous tumors in mice was tested, and the results are as follows: Figure 16 As shown, radiotherapy combined with duracil can reduce the growth rate of tumor volume and improve the survival time of mice; radiotherapy combined with drug delivery system treatment resulted in almost no growth of tumor volume at 26 days, and the survival rate of mice remained as high as 100% at 44 days. At 30 days, the tumors of 6 mice (60%) were completely remitted, which significantly improved the survival time of mice.

[0179] (4) Test the recurrence rate in mice after secondary tumor grafting.

[0180] ①Trial grouping and intervention methods:

[0181] Secondary tumor inoculation control group: Three 7-week-old female C57 mice that underwent the same procedure (1) were selected. They did not receive the first tumor inoculation treatment, but were subcutaneously inoculated with 1×10⁻⁶ mmol / L on day 60. 6 One LL2 tumor cell;

[0182] Secondary tumor inoculation RT+DDS group: In step (3), 6 mice in the RT+DDS group achieved complete tumor remission after day 60. Three of these mice that achieved complete tumor remission were selected for a second subcutaneous inoculation of 1×10⁻⁶ on day 60. 6 LL2 tumor cells were analyzed to assess tumor recurrence.

[0183] ② Monitoring of tumor size and growth status in mice:

[0184] Changes in tumor volume and survival rate in mice after secondary tumor grafting are as follows: Figure 17 As shown, after the second tumor graft, obvious tumors were observed in the control group, with the tumor volume increasing and the survival rate gradually decreasing; while the mice that had undergone radiotherapy combined with drug delivery system treatment did not have obvious tumor recurrence, and no mice died due to excessively large tumors.

[0185] (5) Test the long-term immune memory of mice in complete tumor remission.

[0186] On day 60, spleens were collected from three of the six mice in the RT+DDS group that achieved complete tumor remission, along with three age-matched mice that did not receive any treatment. Single-cell suspensions were obtained and subjected to flow cytometry staining to analyze changes in the number of immune memory cells, namely central memory T cells (Tcm) and effector memory T cells (Tem), in the spleen. The results are as follows: Figure 18 As shown, the proportion of Tem cells and the proportion of Tcm cells in the spleen of mice in the RT+DDS group were significantly increased and significantly decreased, indicating that the prepared drug delivery system combined with radiotherapy can significantly enhance the immune memory capacity of mice after treatment.

[0187] Test Example 5

[0188] The effect of the drug delivery system prepared in Example 1 on improving the tumor microenvironment was evaluated.

[0189] (1) The LL2 subcutaneous tumor mouse model and experimental grouping and intervention methods were consistent with the steps of test case 4(1) and (2);

[0190] (2) Forty-eight hours after the third treatment (i.e., day 14), tumor tissues from mice in each experimental group were collected, digested to obtain single-cell suspensions, and then subjected to flow cytometry staining to analyze the proportion of each type of DC cell and CD3 / CD8 T cell in the tumor microenvironment. Statistical analysis was also performed, and the results are as follows: Figure 19As shown; ELISA was used to detect and statistically analyze the secretion levels of pro-inflammatory cytokines (IL-1β, IL-6) and anti-inflammatory cytokines (IFN-γ, IL-12p40) in the supernatant of tumor tissue. The results are as follows. Figure 20 As shown; tumor tissue sections from mice in each experimental group were prepared using HE and TUNEL staining methods to test the level of cell apoptosis, and the results are as follows. Figure 21 As shown, the results indicate that, at the tissue level, the drug delivery system prepared in Example 1, when combined with radiotherapy, can significantly kill tumor cells and induce apoptosis.

[0191] Test Example 6

[0192] Biosafety of the drug delivery system prepared in Example 1 was tested.

[0193] Mice from the control group (Example 5), the lanthanum-LIP group, the UDDS group, and the DDS group were subjected to three treatments. 48 hours after treatment (day 14), heart, liver, spleen, lung, and kidney organs were collected, fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for observation. The results are as follows: Figure 22 As shown in the figure. Simultaneously, the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) expressed in the liver, and the levels of creatinine and urea nitrogen expressed in the kidneys in mouse peripheral blood serum were detected using AST assay kits, ALT assay kits, creatinine assay kits, and urea nitrogen assay kits, respectively. The results are shown in the figure. Figure 23 As shown, this indicates that there was no significant damage to the liver and kidney functions of the mice.

[0194] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0195] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0196] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A drug delivery system for improving the tumor microenvironment, characterized in that, The drug delivery system includes liposomes, an antitumor drug loaded in the liposomes, and polymer particles encapsulated in the core of the liposomes; The polymer particles consist of PC7A polymer and Poly(I:C) polymer; The antitumor drug is selected from RXRα agonists, ITPR2 inhibitors, or MCU inhibitors; The RXRα agonist is selected from roximate, SR11237, CD3257, GW0791, or isomers of the aforementioned drugs, or pharmaceutically acceptable salts of the aforementioned drugs.

2. The drug delivery system for improving the tumor microenvironment according to claim 1, characterized in that, The ITPR2 inhibitors include 2-aminoethyl diphenylboronic acid ester or Xestospongin-C; Preferably, the MCU inhibitor includes ruthenium red, Ru265, or Ru360.

3. The drug delivery system for improving the tumor microenvironment according to claim 1 or 2, characterized in that, The raw materials for preparing the liposomes include phospholipids, cholesterol, and tumor cell membrane materials; Preferably, the tumor cells include lung cancer cells; Preferably, the tumor cell membrane material is a fragment of the tumor cell membrane; Preferably, the mass ratio of the phospholipids, cholesterol, and tumor cell membrane material is (10-200):(10-20):1, and more preferably (50-100):(10-20):1; Preferably, the phospholipid includes soybean lecithin.

4. The drug delivery system for improving the tumor microenvironment according to any one of claims 1 to 3, characterized in that, The mass ratio of the PC7A polymer to the Poly(I:C) polymer is 1:(0.2-10), preferably 1:(0.5-2); Preferably, the number-average molecular weight of the PC7A polymer is 4000 to 7000; Preferably, the number-average molecular weight of the Poly(I:C) polymer is 500 to 800.

5. The drug delivery system for improving the tumor microenvironment according to any one of claims 1 to 4, characterized in that, The mass ratio of the antitumor drug to the liposomes is 1:(10-20); Preferably, the mass ratio of the polymer particles to the liposomes is (1-10):(1-10).

6. A method for preparing a drug delivery system for improving the tumor microenvironment as described in any one of claims 1 to 5, characterized in that, The preparation method includes: preparing liposomes loaded with antitumor drugs; mixing polymer particles with liposomes loaded with antitumor drugs, extruding the mixture into a film, and obtaining a drug delivery system.

7. The preparation method according to claim 6, characterized in that, The preparation of liposomes loaded with antitumor drugs includes either of the following two methods: (a) The antitumor drug is mixed with phospholipids, cholesterol and tumor cell membrane materials and dissolved in an organic solvent. The solvent is removed by rotary evaporation to form a lipid film. The film is then hydrated to obtain liposomes loaded with the antitumor drug. (b) The antitumor drug is mixed with phospholipids and cholesterol and dissolved in an organic solvent. The solvent is removed by rotary evaporation to form a lipid film. The film is then hydrated and mixed with tumor cell membrane material to obtain liposomes loaded with antitumor drugs.

8. The preparation method according to claim 6 or 7, characterized in that, The method for preparing the polymer particles includes the following steps: PC7A polymer and Poly(I:C) polymer were mixed in water and self-assembled to obtain polymer particles; Preferably, the mixing temperature is 20–40°C and the mixing time is 5–300 s.

9. The preparation method according to any one of claims 6 to 8, characterized in that, The polymer particles are mixed with the liposomes loaded with antitumor drugs at a temperature of 20–40°C for a time of 0.5–10 min.

10. The use of a drug delivery system for improving the tumor microenvironment as described in any one of claims 1 to 5 in the preparation of antitumor drugs.

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